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2018-04-03T00:21:54.265Z | 2008-02-27T00:00:00.000 | 208927680 | {
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} | pes2o/s2orc | 1-Phenyl-3-(pyren-1-yl)prop-2-en-1-one
The title compound, C25H16O, was prepared by the condensation reaction of pyrene-1-carbaldehyde and acetophenone in ethanol solution at room temperature. The phenyl ring forms a dihedral angle of 39.10 (11)° with the pyrene ring system. In the crystal structure, adjacent pyrene ring systems are linked by aromatic π–π stacking interactions, with a perpendicular interplanar distance of 3.267 (6) Å and a centroid–centroid offset of 2.946 (7) Å.
The title compound, C 25 H 16 O, was prepared by the condensation reaction of pyrene-1-carbaldehyde and acetophenone in ethanol solution at room temperature. The phenyl ring forms a dihedral angle of 39.10 (11) with the pyrene ring system. In the crystal structure, adjacent pyrene ring systems are linked by aromaticstacking interactions, with a perpendicular interplanar distance of 3.267 (6) Å and a centroid-centroid offset of 2.946 (7) Å . (Bruker, 2002); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 1997); program(s) used to refine structure: SHELXL97 (Sheldrick, 1997); molecular graphics: SHELXTL (Sheldrick, 1990); software used to prepare material for publication: SHELXTL.
Comment
Chalcone derivatives have always been of interest in the field of inorganic, organic and physical chemists and biology (Strack, 1997) due to their importance in many organic synthetic pathways, biochemical processes and enzymatic mechanisms (Ansari et al., 2005;Pattanaik et al., 2002;Nielsen et al., 2005). In this paper, we report the crystal structure of the title compound, which was obtained by the condensation reaction of pyrene-1-carbaldehyde and acetophenone in ethanol solution at room temperature.
In the title compound, the pyrene ring is substantially planar (maximum displacement 0.011 (4) Å for C12) and forms a dihedral angle of 39.10 (11)° with the phenyl ring. In the crystal packing, adjacent pyrene rings are linked by aromatic π-π stacking interactions, with a centroid-centroid distance of 4.339 (7) Å, a perpendicular interplanar distance of 3.267 (6) Å and a centroid-centroid offset of 2.946 (7) Å.
Experimental
The title compound was prepared by the condensation reaction of pyrene-1-carbaldehyde (0.05 mol) and acetophenone (0.05 mol) in ethanol (20 ml) at room temperature. Single crystals of the title compound suitable for X-ray measurements were obtained by slow evaporation of an ethanol/acetonitrile solution (1:1 v/v) at room temperature.
Refinement
All H atoms were fixed geometrically and were treated as riding on the parent C atoms, with C-H distances of 0.93 Å.
U iso (H) = 1.2 U eq (C). In the absence of significant anomalous scattering effects, Friedel pairs were merged in the final refinement. Fig. 1. The molecular structure of the title compound showing 50% probability displacement ellipsoids and the atom-numbering scheme.
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes.
Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > 2sigma(F 2 ) is used only for calculat-supplementary materials sup-3 ing R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger. | v3-fos-license |
2018-04-03T05:18:09.232Z | 1992-01-25T00:00:00.000 | 26015066 | {
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} | pes2o/s2orc | The functional characteristics of a human apolipoprotein E variant (cysteine at residue 142) may explain its association with dominant expression of type III hyperlipoproteinemia.
Type III hyperlipoproteinemia typically is associated with homozygosity for apolipoprotein (apo) E2(Arg158----Cys). Dominant expression of type III hyperlipoproteinemia associated with apoE phenotype E3/3 is caused by heterozygosity for a human apoE variant, apoE3(Cys112----Arg, Arg142----Cys). However, this apoE3 variant was not separable from the normal apoE3 in these patients' plasma because the two proteins have identical amino acid composition, charge, and molecular weight. Therefore, to determine the functional characteristics of this protein, we used recombinant DNA techniques to produce this apoE variant in bacteria. We also produced a non-naturally occurring variant, apoE(Arg142----Cys), that had only the cysteine substituted at residue 142. These two apoE variants were purified from cell lysates of the transfected Escherichia coli by ultracentrifugal flotation in the presence of phospholipid, by gel filtration chromatography, and by heparin-Sepharose chromatography. Both Cys142 apoE variants bound to lipoprotein receptors on human fibroblasts with only about 20% of normal binding activity. Therefore, cysteine at residue 142, not arginine at residue 112, is responsible for the decreased receptor binding activity of the variants. Cysteamine treatment and removal of the carboxyl-terminal domain had little effect on the binding activity, whereas both modulate the receptor binding activity of apoE2(Arg158----Cys). The mutation at residue 142 decreased the binding activity of apoE to both heparin and the monoclonal antibody 1D7 (this antibody inhibits receptor binding of apoE), whereas apoE2(Arg158----Cys), which is associated with recessive expression of type III hyperlipoproteinemia, binds normally to both. The Arg112, Cys142 variant predominantes 3:1 over normal apoE3 in the very low density lipoproteins of plasma from an affected subject, as assessed by differential reactivity with the antibody 1D7. The unique combination of functional properties of the Arg112, Cys142 variant provides a possible explanation for its association with dominant expression of type III hyperlipoproteinemia.
The Functional Characteristics of a Human Apolipoprotein E Variant (Cysteine at Residue 142) May Explain Its Association with Dominant Expression of Type I11 Hyperlipoproteinemia"
(Received for publication, June 25,1991) Yukio Horie, Sergio Fazio, John R. Westerlund, Karl H. Weisgraber, and Stanley C. Rall, Jr.$
From the Gladstone Foundation Laboratories for Cardiovascular Disease, Cardiovascular Research Institute, University of California, San Francisco, California 94140-0608
Type I11 hyperlipoproteinemia typically is associated with homozygosity for apolipoprotein (apo) E2(Arg1" + Cys). Dominant expression of type 111 hyperlipoproteinemia associated with apoE phenotype E3/3 is caused by heterozygosity for a human apoE variant, apoE3(Cys112 4 Arg, Arg'42 4 Cys). However, this apoE3 variant was not separable from the normal apoE3 in these patients' plasma because the two proteins have identical amino acid composition, charge, and molecular weight. Therefore, to determine the functional characteristics of this protein, we used recombinant DNA techniques to produce this apoE variant in bacteria. We also produced a non-naturally occurring variant, a p~E ( A r g '~~ + Cys), that had only the cysteine substituted at residue 142. These two apoE variants were purified from cell lysates of the transfected Escherichia coli by ultracentrifugal flotation in the presence of phospholipid, by gel filtration chromatography, and by heparin-Sepharose chromatography. Both C Y S '~~ apoE variants bound to lipoprotein receptors on human fibroblasts with only about 20% of normal binding activity. Therefore, cysteine at residue 142, not arginine at residue 112, is responsible for the decreased receptor binding activity of the variants. Cysteamine treatment and removal of the carboxylterminal domain had little effect on the binding activity, whereas both modulate the receptor binding activity of apoE2(ArglS8 + Cys). The mutation at residue 142 decreased the binding activity of apoE to both heparin and the monoclonal antibody 1D7 (this antibody inhibits receptor binding of apoE), whereas apoE2(Arg1" 4 Cys), which is associated with recessive expression of type I11 hyperlipoproteinemia, binds normally to both. The Argil2, Cys"' variant predominates 3:l over normal apoE3 in the very low density lipoproteins of plasma from an affected subject, as assessed by differential reactivity with the antibody 1D7. The unique combination of functional properties of the Argil2, Cys142 variant provides a possible explanation for its association with dominant expression of type 111 hyperlipoproteinemia.
* This work was supported in part by National Institutes of Health Program Project Grant HL41633. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Human apolipoprotein (apo)' E, a 299-residue apolipoprotein that is a component of several classes of lipoproteins, plays a key role in the receptor-mediated uptake of these lipoproteins (1). The three major alleles of apoE code for three major isoforms (E2, E3, E4), which can be distinguished by isoelectric focusing (2); apoE3 is the most frequent and is considered the wild type, and apoE4 and -E2 are considered variants (3). Whereas apoE3 and apoE4 bind normally to receptors, apoE2 is defective in interacting with receptors, and this dysfunction has been shown to be an underlying cause of type I11 hyperlipoproteinemia (3). This protein, apoE2(Arg15' + Cys), has only 1% of the normal activity in in vitro assays (4). Type I11 hyperlipoproteinemia is almost invariably associated with homozygosity for apoE2(Arg15' -+ Cys); however, while about 1% of all individuals have the E2/2 phenotype, only 2-10% of them actually develop type I11 hyperlipoproteinemia (5). This indicates that other factors are required for overt expression of the hyperlipidemia (3,5).
In 1983, Have1 et al. ( 6 ) described members of a family of Salvadoran origin who had type I11 hyperlipoproteinemia but whose apoE phenotype was E3/3. In 1989, Rall et al. (7) reported that this type I11 hyperlipoproteinemia is caused by heterozygosity for an apoE3 variant with arginine at residue 112 and cysteine at residue 142 (Cys"' -+ Arg, ArgI4' + Cys). All the subjects with this variant have @-very low density lipoproteins and other characteristics of type I11 hyperlipoproteinemia in spite of the presence of normal E3. The transmission of this disorder is apparently a dominant trait, whereas typical type I11 hyperlipoproteinemia results from the inheritance of two alleles for apoE2(Arg1" + Cys) and therefore appears to be a recessive trait.
Rall et al. (7) demonstrated that in patients with the Argil2, C Y S '~~ variant the total apoE from the d < 1.02 g/ml lipoprotein fraction of their plasma has about 20% of normal binding activity. However, the variant apoE could not be separated from normal apoE3 because both have an identical amino acid composition, charge, and molecular weight. Therefore, to determine the characteristics of this variant, it was necessary to produce this protein by recombinant DNA techniques. In this study we produced this apoE variant by expression in bacteria and investigated its binding activity to the low density lipoprotein (LDL) receptor. We also tested its binding affinity for heparin and monoclonal antibody 1D7 (which inhibits receptor binding activity of apoE) because their binding sites are located in the region of residues 140-150 (8,9), and arginine a t residue 142 could be one of the most important ' The abbreviations used are: apo, apolipoprotein; VLDL, very low density lipoprotein(s); LDL, low density lipoprotein(s); bp, base pair(s); DMPC, dimyristoylphosphatidylcholine; IDL, intermediate density lipoprotein(s); HDL, high density lipoprotein(s). residues for these interactions. We report that the substitution of cysteine for arginine at residue 142 in apoE causes a reduction in binding to the LDL receptor and to heparin and antibody 1D7. We also demonstrate that the Arg"', C Y S '~~ apoE variant predominates in the very low density lipoproteins (VLDL) from the plasma of an affected subject.
Construction of Human Apolipoprotein E Variant Expression Vec-
tors-Two human apoE variant expression vectors were designed one coded for the naturally occurring variant (Cys"' + Arg, ArgI4' + Cys), and the other coded for a protein whose only mutation was cysteine instead of arginine a t residue 142. This latter variant was produced to enable us to distinguish the relative contributions of residues 112 and 142 to receptor binding. To produce these variant plasmids, mutagenesis linkers were prepared using the polymerase chain reaction. A portion of DNA subcloned from the patient's genomic DNA (7) that included both the residue 112 and 142 mutations was amplified using primer PCREl (10) (nucleotides 3616-3637 of the apoE gene (11)) and PCRE3 (nucleotides 3988-3968 of the apoE gene). The mutagenesis linkers were prepared by digestion of this amplified DNA with specific restriction enzymes. The mutagenesis linker contains both mutations (Argil2 and Cys14') when amplified DNA is digested with Sty1 and NarI. On the other hand, the mutagenesis linker contains only the mutated codon for CysI4' when digested with Sac11 and NarI (all restriction enzymes were purchased from New England BioLabs, Beverly, MA). The apoE3 expression vector PTV194 (12), which was digested with the same enzymes as the linker, was ligated with the linker and transformed into Escherichia coli cells.
To produce the expression vector for the amino-terminal, 22-kDa fragment (residues 1-191) of normal apoE3, a stop codon was inserted into PTV194 at the position corresponding to amino acid 192. The same method was used to produce the vector for the 22-kDa fragment of the Arg"', CYS'~' variant.
Screening of Colonies-After transformation, colonies resistant to ampicillin were screened by several methods. First, plasmids extracted from transfected cells were digested with the restriction enzyme FspI. There is one FspI site in the normal apoE sequence that corresponds t o amino acid 142 when arginine is present, and there is one site in the plasmid itself. Therefore, this enzyme digests the normal apoE vector into two fragments, while vectors that contain the mutation a t residue 142 are only linearized because of the loss of this restriction site. Second, bacterial pellets were examined for their expression of apoE protein by immunoblotting of sodium dodecyl sulfate-polyacrylamide gels with a polyclonal antibody against human apoE. Third, a portion of apoE DNA was amplified from total DNA extracted from these plasmids using primers PCREl and PCRE2 (10) (nucleotides 3914-3893 of the apoE gene) and then digested with restriction enzyme HhaI. Digestion sites of HhaI include sites corresponding to amino acids 112 and 142 when arginine is present a t these sites (13). As shown in Fig. L4, the 66-base pair (bp) fragment is specific for the mutation at residue 142 and the 72-bp fragment for that at residue 112. Therefore, the HhaI genotyping method for apoE (13) can be used to identify this rare apoE variant in screening studies, based on the simultaneous presence of the 72-and 66-bp fragments. As shown in Fig Finally, the entire DNA sequence of the linker portion of DNA from all positive colonies was sequenced by the dideoxynucleotide chain termination method (14).
Production and Purification of Apolipoprotein E Variants and Protein Analysis-The E. coli expressing the apoE variant protein was grown in 10 liters of LB medium a t 30 "C and then induced by raising the temperature to 42 "C for 20 min (12). After 20 min of induction, the fermented medium was cooled by adding crushed dry ice and then centrifuged a t 4000 X g for 25 min. The pellet was collected and then lyophilized. This lyophilized cell "cake" was sonicated in the presence of dipalmitoylphosphatidylcholine, and the phospholipid-bindingproteins were floated by ultracentrifugation at a density of 1.21 g/ml, as described (15). The floated sample was dialyzed, lyophilized, and delipidated. The delipidated protein was purified by gel filtration chromatography and heparin-Sepharose chromatography. To produce 22-kDa fragments, the induction was carried out for 45 min. Analytic protein methods, including isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were performed as described (16). Charge modification with cysteamine was performed as described (16).
Receptor Binding Assays-Receptor binding assays of isolated apoE-dimyristoylphosphatidylcholine (DMPC) complexes were performed using cultured human fibroblasts with '"1-LDL as competitor (17). Charge modification with cysteamine was performed as described (18). Either bacterially produced apoE3 or apoE3 obtained from human plasma was used as a control because both have nearly identical receptor binding activities (12). Direct binding assays were performed as described (19), using apoE radioiodinated to specific activities similar to those previously reported (19).
Heparin Binding and Monoclonal Antibody 1 0 7 Binding-The affinity of apoE for heparin was measured as the concentration of NH4HCOa at which the protein eluted from a heparin-Sepharose column (9). Briefly, the test protein was bound to the heparin-Sepharose column, washed with 15 mM NH4HC03 containing 0.1% P-mercaptoethanol, and eluted with a 15-750 mM NH4HCOs gradient containing 0.1% P-mercaptoethanol. The elution profile was recorded a t 280 nm, and 2-ml samples were collected. The concentration of NH4HC03 was calculated from the conductivity of the fractionated sample.
The binding affinity of the 22-kDa fragment of the variant apoE for monoclonal antibody 1D7 was measured in a competitive binding assay against the 22-kDa fragment of normal apoE3, which was coated in the wells as described (8). Immunoblot analysis with 1D7 was conducted using '"I-sheep anti-mouse immunoglobulin G as a second antibody.
For both heparin binding and 1D7 competition experiments, bacterially produced 22-kDa fragments were used. This was done to avoid any possible difficulties due to the intact protein being tetrameric in free solution (the 22-kDa fragment is monomeric) (20) or to the existence of a second heparin-binding site in the carboxyl-terminal domain (9).
Lipoprotein Distribution of the Apolipoprotein E Variant in the Plasma of an Affected Subject-Plasma was obtained from one of the affected subjects (111-4 in Refs. 6 and 7) by centrifugation at 2000 rpm for 15 min a t 4 "C. A 2 0 0 4 sample of plasma was subjected to Superose 6 chromatography (1 X 30 cm, Pharmacia LKB Biotechnology Inc.). Lipoproteins were separated at a flow rate of 0.5 ml/ min at ambient temperature in 150 mM NaCl, 10 mM sodium phosphate, 1 mM EDTA, pH 7.4. Fifty-five 0.5-ml fractions were collected. The VLDL, intermediate density lipoprotein (IDL)/LDL, and high density lipoprotein (HDL) fractions were determined by measurement of cholesterol (Spectrum cholesterol reagent, Abbott Laboratories) and triglycerides (Triglycerides GPO Reagent Set, Boehringer Mannheim). The pooled VLDL, IDL/LDL, and HDL fractions were concentrated using Centricon filters (Amicon). Lipid-binding proteins were precipitated with Cab-0-Si1 (Sigma) and eluted with gel loading buffer (21). Approximately equal amounts of total apoE from each fraction were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel (12%). Proteins were transferred to nitrocellulose and immunoblotted first with antibody 1D7 and "'I-sheep anti-mouse immunoglobulin G and autoradiographed for 16 h. The same nitrocellulose filter was then immunoblotted with polyclonal rabbit antihuman apoE antisera and 12sI-goat anti-rabbit immunoglobulin G and autoradiographed for 2 h. The autoradiographs were then subjected t o densitometric analysis.
Purification of Apolipoprotein E Variants and Protein
Analysis-The intact apoE variants were purified as summarized in Fig. 2 A . In the bacterial pellet, an apoE band could not be distinguished clearly (lane 1 ). After phospholipid flotation, apoE became a major component (lane 2). Pure apoE was obtained after further steps of gel filtration (lane 3 ) and heparin affinity chromatography (lane 4 ) . The 22-kDa fragment was purified by the same methods (Fig. 2B); however, it did not need the final heparin affinity chromatography step because the 22-kDa fragment of the apoE variant was pure after gel filtration (Fig. 2B, lane 3 ) . To ascertain the isoelectric focusing position and the number of cysteines of these variants, isoelectric focusing was performed without and with cysteamine treatment. These variants showed the expected charge and cysteine number (data not shown).
Receptor Binding Assays-Receptor binding activities of the variants were determined in an in vitro competition assay, using the 50% competition point from logit-log plots of the binding data as a measure of activity. Both the naturally occurring Arg"', CYS'~' variant and the non-naturally occurring Cys"', CYS'~' variant were defective in binding to lipoprotein receptors on cultured human fibroblasts. A representative competition experiment is shown in Fig. 3, and a summary of all experiments is presented in Table I. The Arg"', Cys14' variant averaged 21% of normal binding, and the Cys"', CYS'~' variant slightly less; the difference was mostly due to one experiment (no. 2 in Table I).
Direct binding assays were performed with Iz5I-apoE. DMPC complexes to confirm the competition data. From Scatchard analysis of the binding data, normal apoE3 had a Kd = 0.10 nM, the same as previously reported by Pitas et al. (19). In contrast, the Arg"', CYS'~' variant had a Kd = 0.27 nM and the CYS'~', Cyd4' variant had a Kd = 0.24 nM. The variants had slightly higher BmaX compared with normal apoE3 (1.4 ng/35-mm dish uersus 1.1 ng/35-mm dish). Therefore, the variants display a reduced affinity for binding to LDL receptors.
We also measured the receptor binding activities of a 1:l mixture of each variant apoE and normal E3 because Rall et al. (7) had shown that the total apoE purified from the d < 1.02 g/ml lipoprotein fraction from the plasma of patients had 19-26% of normal activity. This artificial 1:l mixture exhibited only -40% of control activity (Table I), although this activity was higher than that of the previously reported plasma mixture. To determine whether the manner by which the mixture was prepared had an effect on receptor binding activity, we made two further 1:l mixtures: a 1:l protein mixture that had been denatured by guanidine before recombination with DMPC, and a 1:1 mixture of DMPC complexes previously formed with either apoE3 or the apoE variant. All three mixtures bound almost equally, indicating that the method of mixing had no impact on binding activity (data not shown).
To assess the effect of cysteamine treatment on receptor binding, the apoE variants were incubated with cysteamine before being complexed with DMPC. Modifications were verified by isoelectric focusing (data not shown). Cysteamine treatment increased the binding activity of the Arg"', CysI4 variant by 68% (n = 2) and of the C~S "~, CYS'~' variant by 45% (n = 2), values similar to the 30% activation for control apoE3 (4). Thus, cysteamine has little effect on these variants, in marked contrast to its 14-fold effect (n = 1) on apoE2, a finding in agreement with previous data (4,18).
To assess the effect of removing the carboxyl-terminal domain, purified 22-kDa fragments were tested for receptor binding activity. The binding activity of the 22-kDa fragment of the Arg'l', CYS'~' variant was only 47% (n = 3) of that of the normal apoE3 22-kDa fragment. Cysteamine treatment of the variant 22-kDa fragment did not increase the activity significantly (21%) (n = 3). This also contrasts markedly with cysteamine treatment of the 22-kDa fragment of apoE2(Arg15' + Cys), whose binding activity increased over 6-fold (i.e. to normal) after cysteamine treatment (18).
Heparin Biding Affinity-To assess the effect of the mutation at residue 142 on heparin binding, heparin-Sepharose gradient chromatography of the 22-kDa fragments of normal apoE3 and the Arg'l', CYS'~' variant was conducted. The 22-kDa fragment of normal apoE3 eluted at a salt concentration of 105 mM NH,HCO, (Fig. 4, upper panel), whereas the variant 22-kDa fragment eluted at 63 mM NH4HC03 (Fig. 4, lower panel). This indicated that the presence of Cys14' leads to a weaker affinity for heparin and that Arg14' is a major contributor to apoE-heparin binding. Antibody 1 0 7 Binding Affinity-To assess the effect of the mutation at residue 142 on the binding to monoclonal antibody 1D7, a competition study was performed using antibody 1D7 and the 22-kDa fragments of apoE. The mutation at residue 142 substantially decreased the binding activity of apoE to 1D7 (Fig. 5A). The concentration at which the Argil', Cys14' variant 22-kDa fragment displaced 50% of the 1D7 from the immobilized apoE3 22-kDa fragment was 23.2 pg/ ml (n = 4), compared with 0.81 pg/ml (n = 4) for the 22-kDa fragment of normal apoE3. Therefore, this apoE variant has only about 3.5% of normal affinity for antibody 1D7. Furthermore, immunoblot analysis indicated that antibody 1D7 failed to detect the variant apoE 22-kDa fragment under the incubation and washing conditions used (Fig. 5B).
Lipoprotein Distribution of the Arg"' , Cys14' Variant-To measure the relative amounts of the Arg"', Cys14' variant and normal apoE3 directly in the lipoprotein fractions from a type I11 hyperlipoproteinemic subject heterozygous for the variant (7), we took advantage of the immunoreactivity difference of the variant for antibody 1D7 (see Fig. 5). As shown in Fig. 6, antibody 1D7, which detects essentially only normal apoE, detected much less apoE in the VLDL fraction compared with either the IDL/LDL or HDL fractions. The polyclonal antibody, which detects equally the variant and normal apoE, was used to normalize any differences in the total amount of apoE In both panels, the absorbance peak at fractions 7-8 contained very little protein as assessed by sodium dodecyl sulfate-polyacrylamide gels or colorimetric assays but was reproducibly seen at the beginning of the gradient. The absorbance was likely due to a nonprotein contaminant in the preparations. among fractions (Fig. 6). Densitometric comparisons indicated that normal apoE3 accounted for 27% of the total apoE in VLDL, 50% in IDL/LDL, and 55% in HDL. Therefore, the Argl'', Cys14' variant is overrepresented in the VLDL fraction from the type I11 hyperlipoproteinemic subject, there being almost three times more variant than normal apoE3 in this fraction.
DISCUSSION
A human apoE variant with cysteine substituted for arginine at residue 142 is known to be associated with dominant transmission of the genetic lipid disorder type I11 hyperlipoproteinemia (7). We have produced this variant in bacteria and investigated the impact of this particular substitution on several important interactions: apoE binding to LDL receptors; its affinity for the monoclonal antibody 1D7, which blocks apoE binding to those receptors; and its affinity for heparin. Arginine 142 is one in a cluster of six basic residues in the 140-150 region of apoE that has previously been implicated in these interactions (1,8,9). Crystallographic analysis has shown that these basic residues occur in an After staining with Coomassie Blue, the protein in the gel was transferred to nitrocellulose paper. Transferred proteins were incubated with antibody ID7 followed by 12sI-sheep antibody against mouse immunoglobulin G. Autoradiography was performed for 16 h. polyclona! FIG. 6. Distribution of the Arg"', Cys'"' variant among an affected subject's plasma lipoproteins. Plasma was fractionated on a Superose 6 column. The resultant VLDL, IDL/LDL, and HDL fractions were pooled, concentrated, and the apoproteins precipitated with Cab-0-Sil. After dissolution in gel running buffer, the proteins were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel (12%) and blotted to a nitrocellulose filter. After incubation with antibody 1D7 and autoradiography for 16 h (upper panel), the filter was incubated with the polyclonal antibody and autoradiographed for 2 h (lowerpanel). Because of the much shorter exposure time required for the polyclonal antibody, the contribution of radioactivity from the 1D7 incubation (upper panel) contributes only minimally to the radioactivity in the lower panel. The band below apoE in the upper panel is recognized nonspecifically by the "'1-sheep anti-mouse secondary antibody since that band is present when incubation with 1D7 is omitted. extended a-helical segment in which all their side chains are exposed to the aqueous environment (22), making it possible for them to participate in binding to the clusters of negatively charged amino acid residues of the LDL receptor.
Our results demonstrate that the cysteine-for-arginine sub-stitution at residue 142',has a profound effect on all three interactions. This substitution reduces receptor binding to about 20% that of normal apoE3. Because the naturally occurring human variant also has a substitution of arginine for cysteine at residue 112, we also produced a Cys112, C Y S '~~ variant so that we also could assess the role of residue 112 in receptor binding. This latter variant was only slightly less active than the Arg"', C Y S '~~ variant, indicating that most, if not all, of the receptor binding defect is due to the presence of C Y S '~~ and not to the substitution at residue 112. This result is in agreement with previous findings that showed that there was no difference in receptor binding between apoE3 (cysteine at residue 112) and apoE4 (arginine a t residue 112) (4). With the exception of apoE2(ArglSR + Cys), which has about 1% of normal activity, the -20% receptor binding activity of the Arg'12, Cys14' apoE variant is the lowest of the naturally occurring variants. Using site-specific mutagenesis, Lalazar et al. (15) reported two apoE variants that have lower binding activity than these variants with CYS'~~: one with a substitution of alanine for lysine at residue 143 (9%), and the other with proline for leucine a t residue 144 (13%). This .suggests that residues 142-144 are especially crucial for binding to the LDL receptor.
We also determined the receptor binding activities of the cysteamine-treated Argil2, CYS'~' variant and its 22-kDa fragment, because cysteamine treatment and removal of the carboxyl-terminal domain have a profound effect on the receptor binding activity of apoE2(ArglS8 4 Cys) (18). Both of these modifications enhanced the receptor binding of ap0E2(Arg"~ + Cys) by an order of magnitude, and the cysteamine-treated 22-kDa fragment of apoE2(ArgI5' + Cys) actually had normal binding (18). In contrast, the binding activity of the Arg"', C Y S '~~ variant was enhanced relatively little by either modification, and cysteamine treatment of its 22-kDa fragment failed to normalize binding activity. This relative lack of susceptibility to modulation of receptor binding activity supports the notion that the cluster of basic residues in the 140-150 region binds through direct ionic interaction with the negatively charged residues of the ligand-binding domain of the LDL receptor (l), whereas the substitution of cysteine at residue 158 in apoE2(Arg1"+ Cys) indirectly affects receptor binding by local conformational perturbations (18).
It has been reported that the total apoE from the subjects heterozygous for the Arg"*, Cys14' variant had 19-26% of normal receptor binding activity (7). Based only on peptide yields from sequence data, Rall et al. (7) inferred that the total apoE isolated from the d < 1.02 g/ml lipoproteins from the plasma of subjects with this variant was approximately a 1:l mixture of the variant and normal apoE3. Because our results indicated that the isolated Argil2, C Y S '~~ variant had 20% of normal binding by itself, we also determined the binding activity of 1:l mixtures of the variant and normal apoE3. These artificial mixtures had approximately double the binding activity of the variant alone, suggesting that it is unlikely that the total apoE from the subjects is in a 1:l ratio; instead, it is probable that the defective variant apoE predominates, at least in the d < 1.02 g/ml lipoproteins, in spite of the previous estimation (7). This conclusion was confirmed directly by the data in Fig. 6, in which the defective variant apoE predominates almost 3:l over normal apoE3 only in the VLDL fraction from the plasma of an affected subject. We have demonstrated that the substitution at residue 142 also reduces apoE affinity for heparin (Fig. 4). Previously, Weisgraber et al. (9) showed that the high affinity heparinbinding site on apoE coincided with the receptor-binding site, with the cluster of basic residues again being implicated as Characteristics of ApoE3(Cys112 --., Arg, Arg142 4 Cys) 1967 crucial for the interaction. Our results also support this contention. The physiological significance of the heparin interaction with apoE is uncertain, but heparin is known to interact with lipoproteins, and heparin-like molecules on the cell surface of the vascular endothelium may play a role in lipoprotein processing (23) and even in the development of atherosclerosis (24). If this is the case, then the reduced affinity of the ArgilZ, Cys"' variant for heparin-like molecules may interfere with normal processing of triglyceride-rich lipoproteins and contribute to the accumulation of these lipoproteins in the plasma of affected subjects.
It has been shown that the epitope for monoclonal antibody 1D7 lies in the 130-150 region of apoE, and it was postulated that the important residues of the epitope are the cluster of basic residues at positions 142, 143, 145, 146, and 147 (8). In support of this, Weisgraber et al. (8) determined that natural apoE variants with single substitutions at residue 145 or 146 had 49 or 32%, respectively, of normal apoE3 affinity for antibody 1D7. The results of the present study indicate that the substitution of cysteine at residue 142 leads to a dramatic reduction in apoE affinity for antibody 1D7, to less than 5% of normal (Fig. 5). Thus, Arg"' is a critical determinant of the antibody 1D7 epitope. The functional characteristics of the Arg"' , Cys14' variant provide a possible explanation for its association with dominant expression of type I11 hyperlipoproteinemia. The receptor binding defect alone cannot be the explanation for dominant transmission. Although this variant is defective in receptor binding, it is less defective than apoE2(Arglm 4 Cys) in in vitro assays, and Chappell (25) has demonstrated that j3-VLDL from subjects with the Argil', Cys14' variant has higher binding affinity than does j3-VLDL from E2/2 subjects.
Part of the explanation for dominant expression may lie with the distribution of the variant apoE. Because any individual triglyceride-rich lipoprotein particle will have multiple copies of apoE and because in heterozygous subjects the normal and defective apoE probably distribute randomly, some lipoprotein particles will have more of the variant than of the normal apoE and vice versa. Over time, those particles with more of the normal apoE will be cleared from the circulation more efficiently, whereas the clearance of particles with more of the receptor binding-defective apoE variant will be retarded. Although this differential distribution of normal and variant apoE also occurs in E3/2 subjects, apoE2(Arglm + Cys) is only slightly increased relative to apoE3 in their triglyceride-rich lipoproteins (5). However, this differential distribution is not nearly as pronounced as the 3:l ratio demonstrated here for the Arg"', Cys14' variant. It is known that the presence of arginine at residue 112, as occurs in this variant, confers upon apoE a preference for association with triglyceride-rich lipoproteins (26). This could account for the observed selective accumulation of the Argil2, Cys14' variant in the triglyceride-rich lipoproteins of affected subjects. Thus, it is likely that the natural preference of apoE with Arg"' for triglyceride-rich lipoproteins, in conjunction with the receptor binding defect, is a major part of the explanation for this increased selective accumulation. This may also be the case for apoE-Leiden, a variant with Arg"' that demonstrates this same selective accumulation and is associated with dominant expression of type I11 hyperlipoproteinemia (27).
Finally, if the interaction of apoE with heparin-like molecules is important for lipoprotein processing and/or catabolism in uiuo, then the lower affinity of the Arg"' , Cys14' variant for heparin may also be a contributing factor to the accumulation of 8-VLDL. This is probably not the case for apoE2(Arglfi8 4 Cys), which binds normally to heparin (9), but might be the case for any other apoE variant that has a substitution for a basic residue in the region of the heparin-/ receptor-binding site (residues 135-150). It is already known that some of these latter variants are associated with dominant expression of type I11 hyperlipoproteinemia (28).
In summary, we believe that the unique combination of functional properties of the Arg"', Cys14' apoE variant, i.e. defective receptor binding, reduced affinity for heparin, and especially its preference for association with triglyceride-rich lipoproteins, leads to excessive accumulation of j3-VLDL, which in turn is manifested in dominant expression of type I11 hyperlipoproteinemia. | v3-fos-license |
2020-12-17T09:11:42.400Z | 2021-01-01T00:00:00.000 | 229939022 | {
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} | pes2o/s2orc | High mobility group box 1 regulates gastric cancer cell proliferation and migration via RAGE-mTOR/ERK feedback loop
Gastric cancer (GC) is a common malignancy tumour in China. Despite various therapeutic approaches to improve the survival rate of GC patients, the effectiveness of currently available treatments remains unsatisfactory. High mobility group box 1 (HMGB1) is reported to play a role in tumour development. However, the molecular mechanisms involved in HMGB1-mediated regulation of proliferation and migration of GC cells remain unclear. In the present study, we demonstrated that HMGB1 is highly expressed in GC cells and tissue. In HGC-27 GC cells, HMGB1 overexpression or HMGB1 RNA interference both demonstrated that HMGB1 could promote GC cell proliferation and migration. Investigation of the underlying molecular mechanisms revealed that HMGB1 enhanced cyclins expression, induced epithelial-to-mesenchymal transition and matrix metalloproteinase (MMPs) expression and promoted RAGE expression as well as RAGE-mediated activation of Akt/mTOR/P70S6K and ERK/P90RSK/CREB signalling pathways. We also found that inhibition of ERK and mTOR using specific inhibitors reduced recombinant human HMGB1-induced RAGE expression, suggesting that the RAGE-mTOR/ERK positive feedback loop is involved in HMGB1-induced GC cell proliferation and migration. Our study highlights a novel mechanism by which HMGB1 promotes GC cell proliferation and migration via RAGE-mediated Akt-mTOR and ERK-CREB signalling pathways which also involves the RAGE-mTOR/ERK feedback loop. These findings indicate that HMGB1 is a potential therapeutic target for GC.
Introduction
Gastric cancer (GC) is a common cancer in China, and has high morbidity and mortality rates [1]. Due to a lack of early diagnostic markers and easy metastasis, the effects of GC therapy remain unsatisfactory [2]. Therefore, it is important to identify a biomarker for effective GC diagnosis and treatment.
High mobility group box 1 (HMGB1) is a highly conserved chromosomal protein located mainly in nuclei and is involved in transcription-level regulation of various genes [3]. HMGB1 can be actively or passively released into the extracellular environment, where it binds with its receptors, including receptor of advanced glycation end-product (RAGE) and toll-like receptors and regulates the development of various types of tumour [4,5]. The effect of HMGB1 on malignancy is complicated since it both promotes and counteracts malignancy [4]. Zuo
Ivyspring
International Publisher et al. showed that HMGB1 inhibits lung cancer cell metastasis by suppressing the activation of CREB and nWASP expression [6]. Luan et al. reported that HMGB1 is negatively correlated with the development of endometrial carcinoma and prevents cancer cell invasion and metastasis by inhibiting epithelial-to-mesenchymal transition (EMT) [7]. However, some studies have suggested that HMGB1 promotes cancer cell development and progression [8,9]. HMGB1/TLR4/myeloid differentiation factor 88 signalling promotes progression of GC [10]. HMGB1 promotes GC cells proliferation and migration by activating the NF-кB and ERK signal pathways [11,12]. Although increasing evidence has shown that HMGB1 can induce the progression of GC [13], the underlying molecular mechanisms remain unclear.
We previously reported that HMGB1 expression levels were significantly higher in GC cells than in normal gastric epithelial cells, and knockdown of HMGB1 enhanced aloin-induced GC apoptosis [14]. This finding indicated that HMGB1 was involved in GC progression. However, the exact mechanisms of HMGB1 in the proliferation and migration of GC remain elusive.
The present study investigated the effects of HMGB1 on GC proliferation and migration and explored the underlying molecular mechanisms involved. Our results showed that HMGB1 was expressed at higher levels in GC tissues and cells compared with adjacent non-tumour tissues and normal gastric epithelial cells, respectively. HMGB1 promoted GC HGC-27 cell proliferation and migration via RAGE-mediated Akt-mTOR and ERK-CREB signalling pathways. Furthermore, our data indicated that suppression of mTOR or ERK activation reduced the expression levels of RAGE, suggesting that the RAGE-mTOR/ERK feedback loop is involved in HMGB1-induced GC proliferation and migration. Taken together, our results highlight HMGB1 as a potential therapeutic target for GC and provide a novel perspective for the effect of HMGB1 on GC cell proliferation and migration.
Tissue microarray and immunohistochemistry (IHC) staining
GC adenocarcinoma and adjacent non-tumour tissue samples were obtained from 36 patients that underwent curative gastric cancer resection in the First Affiliated Yijishan Hospital of Wannna Medical College (Wuhu, Anhui, China). No patient received anti-tumor treatment before surgery. Sample preparation and experiment operation were conducted in accordance with ethical and legal standards, and the study was consented by the Yijishan Hospital of Wannan Medical College Ethics Committee. GC tissue microarray was produced by Service Biotechnology Co., Ltd. (Wuhan, China). HMGB1 levels were measured using HMGB1 antibody (diluted 1:200). A tissue chip scanner (Pannoramic MIDI, 3D HISTECH) was used to obtain tissue information. Briefly, Quant center is the analysis software for the Pannoramic viewer. After image scanning, the software densito Quant in Quant center was used to automatically identify and set all dark brown tissues to be strongly positive, brown yellow to be moderately positive, light yellow to be weakly positive and blue nuclei to be negative. The areas (in pixels) of strong positive, moderate positive, weak positive and negative, and the percentage of positive, were identified in each tissue point, and then histochemistry score (H-SCORE) was performed. H-SCORE = ∑(Pi×I) = (percentage of cells of weak intensity ×1) + (percentage of cells of intensive intensity ×2) + (percentage of cells of strong intensity×3), in which Pi indicates the percentage of positive cells in the total number of cells in the section; I represents the staining intensity. HMGB1 expression was semi-quantitatively estimated using the H-SCORE. The score ranges from 0 to 300, with higher scores representing a strong positive result. HMGB1 expression in GC tissue was categorised in advance into two groups: low expression HMGB1 (H-SCORE, <100) and high expression HMGB1 (H-SCORE, 100-300).
Clinical parameters and data analysis
The clinic parameters of enrolled patients were shown in Table 1. The association of HMGB1 expression and the clinical parameters of GC patients were analysed using Fisher's exact test.
Colony formation assay
After transfection, HGC-27 cells were seeded in 6-well plates and cultured in a humidified atmosphere with 5% CO 2 at 37°C for 2 weeks. The culture supernatants were discarded and the cell colonies were fixed in the wells using 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 30 min. After washing with phosphate-buffered saline (PBS), the cell colonies were observed and the numbers of colonies (≥25 cells/colony) were counted under a light microscope (Olympus, Japan).
EdU assay
HGC-27 cells were seeded in 24-well plates and the cell proliferation ability was detected by EdU assay according to the manufacturer's instructions. Cells were observed using an inverted fluorescence microscopy (100× magnification; Olympus, Tokyo, Japan). The ratio of EdU-positive stained cells (red fluorescence) to DAPI-stained cells (blue fluorescence) was calculated.
Wound healing assay
After treatment, HGC-27 cells were seeded in 12-well plates. When the cells reached monolayer confluence, the monolayer was scratched using a clean 200-μL pipette tip and the detached cells were removed by gently washing with PBS. Images were taken at 0 h and 24 h under a light microscopy (100× magnification; Olympus, Tokyo, Japan). The wound area was analysed using Image J version 1.52 software.
Transwell assay
After treatment, HGC-27 cells were suspended in serum-free 1640 medium. A 200-μL cell suspension containing 2 × 10 4 cells was seeded in the upper chamber of transwell places (Millipore, Billerica, MA, USA) and 600 μL of 1640 medium supplemented with 20% FBS was placed in the lower chamber. After culturing for 24 h at 37°C and 5% CO 2 , cells on the upper surfaces were gently removed using a cotton swab and cells that migrated to the lower surface were fixed using 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 30 min at room temperature. Five randomly chosen fields were captured and photographed under an inverted microscope (100× magnification; Olympus, Japan). The number of migrated cells were counted using Image J version 1.52 software.
Western blotting
Western blotting was performed according to the methods described previously by Wang et al. [15]. Total protein was extracted from cells using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with phenylmethylsulfonyl fluoride (PMSF). After 30 min lysing on ice, the amount of total protein was determined using a bicinchoninic acid (BCA) kit. Equal amounts of protein in each sample were separated using 12% SDS-PAGE and transferred to nitrocellulose membranes (Pall Corporation, Port Washington, USA). The membranes were then blocked with 5% skimmed milk at room temperature for 1 h, rinsed three times with TBST and incubated with the indicated primary antibodies overnight at 4°C. The membranes were then incubated with secondary antibody for 2 h at room temperature. Antibody-antigen complexes were visualised using either a LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences) or a chemiluminescence imaging system (Clinx, Shanghai, China). Protein levels were semi-quantified using Image J 1.52 software.
Statistical analysis
Experiments were performed in triplicate and data were shown as mean ± SD. The statistical significance of differences was performed using Student's t test for comparison of two groups or one-way analysis of variance (version 17.0 SPSS, Chicago, IL, USA) for comparison of more than two groups followed by Tukey's multiple comparison test. Fisher's exact test was used to analyse correlations between HMGB1 and clinicopathological factors of patients with GC. All p-values <0.05 were considered significant.
HMGB1 was highly expressed in GC tissues and cells
Tissue microarrays including 36 pairs of tumour and adjacent normal tissues were evaluated by IHC. The positive immunohistochemical staining of HMGB1 was dark brown, and the staining intensity and number of positive cells in cancer tissues was significantly higher than that of adjacent tissues. Semi-quantitative estimation using the H-SCORE showed that the positive expression of HMGB1 was higher in GC tissues than in the adjacent normal tissue (Fig. 1A). We also measured levels of HMGB1 in GC HGC-27 and normal gastric epithelial cells GES-1 using Western blotting. Expression of HMGB1 was significantly higher in GC cells compared with normal gastric epithelial cells (Fig. 1B), suggesting that HMGB1 may regulate GC progression.
Correlation between HMGBl expression and Clinical parameters
GC patients were classified according to HMGBI immunoreactive intensity as low and high HMGB1 groups. The association between HMGB1 expression and the clinical parameters of the GC patients was analysed. As summarised in Table 1, our data indicated that there was no significant correlation between HMGB1 level and the clinicopathologic factors, such as age, gender, tumour size, TNM stage and histological grade (p > 0.05 for all).
HMGB1 promotes colony formation and proliferation in GC cells
To investigate the effects of HMGB1 on GC, HGC-27 cells were transfected with GFP-labelled HMGB1 overexpression plasmids (GFP-HMGB1) or HMGB1 shRNA interference plasmids (HMGB1 shRNA). The transfection efficiencies were verified using Western blotting. We selected 3 µg of HMGB1-GFP plasmid to transfect HGC-27 cells in subsequent experiments ( Fig. 2A). Three shRNAs targeting HMGB1 (nos. 3616, 3618 and 3619) were constructed and transfected to HGC-27 cells. The interference efficiencies of the different plasmids are shown in Fig. 2B. HMGB1 shRNA no. 3618 exhibited obvious inhibitory effects; therefore, this plasmid was used for subsequent experiments. Ta-T1 11 3 8 0.343 T2-T4 25 3 22 Next, the effects of HMGB1 on GC proliferation were assessed using EdU and colony formation assays. EdU (5-ethyl-2'-deoxyuridine) is a thymine nucleoside analogue, which can replace thymine (T) and infiltrate into the replicating DNA molecules during the cell proliferation stage. EdU positive cells (red staining) represent proliferative cells, blue staining for total cells, the percentage of red staining cells in total cells represent GC proliferation ability. The data showed that overexpression of HMGB1 enhanced HGC-27 cell proliferation compared with cells transfected with the negative plasmids. Furthermore, downregulation of HMGB1 exhibited the opposite effect (Fig. 2C, D). These results suggested that HMGB1 enhanced the proliferation ability of HGC-27 cells.
HMGB1 enhances GC cells migration
Wound healing and transwell assays were used to evaluate the effects of HMGB1 on GC migration. As shown in Fig. 3A, the healing rate of scratches clearly increased after GFP-HMGB1 transfection and the migration of HGC-27 cells was also increased following HMGB1 overexpression (Fig. 3B). However, HMGB1 knockdown noticeably decreased the wound healing rate and migration of cells (Fig. 3C, D). These findings indicated that HMGB1 increased HGC-27 cell migration.
HMGB1 affects the expression levels of cyclins, MMPs and EMT markers
To explore the mechanisms of HMGB1-mediated promotion of GC proliferation and migration, levels of cyclin D1, cyclin E1, PCNA, MMP-2, MMP-9, N-cadherin and E-cadherin were measured using Western blotting. The expression levels of cyclin D1, cyclin E1 and PCNA were increased following HMGB1 overexpression, but were attenuated following HMGB1 knockdown compared with cells transfected with negative plasmids (Fig. 4A, B). Measurement of MMP-2, MMP-9, N-cadherin and E-cadherin expression showed that overexpression of HMGB1 upregulated expression levels of N-cadherin and downregulated the expression levels of E-cadherin, MMP-2 and MMP-9, whereas knockdown of HMGB1 showed the opposite effect (Fig. 4C, D). Taken together, our data indicate that HMGB1 is able to promote the expression of cyclins and MMPs and enhance EMT in HGC-27 cells.
HMGB1 promotes activation of Akt/mTOR/ P70S6K and ERK/P90RSK/CREB signalling pathways
Akt and ERK play an important role in cancer initiation and progression [16]. To investigate the potential molecular mechanisms of HMGB1mediated regulation of GC cell proliferation and migration, we measured the activation of Akt and ERK signalling pathways using Western blotting. Phosphorylation of Akt/mTOR and ERK/P90RSK/ CREB were increased in HMGB1-overexpressed HGC-27 cells compared with cells transfected with the control plasmid, while the activation of these signalling pathways showed the opposite effects after transfection with HMGB1 shRNA. Phosphorylation of P70S6K and S6 were enhanced in response to HMGB1 overexpression, but there was no statistical difference compared with cells transfected with the control plasmid (Fig. 5A, B). Taken together, these data suggest that HMGB1 promotes GC cells proliferation and migration by enhancing the phosphorylation of Akt/mTOR and ERK signalling pathways in HGC-27 cells. with HMGB1 shRNA and negative plasmids for 48 h and HMGB1 levels were measured by Western blotting. β-actin was used as a loading control. HGC-27 cells were transfected with 3 µg of GFP-HMGB1 for 24 h or HMGB1 shRNA plasmids for 48 h, with an equivalent negative plasmid as control respectively. EdU assay (magnification, 100×) (C) and colony formation assay (D) were used to measure cell proliferation. Experiments were repeated three times and data represent mean ± SD. *p < 0.05 and ** p < 0.01 vs cells transfected with negative plasmid.
HMGB1 upregulates the expression of RAGE but not TLR2 and TLR4
Extracellular HMGB1 participates in regulating a variety of biological behaviours by binding with receptors such as RAGE, TLR2 and TLR4 [5,10]. We explored the effect of HMGB1 on its receptor expression. HGC-27 cells were transfected with HMGB1 overexpression or control plasmids and Western blotting was used to determine the levels of TLR2, TLR4 and RAGE. Overexpression of HMGB1 increased RAGE expression but not TLR2 and TLR4 (Fig. 6A), whereas knockdown of HMGB1 decreased the expression level of RAGE, but did not affect the expression of TLR2 and TLR4 (Fig. 6B). These results suggested that RAGE may mediate the activation of Akt and ERK signalling pathways induced by HMGB1.
RAGE mediates HMGB1-induced Akt and ERK signalling pathway activation
To determine whether RAGE receptor was responsible for HMGB1-induced Akt and ERK signalling pathway activation, HGC-27 cells were treated with rhHMGB1 (2 μg/mL) in the presence of the RAGE inhibitor, FPS-ZM1. rhHMGB1 enhanced the phosphorylation of Akt/mTOR/P70S6K and ERK/P90RSK signalling pathways and FPS-ZM1 pretreatment attenuated rhHMGB1-induced phosphorylation of these signalling pathways (Fig. 7A). Phosphorylation of Akt, mTOR, P70S6K, S6, ERK and P90RSK were detected by Western blotting. Their relative levels were normalised to total protein respectively. (B) HGC-27 cells were pretreated with LY29402, rapamycin and U0126 for 1 h and then treated with rhHMGB1 for 12 h and RAGE expression and of Akt, mTOR and ERK were detected by Western blotting. The relative levels of RAGE were normalised to β-actin. Data represent mean ± SD. #p < 0.05 vs control group, *p < 0.05 and **p < 0.01 vs rhHMGB1-treated cells.
Inhibition of mTOR and ERK activation attenuates rhHMGB1-induced RAGE expression
Although our results suggested that HMGB1 induced RAGE expression, the underlying molecular mechanism remained unclear. HGC-27 cells were pretreated with either LY294002, rapamycin or U0126 (specific inhibitors of Akt, mTOR and ERK, respectively) for 1 h prior to stimulation with rhHMGB1 for 12 h and measurement of RAGE level using Western blotting. Expectedly, our data showed that inhibition of mTOR and ERK activation significantly downregulated rhHMGB1-induced RAGE expression (Fig. 7B).
Inhibition of Akt and ERK activation attenuates rhHMB1-induced HGC-27 cell proliferation and migration
In order to further verify the role of Akt/mTOR and ERK in rhHMGB1-induced HGC-27 cell proliferation and migration, the effects of LY294002, rapamycin and U0126 on rhHMGB1-induced GC cell proliferation and migration were examined using EdU, colony formation, wound healing and transwell assays. Treatment with the inhibitors suppressed rhHMGB1-induced HGC-27 cell proliferation and migration (Fig. 8A-D). Taken together, the results suggested that HMGB1 regulated GC cell proliferation and migration via Akt/mTOR and ERK signalling pathways.
Discussion
GC is one of the leading causes of cancer-related mortality worldwide [17]. Its high aggressiveness and poor diagnosis are the main reasons for unsatisfactory therapeutic effects [18,19]. HMGB1 is reported to be highly expressed in many malignant tumours and is closely related to apoptosis, proliferation and migration of cancer cells [3,13]. Therefore, anticancer therapy targeting HMGB1 is attracting increased attention [20,21].
In the present study, we measured the expression levels of HMGB1 in GC tissues and cells. As previously reported [22,23], we observed that HMGB1 was highly expressed in GC tissue compared with adjacent non-cancerous tissue (Fig. 1A). This suggested that HMGB1 was involved in the progression of GC. Therefore, we examined the association between HMGB1 and clinicopathological factors, but found no correlation (Table 1). This result was consistent with the findings reported previously by Zhang et al. [11]. However, Suren et al. found that HMGB1 expression was significantly correlated with T stage and tumour differentiation [23]. These differences may be due to differences in the evaluation of staining and the number of cases studied. HMGB1 expression levels in GC cells were also measure by Western blotting. Our results showed that HMGB1 expression was higher in GC cells compared with normal gastric epithelial cells (Fig. 1B).
Due to the high expression levels of HMGB1, HGC-27 cells were used in subsequent experiments.
To determine the roles of HMGB1 in GC proliferation and migration, GFP-HMGB1 overexpression and HMGB1 shRNA plasmids were transfected in cells. Our results revealed that overexpression of HMGB1 promoted colony formation and proliferation ability in GC cell, whereas HMGB1 knockdown showed the opposite effects (Fig. 2). Both wound healing and transwell assays showed that the migration of HGC-27 cells was also enhanced by HMGB1 overexpression. Furthermore, downregulation of HMGB1 reduced the migration capacity of GC cells (Fig. 3). Our results are consistent with the findings from previous studies [11,24].
PCNA plays a key role in the initiation and extension of replication and is an excellent inhibition target to shut down highly proliferative cells [25]. Cyclins are essential proteins involved in cell cycle regulation and inhibition of cyclins expression reduces cell proliferation [26]. To further examine the effects of HMGB1 on cell proliferation and migration, we determined the expression levels of cyclins and PCNA in HGC-27 cells. Overexpression of HMGB1 increased the expression levels of cyclin D1, E1 and PCNA, whereas HMGB1 knockdown reduced their expression (Fig. 4A, B). These data suggested that HMGB1 enhanced GC cell proliferation by regulation of cyclins expression and DNA replication. EMT is a key feature of tumour metastasis [27] and downregulation of MMP-2 and MMP-9 expression indicates cancer cell invasion and metastasis [28,29]. The expression levels of MMP-2, MMP-9 and EMT marker proteins were also measured in the present study. Our data showed that overexpression of HMGB1 enhanced MMP-2, MMP-9 and N-cadherin levels and reduced E-cadherin expression, whereas knockdown of HMGB1 showed the opposite effects (Fig. 4C, D). Taken together, these results indicate that HMGB1 promotes GC cell migration by regulating EMT and MMP expression. HGC-27 cells were treated with LY29402, rapamycin and U0126 for 1 h and stimulated with rhHMGB1 for 12 h. EdU assay (A) and colony formation assay (B) were used to detect cell proliferation and wound healing (C) and transwell (D) assays were used to determine cell migration, magnification, 100×. All experiments were repeated three times and data represent mean ±SD. # p < 0.05, ## p < 0.01 vs control group, *p < 0.05 and **p < 0.01 vs rhHMGB1-treated cells.
Extracellular
HMGB1 mediates various responses by interacting with its cell surface receptors and triggering diverse biological effects, such as cell proliferation, migration, differentiation and apoptosis [30,31]. Akt/mTOR and ERK signalling pathways play an important role in cell proliferation, survival, growth and apoptosis [32][33][34]. We examined the effects of HMGB1 on Akt/mTOR and ERK/CREB signalling pathway activation. Upregulation of HMGB1 enhanced phosphorylation of the Akt/mTOR and ERK signalling pathways. However, knockdown of HMGB1 caused a clear decrease in the activation of these signalling pathways (Fig. 5). HMGB1 overexpression enhanced the phosphorylation of P70S6K and S6, but was not significantly different. These may be due to high expression of HMGB1 in HGC-27 cells. Activation of P70S6K and S6 induced by endogenous HMGB1 is close to the maximum level.
To explore which receptor mediate the activation of Akt/mTOR and ERK signalling pathways induced by HMGB1, expression levels of RAGE, TLR2 and TLR4 were measured. Overexpression and knockdown of HMGB1 in HGC-27 cells revealed that HMGB1 upregulated expression levels of RAGE, but not TLR2 and TLR4 (Fig. 6). This suggested that RAGE may mediate HMGB1-induced Akt and ERK signalling pathways activation. To test this hypothesis, HGC-27 cells were pretreated with the RAGE inhibitor, FPS-ZM1, then stimulated using rhHMGB1 and measured phosphorylation of Akt and ERK signalling pathways using Western blotting. As expected, inhibition of RAGE suppressed rhHMGB1-induced Akt/mTOR and ERK signalling pathways activation (Fig. 7A). These data suggested that HMGB1 promoted HGC-27 cell proliferation and migration via RAGE-mediated Akt and ERK signalling pathways activation.
HMGB1 was reported to induce endothelial progenitor cell (EPC) migration via a RAGEdependent PI3K/Akt signalling pathway; however, the ERK signalling pathway was not involved in HMGB1/RAGE-induced EPC migration [35]. This difference in the signalling pathway may due to the different cell type studied. Consistent with our findings, HMGB1 was shown to promote prostate cancer development and metastasis by activating the Akt signalling pathway [35]. To clarify the signalling pathway involved in HMGB1-induced RAGE expression, we used specific inhibitors of Akt, mTOR and ERK to block the activation of these signal molecules. Blocking mTOR or ERK activation clearly attenuated rhHMGB1-induced RAGE expression (Fig. 7B). This result indicated that rhHMGB1 induced RAGE expression via the mTOR and ERK signalling pathways. Thus, our data indicated that a RAGE-mTOR/ERK feedback loop was involved in HMGB-induced GC cell proliferation and migration.
To further demonstrate that the Akt/mTOR and ERK signalling pathways are involved in HMGB1-induced HGC-27 cell proliferation and migration, HGC-27 cells were treated with specific inhibitors of Akt, mTOR and ERK. Our result revealed that inhibition of Akt, mTOR and ERK reduced rhHMGB1-induced GC cells proliferation and migration (Fig. 8).
The biological effects of extracellular HMGB1 require its translocation from the nucleus to the cytoplasm; however, it is not known how HMGB1 transfers to the cytoplasm. It is unclear whether the tumour microenvironment affects the nuclear translocation of HMGB1 and whether nuclear translocation of HMGB1 is related to its post-translation modification. Future studies are required to elucidate the mechanisms.
In conclusion, our findings indicate that HMGB1 regulates GC cell proliferation and migration via activation of RAGE-mediated Akt/mTOR and ERK signalling pathways and a RAGE-mTOR/ERK feedback loop (Fig. 9). Our study provides a new perspective for the effect of HMGB1 on proliferation and migration of GC cells. Macro-molecules Research Provincial Key Laboratory Project (grant nos. 1306C083008), National college Students' innovation and Entrepreneurship training program project (grant nos. 201910368005).
Author contributions
ZQ, SQ and YZ designed the experiments. TT, SW, TC, ZC and YM performed the experiments. TT, SW and YM analysed the data. TT and ZQ contributed to the writing. All authors reviewed the manuscript.
Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request. | v3-fos-license |
2018-04-03T03:26:25.981Z | 2018-02-20T00:00:00.000 | 3428198 | {
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} | pes2o/s2orc | Angiotensin-converting enzyme inhibitor reduces scar formation by inhibiting both canonical and noncanonical TGF-β1 pathways
Angiotensin-converting enzyme inhibitors (ACEIs) can improve the fibrotic processes in many internal organs. Recent studies have shown a relationship between ACEI with cutaneous scar formation, although it has not been confirmed, and the underlying mechanism is unclear. In this study, we cultured mouse NIH 3T3 fibroblasts with different concentrations of ACEI. We measured cell proliferation with a Cell Counting Kit-8 and collagen expression with a Sirius Red Collagen Detection Kit. Flow cytometry and western blotting were used to detect transforming growth factor β1 (TGF-β1) signaling. We also confirmed the potential antifibrotic activity of ACEI in a rat scar model. ACEI reduced fibroblast proliferation, suppressed collagen and TGF-β1 expression, and downregulated the phosphorylation of SMAD2/3 and TAK1, both in vitro and in vivo. A microscopic examination showed that rat scars treated with ramipril or losartan were not only narrower than in the controls, but also displayed enhanced re-epithelialization and neovascularization, and the formation of organized granulation tissue. These data indicate that ACEI inhibits scar formation by suppressing both TGF-β1/SMAD2/3 and TGF-β1/TAK1 pathways, and may have clinical utility in the future.
Scar formation after injury is an unavoidable outcome of wound healing in adult mammal, and is characterized by persistent changes in the normal structure and function of the skin 1 . The overproliferation of fibroblasts and the overproduction of nonfunctional extracellular matrix components have deleterious consequences [1][2][3] . Skin scars, which range from barely visible fine white lines to disfiguring hypertrophic scars or keloids, are well documented [4][5][6][7] . A scar can cause cosmetic problems, but scarring can also result in the loss of joint function or hinder growth in children 8,9 . Psychological distress, including anxiety and depression, also often occurs 6,8,10,11 . Therefore, both patients and physicians should welcome even small improvements in scar management.
The lessons learnt from the treatment of fibrosis in internal organs have markedly advanced our understanding of scar management. It has been reported that angiotensin-converting enzyme inhibitors (ACEIs) improve the fibrotic processes in the heart, lung, liver, and kidney [12][13][14][15] . Two case studies also reported that low-dose enalapril (ACEI) improved postsurgical abdominal keloid scarring 16 . Hakan Uzun et al. 17 reported that the early application of enalapril after dermal injury reduced scar formation in a rabbit ear wounding model. These results suggest that ACEI is an ideal treatment for scar management, but the underlying mechanism is not well understood.
The transforming growth factor-β (TGF-β) superfamily regulates many cellular functions, including cell growth, differentiation, adhesion, migration, and apoptosis 18 . Abnormal TGF-β signaling has been detected in increasing numbers of fibrotic and inflammatory conditions, including liver cirrhosis, renal fibrosis, systemic sclerosis, and hypertrophic scars 2,[19][20][21][22] . TGF-β1 occurs almost ubiquitously in mammalian tissues, and the development of tissue fibrosis is primarily attributed to this protein 23 . There are two major pathways through which TGF-β1 propagates its signals. The canonical pathway involves the downstream effectors SMAD family member 2 and SMAD family member 3 (SMAD2/3), whereas the noncanonical pathway is closely associated with TGF-β-activated kinase 1 (TAK1) 24,25 . Because TGF-β1 signaling is so important in scar formation, we hypothesized that ACEI reduces scar formation by modulating TGF-β1-related pathways. Therefore, we investigated the underlying mechanism in vitro and in vivo.
Fibroblasts are the ultimate effector cells in scar formation 26 . Heemskerk et al. confirmed the presence of angiotensin-converting enzyme (ACE) in a NIH 3T3 fibroblast model 27 . Therefore, we first used mouse NIH 3T3 fibroblasts cultured with different concentrations of ACEI to evaluate the levels of fibrosis induced and the role of TGF-β1 signaling. We also confirmed our findings in a rat model of full-thickness skin wounds. Our results showed that the appropriate dose of ACEI induced the extracellular matrix to suppress the TGF-β1/SMAD2/3 and TGF-β1/TAK1 pathways, leading to the inhibition of scar formation. These findings suggest that ACEI can be used clinically to prevent scar formation.
ACEI reduced fibroblast proliferation and collagen expression.
To determine whether ACEI plays a significant role in cutaneous scar formation, we first studied mouse NIH 3T3 fibroblasts. The cells were cultured with various concentrations (0, 1, 10, 100 μM) of lisinopril for 24 hours. Cell proliferation measured with a Cell Counting Kit-8 (CCK-8). Lisinopril at a concentration of 100 μM inhibited cell proliferation more strongly than concentrations of 0, 1, or 10 μM, whereas the effect of 0, 1, and 10 μM lisinopril did not differ significantly (Fig. 1A). To better simulate physiological wounding, several groups of cells were also treated with 1.0 ng/mL TGF-β1 to simulate the traumatic stimulus. Interestingly, with the addition of TGF-β1, cell proliferation was also inhibited significantly by 10 μM lisinopril (Fig. 1B).
The total collagen in NIH 3T3 cells were detected with a Sirius Red Collagen Detection Kit. After culture for 72 h, the secretion of total collagen from the cells without lisinopril was higher than that from cells treated with lisinopril, with the stimulation of TGF-β1. However, the significant differences could not be found between the two groups without the stimulation of TGF-β1 (Fig. 1C).
ACEI suppressed phosphorylation of SMAD2/3 and TAK1 in vitro.
To further clarify the mechanism underlying the ACEI-related downregulation of cell proliferation and collagen secretion, we used fluorescence-activated cell sorting (FACS) to quantify TGF-β1 expression. TGF-β1 decreased significantly in NIH 3T3 cells in the presence of 10 μM lisinopril ( Fig. 2A and Supplementary Fig. 1). Because the canonical pathway of TGF-β1 signaling is closely associated with SMAD2/3 28-30 , we analyzed the levels of phosphorylated SMAD2/3 (p-Smad2/3), the biologically active form of the proteins. As expected, FACS showed that p-SMAD2/3 decreased in the cells in the presence of 10 μM lisinopril ( Fig. 2B and Supplementary Fig. 2).
Experiments were performed to determine the involvement of both the canonical and noncanonical TGF-β1 signaling pathways in this phenomenon. One group of cells was cultured in normal nutrient medium supplemented with 10 μM lisinopril and stimulated with 2 ng/mL TGF-β1 for 30 minutes before the cell proteins were extracted. One group was cultured in normal nutrient medium supplemented with 10 μM lisinopril, without stimulation, and another group was cultured in normal nutrient medium without lisinopril but with TGF-β1 stimulation. The last group was cultured in normal nutrient medium with nothing added. Consistent with the FACS results, a western blotting analysis showed that with TGF-β1 stimulation, less p-SMAD2/3 was produced in the NIH 3T3 cells treated with lisinopril than that in the blank control group, whereas no p-SMAD2/3 was detected without TGF-β1 stimulation. The quantity of p-TAK1 was also lower in the NIH 3T3 cells treated with lisinopril than in the blank control group, with or without TGF-β1 stimulation ( Fig. 2C and Supplementary Fig. 3).
ACEI inhibited fibrosis and scarring in rats with acute dermal wounds. Based on the antifibrotic property of ACEI in NIH 3T3 cells, we next investigated the potential antifibrotic activity of ACEI in a rate model of acute dermal wounding. Two sections of skin were surgically removed from the backs of rats (Fig. 3A). Because the early application of a drug improved its performance 17 , the rats were randomly assigned to receive either ramipril (ACEI), losartan (angiotensin receptor blocker, ARB) or hydralazine (another type of blood-pressure-lowering agent) immediately after dermal injury. The control rats received only vehicle (water). We used lisinopril to treat the cultured cells because it is more soluble in water than other ACEIs, whereas we chose ramipril for the in vivo experiments because it has a relatively longer half-life than other ACEIs. The wound and scar widths were measured in all four groups throughout the healing process. The scar width did not differ significantly in all four groups on postoperative days 2, 6, or 10, but in postoperative days 12 and 14, the scar width was significantly narrower in the ramipril and losartan groups than in the other groups (Fig. 3B).
The wounds in all four groups were completely epithelialized within 14 days, after which the animals were killed and the scar tissues were collected (Fig. 3C). A microscopic examination on the final day revealed that the scars in the ramipril and losartan groups were not only narrower, but also showed better re-epithelialization and neovascularization than those in the other groups, and the formation of organized granulation tissue was apparent (Fig. 4A). Masson staining showed that the ramipril and losartan groups had loosely arranged collagen fibers and fewer fibroblasts, whereas the hydralazine and blank control groups had dense, irregular collagen fibers and more fibroblasts (Fig. 4B). Consistent with the gross measure of scar widths, the relative scar area and width determined with a histological examination were smaller in the groups treated with ramipril or losartan (Fig. 4C).
ACEI inhibited SMAD2/3 and TAK1 pathways in vivo.
Having established the ACEI-associated downregulation of TGF-β1 signaling in murine fibroblasts, we then investigated the potential mechanism in vivo. Real-time reverse transcription (RT)-PCR showed that the levels of SMAD2/3 and TAK1 mRNAs were significantly lower in the scar tissue of the ramipril and losartan groups than in those of the hydralazine and blank Scientific RepoRTs | (2018) 8:3332 | DOI:10.1038/s41598-018-21600-w control groups (Fig. 5A). A western blotting analysis also showed that the levels of p-SMAD2/3 and p-TAK1 proteins were lower in the groups with less scar formation ( Fig. 5B and Supplementary Fig. 4). As expected, the mRNA and protein levels of TGF-β1, collagen I and collagen III were statistically lower in the tissues of the ramipril and losartan groups than in those of the other two groups.
Discussion
Here, we have presented strong evidence of the effect of ACEI on wound healing and scar formation in rat dorsal wounds, and have demonstrated the crosstalk between ACEI and TGF-β1 both in vitro and in vivo. The improvement in scar formation was associated with reductions in fibroblast proliferation, collagen deposition and TGF-β1 expression and led to a more normal structure of the healing skin. The improved skin parameters were associated with the downregulated phosphorylation of SMAD2/3 and TAK1, which indicated the inhibition of both the canonical and noncanonical TGF-β1 pathways. Our results are consistent with previous reports on the effects of the RAS on myocardial infarction, which demonstrate the efficacy of ramipril in reducing fibrosis and collagen accumulation 31,32 . Our results also suggest that the beneficial effects of the RAS blockade induced by ACEI probably also extend to ARBs, such as losartan. Most cells are involved in wound healing, including lymphocytes, neutrophils, macrophages, and fibroblasts. Fibroblasts are the major mesenchymal cell type in connective tissue, and are recruited to the injured site where they deposit collagen and elastic fibers 33 . They provides many regulatory mediators to the microenvironment and thereby contribute to the maintenance of wound healing and scar formation 34,35 . Our first study suggested that ACEI reduced the proliferation of murine NIH 3T3 fibroblasts, especially when the cells were stimulated with TGF-β1. During physiological wound repair, but not in tissue fibrosis, myofibroblasts are transiently present and are removed by the initiation of the apoptotic machinery 36 . Therefore, the proper inhibition of fibroblast proliferation can reduce abnormal wound healing and the development of fibrotic diseases 37 .
TGF-β1 signaling is significantly involved in many phases of wound healing, including inflammation and angiogenesis [38][39][40] . Our study has shown that ACEI inhibits the phosphorylation of SMAD2/3 and TAK1, which are two mediators of TGF-β1 signaling. The canonical signaling pathway for TGF-β1 involves the SMAD family 25 . Upon phosphorylation by TGF-β1 receptors, SMAD2 and SMAD3 form heteromeric complexes with co-SMAD or SMAD4. The SMAD2-SMAD3-SMAD4 complex is then translocated into the nucleus, where it regulates the transcription of TGF-β1 target genes 25,41,42 . Distinct from the activation of SMAD-dependent cascades, a predominantly SMAD-independent signaling pathway that mediates the profibrotic effects of TGF-β1 operates via TAK1 24 . TAK1 is involved in the TGF-β1-induced expression of type I collagen and fibronectin by activating the MAPK kinase(MKK)3/p38 and MKK4/JNK signaling cascades, respectively 24 . Our study demonstrated that ACEI inhibits both pathways of TGF-β1 signaling, reducing fibroblast proliferation and collagen deposition, leading to a more normal structure of the healing skin.
TGF-β1 is considered as the most important target in scar management because it supports excessive disorganized collagen deposition 43 , which is consistent with our findings in microscopic observation. Although reducing the expression of TGF-β1 by gene transfection or antibodies has been demonstrated experimentally 1,44 , no medicinal product is available for routine use. TGF-β1 regulates the expression of multiple genes related to fibrosis via both the canonical and noncanonical pathways. Therefore, the simultaneous inhibition of SMAD2/3 and TAK1 by ACEI is a promising strategy for blocking TGF-β1 signal transduction. No significant differences in bodyweight or physical condition were observed after the administration of different drugs to the rats in this study. These results indicate that ACEI significantly inhibits TGF-β1-induced scar formation in vivo, but does not significantly affect the general health of rats.
Our in vivo study also investigated the impact of ARBs, specifically losartan. Clinically, both ACEIs and ARBs have yielded similar results in terms of blood pressure control and cardiovascular protection 45,46 . ACEIs and ARBs differ pharmacologically in their mechanism of action and the levels at which they block the RAS. Although ARBs block RAS distally, at the level of the angiotensin II type-1 (AT1R), ACEIs block the conversion of angiotensin I to angiotensin II and thus reduce the amount of available angiotensin II to bind to either AT1R or angiotensin II type-2 receptor (AT2R) 47 . Both AT1R and AT2R are upregulated in human cutaneous wounds 48,49 , and AT2R is expressed more strongly than AT1R within the area of scarring 49 . Enhanced ACE expression is still detectable in cutaneous human scars 3 months after wounding 48 . However, the beneficial effects of RAS blockade are quite confusing. In AT1R-knockout mice, wound healing was delayed relative to that in the controls 50 , perhaps resulting from the disruption of the inflammatory phase and the impairment of the transition to proliferation and remodeling [51][52][53] . The knockout of AT2R accelerated healing but impaird quality 54 . The effect of AT2R on rate of wound closure may depend on the phase of wound healing in which it is applied. Recently, Abadir et al. suggested that the use of topical ARBs is an effective treatment for chronic wounds, whereas ACEI was not 47 . However, our study shows that both ACEI (ramipril) and ARB (losartan) positively affected wound healing and scar formation when orally administered. Our results are also supported by other recent studies that have shown that the oral treatment of diabetic rats or mice with ACEI or ARB accelerated wound healing 55,56 . The discrepancies among these studies might be explained by the differences in the metabolism of the drug and their tissue distributions, as well as by their systemic effects (e.g., on blood pressure, heart rate and the immune system).
In conclusion, our findings provide powerful insights into the inhibition of scar formation by ACEI. Orally administered ACEI normalized the structure of healing skin, resulting from the dual inhibition of SMAD2/3 and TAK1 signaling. The effects of ramipril on the deposition and arrangement of collagen in healing wounds may open a new avenue for the use of ACEI in fibrotic skin diseases.
Methods
Cells preparation. Mouse NIH 3T3 fibroblasts (American Type Culture Collection) were incubated at 37 °C under 5% CO 2 /95% air. The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS). The concentration of FBS was reduced to 1% at 24 h or 72 h before testing. Some groups of cells were cultured with ramipril (Sigma-Aldrich, St. Louis, MO, USA) and/ Rat animal model. The rat was anesthetized with 7% Chloral Hydrate (0.5 ml/100 g) using peritoneal injection. A thin rectangle of skin (3 × 0.3 cm 2 ) parallel to but 1.5 cm away from the midline, was excised on both sides of dorsal skin, so each rat had two small portions of skin removed. The incision was made through the dermis and subcutaneous fascia, exposing the underlying muscle (which is not cut). A 3 cm (length) × 4 mm (width) × 5 mm (height) trimmed gelatin sponge was inserted into the excised wound. Wound care with 70% ethanol was performed on days 2 and 4 after surgery, and the widths of the wounds or scars were measured with a sliding caliper after surgery. Fourteen days after the operation, the rats were anesthetized with isoflurane inhalation, and the scars were harvested for analysis. The scar widths were grossly measured, and we also calculated the relative scar areas and relative widths with microscope observation. The tissue were stained with hematoxylin and eosin, and images were obtained at 40× magnification with an Olympus CKX41SF Inverted Phase Contrast Microscope. The areas were calculated with the Image-Pro Plus v. 6.0 software (Olympus, Japan).
Real-time RT-PCR.
To analyze the mRNA levels in the scar tissues, total RNA was extracted using the RNeasy Mini Plus kits (Qiagen, Valencia, CA, USA). The purified RNA (200 ng) was reverse transcribed with a high-capacity iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). The expression levels of TGF-β1, Scientific RepoRTs | (2018) 8:3332 | DOI:10.1038/s41598-018-21600-w SMAD2/3, TAK1, collagen I and collagen III, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs (with primers from Applied Biosystems) were analyzed with quantitative real-time RT-PCR using an ABI OneStepPlus TM Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). GAPDH was used as the internal control.
Histological analysis. Wound beds surrounded by a margin of unwounded skin were collected at day 14 after injury (n = 12 wounds in six rats in each group). The wounds were divided in half in the least-healed portion. They were then fixed overnight at 4 °C in 60% methanol, 30% chloroform and 10% acetic acid. The tissues were processed through a graded series of ethanol and embedded in paraffin blocks. Tissue sections of 5 μm were stained with stained with hematoxylin-eosin (H&E) or Masson's trichrome to visualize neotissue formation, collagen deposition and the amount of neovasculature.
Statistical analysis. The in vitro experiments were repeated with at least five batches of NIH 3T3 fibroblasts and the in vivo experiments were repeated three times, and qualitatively similar data were obtained in all repetitions. Data were analyzed with the SPSS, version 19.0 software (SPSS Inc., Chicago, IL, USA). Values are expressed as the mean ± SEM. Comparisons among groups were made with one-way analysis of variance (ANOVA), followed by a least-significant-difference (LSD) or Student-Newman-Keuls (SNK) test (more than 2 groups) or a 2-tailed unpaired Student's t-test (two groups). A value of P < 0.05 was considered significant. As stated above, observers and statisticians were blinded to the group identity during all quantifications. | v3-fos-license |
2018-05-21T20:56:47.476Z | 2018-01-01T00:00:00.000 | 13673325 | {
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} | pes2o/s2orc | Exosomes Secreted by Normoxic and Hypoxic Cardiosphere-derived Cells Have Anti-apoptotic Effect.
Cardiosphere-derived cells (CDCs) have emerged as one of the most promising stem cell types for cardiac protection and repair. Exosomes are required for the regenerative effects of human CDCs and mimic the cardioprotective benefits of CDCs such as anti-apoptotic effect in animal myocardial infarction (MI) models. Here we aimed to investigate the anti-apoptotic effect of the hypoxic and normoxic human CDCs-derived exosomes on induced apoptosis in human embryonic stem cell-derived cardiomyocytes (hESC-CMs). In this study, CDCs were cultured under normoxic (18% O2) and hypoxic (1% O2) conditions and CDC-exosomes were isolated from conditioned media by differential ultracentrifugation. Cobalt chloride as hypoxia-mimetic agents at a high concentration was used to induce apoptosis in hESC-CMs. The caspase-3/7 activity was determined in apoptosis-induced hESC-CMs. The results indicated that the caspase-positive hESC-CMs were significantly decreased from 30.63 ± 1.44% (normalized against untreated cardiomyocytes) to 1.65 ± 0.1 and 1.1 ± 1.09 in the presence of normoxic exosomes (N-exo) at concentration of 25 and 50 μg/mL, respectively. Furthermore, hypoxic exosomes (H-exo) at concentration of 25 and 50 μg/mL led to 8.75 and 12.86 % reduction in caspase-positive cells, respectively. The anti-apoptotic activity of N-exo at the concentrations of 25 and 50 μg/mL was significantly higher than H-exo. These results could provide insights into optimal preparation of CDCs which would greatly influence the anti-apoptotic effect of CDC-exosomes. Totally, CDC-secreted exosomes have the potential to increase the survival of cardiomyocytes by inhibiting apoptosis. Therefore, CDC-exosomes can be developed as therapeutic strategy in ischemic cardiac disease.
Introduction
Cardiovascular disease is one of the leading pathological causes of mortality worldwide (1). Cardiosphere-derived cells (CDCs) currently are in phase 2 clinical trials to reverse postmyocardial infarction (MI) injury. The results of studies in various animal models and also a phase 1 human study have shown that the CDCs have the ability to decrease scar mass, increase viable mass, and halt adverse remodeling (2). It was demonstrated that exosomes secreted by CDCs replicate the cardioprotective and regenerative effects of CDCs, including anti-apoptotic effect (3).
Exosomes, 30-150 nm in diameter lipid bilayer vesicles, are secreted by many cell types and contain a wide range of functional proteins, mRNAs, and miRNA. They are the key transporters of paracrine factors and are able to mediate cell-cell communication. (4). Exosomes as therapeutic agent for repairing damaged myocardium could overcome many obstacles associated with stem cell-based therapy (3,5).
Immediately after coronary arterial occlusion in acute myocardial infarction, cardiomyocytes are stressed by hypoxia which leads to undergo apoptosis (6). Hypoxia-inducible factor-1 (HIF-1) is the master regulator of the cellular adaption to hypoxic stress (7).
Recent reports suggested that hypoxia as an in-vitro environmental stressors can modify the composition of cardiac progenitor cell-derived exosomes (CPC-exo). These studies concluded that hypoxia have beneficial effect on the cardiac response through paracrine signaling (8,9).
In the present study, we aimed to investigate the anti-apoptotic effect of CDCs exosomes in cardiomyocytes protection against CoCl 2induced apoptosis. We assessed the antiapoptotic effect of the isolated exosomes from the media of hypoxia (1% O 2 )-and normoxiatreated CDCs on human embryonic stem cellderived cardiomyocytes (hESC-CMs) in terms of caspase-3/7 activation.
Cell culture
Human CDCs (obtained from Iranian pediatric patients diagnosed with a congenital heart disease) were provided by Royan Cell Bank Services. The Patientsꞌ parents gave their informed consent for study participation and research use. Ethical approval was granted by the Royan institute Ethical Committee (10). CDCs were then cultured in proliferation medium [Iscoveꞌs Modified Dulbeccoꞌs Medium (IMDM), Sigma, USA] supplemented with 1% L-glutamine (Invitrogen, USA), 1% penicillin/ streptomycin (Invitrogen, USA), 10% fetal bovine serum (FBS, Gibco, USA) with 10 ng/ mL basic fibroblast growth factor (bFGF, Royan Biotech) at 37 ºC and 5% CO 2 in 95% humidity.
Cardiac differentiation in static suspension culture
For hESC-CM production in static suspension culture, 5-day-old hESC spheroids with the size of 175 ± 25 µm were transferred to 60 mm nonadhesive bacterial plates (Sigma-Aldrich, USA) in 5 mL of differentiation medium (DM) which contains Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, USA) supplemented with 2% B27 without retinoic acid (Gibco, USA), 0.1 mM β-mercaptoethanol, 2 mM L-glutamine, 1% nonessential amino acid. First, the aggregates were treated for one day with 12 μM CHIR99021, a glycogen synthase kinase 3-β inhibitor. Next, spheroids were washed with Dulbecco's phosphatebuffered saline (DPBS) and were transferred to DM without CHIR99021 for one day. In the next step, the spheroids were transferred to DM containing 5-µM purmorphamine (Stemgent, USA) as the sonic hedgehog agonist, 5 µM IWP2 (Tocris Bioscience, UK) as a Wnt antagonist, 5 µM SB431542 (Sigma-Aldrich, USA) as the inhibitor of transforming growth factor beta (TGF-β) super family type I activin receptorlike kinase receptors. The aggregates were maintained in the medium for two days. On day five, the medium was changed and the spheres transferred to DM after they had been washed with DPBS. The medium was changed every two days. On the seventh day, beating started and reached its highest on the tenth day.
Exosome purification
Exosomes were removed from FBS by ultracentrifugation at 120,000 ×g (Type 45 Ti rotor, 32128 rpm, k-factor 133, L-100XP ultracentrifuge, Beckman Coulter, USA) for 18 hours. After discarding the pellet, the supernatant of FBS was filtered through 0.2 µm filters (Techno Plastic Products, Switzerland) and then used in cell cultures (11). Then, CDCs at the fifth passage were cultured in complete media containing IMDM, 10% exosome-depleted FBS, 1% penicillin-streptomycin, and 1% L-glutamine under normoxic (18% O 2 , 5% CO 2 ) and hypoxic (1% O 2 , 5% CO 2 and 94% N 2 ) conditions in two distinct incubator (Labotec C200, Germany). The conditioned media was collected 48 h later, and then the exosomes were harvested by differential ultracentrifugation (12, 13). The purified exosome pellet was resuspended in 200 μL PBS and stored at -80 °C. The protein content of the exosome suspension was analyzed by Pierce™ BCA Protein Assay kit (Thermo Scientific, USA). The size of exosomes was determined by dynamic light scattering (DLS) with a Zetasizer nanoseries instrument (Malvern Nano-Zetasizer, UK). The morphological characteristics of exosomes were observed under scanning electron microscopy (SEM, KYKY-EM3200, USA) and flow cytometry was used to analyze surface protein markers of exosomes (detailed explanation of these methods and their results will be reported elsewhere).
Immunostaining hESC-CMs were obtained using a protocol described previously (14). To confirm the differentiation of hESC-CM, cardiac specific markers were stained. To achieve dissociated single cardiomyocytes, the beating spheroids at day 14 of differentiation were washed and maintained in DPBS for 5 min. Then, Accumax cell dissociation solution (Sigma, USA) was added and incubated for 10 min. After that, 5×10 4 cells/well of the single cardiomyocytes were seeded into 4-well matrigel-coated plates contained fresh DM. Two days later, after washing with DPBS, the attached cells were fixed with 4% (w/v) paraformaldehyde for 20 min at 4 °C and washed with PBS/0.1% Tween 20. Then, the cells were permeabilized with 0.5% Triton X-100 in DPBS for 30-45 min at room temperature. Blocking was performed with 5% (v/v) goat serum for 1h. Next, the cells were incubated with diluted primary antibodies (1:100) in blocking buffer overnight at 4 °C. Primary antibodies used were antibodies against cardiac specific markers: cardiac troponin T (cTnT, Abcam, UK), myosin light chain 2v (MLC2v, Santa Cruz, USA), actinin (Sigma, USA). After three rounds of washing with PBS/0.1% Tween 20 for 5 min each time, the cells were incubated with secondary antibodies [Alexa Fluor 488 goat anti-mouse IgG antibody (Abcam, UK) Alexa Fluor 546 goat anti-mouse IgG antibody (Abcam, UK)] at a dilution of 1:500 in blocking buffer for 45 min at room temperature. Finally, the cardiomyocytes were washed with PBS/0.1% Tween 20 three times. 4ʹ, 6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei for 5-10 min at room temperature. The cells were examined using fluorescence microscopy (Olympus, IX71, USA). The positive hESC-CMS for cardiac specific markers were counted manually in at least five images from different areas of each sample in three independent experiments.
Apoptosis induction
Cobalt chloride (CoCl 2 , Sigma, USA) was used for induction of apoptosis in hESC-CMs. The appropriate concentration of CoCl 2 in order to induce apoptosis in the hESC-CMs was determined. In brief, hESC-CMs were seeded on 3 cm 2 matrigel-coated plates. Two days later, the cells were treated with different concentrations of CoCl 2 (1, 2 and 3 mM) for 3 h (3,15,16). Then, the caspase-3/7 activity was measured using CellEvent ® Caspase-3/7 Green Ready Probes ® Reagent (Life technologies, USA) according to manufacturer's instructions. The samples were analyzed by the flow cytometer (FACS Calibur; BD Biosciences, USA) using Flowing software, version 2.5.1 (BD Biosciences, USA).
In-vitro apoptosis assay
In order to determine the anti-apoptotic effect of exosomes, hESC-CMs were cultured on 3 cm 2 matrigel-coated plates. 24 h later, the cells were treated with 10, 25 and 50 μg/mL normoxic as well as hypoxic exosomes. Apoptosis was induced in hESC-CM by addition of a selected CoCl 2 concentration after 24 h. The activity of caspase-3/7 was measured 3 h later as described earlier. Apoptosis-induced hESC-CMs with no treatment were used as the positive control of apoptosis induction.
Statistical Analysis
GraphPad Prism software (version 6, USA) was used for statistical analyses. Two independent groups were compared using unpaired student's T-test. One-way ANOVA followed by Tukey post-test was used to perform multiple group comparisons. The differences with a p <0.05 were determined to be statistically significant.
CDCs-derived exosome isolation and characterization
CDCs were cultured under hypoxic or normoxic conditions and the exosomes were isolated from conditioned media by ultracentrifugation after 48 h. The DLS analysis was used to define the size of these exosomes. The mean hydrodynamic diameter of exosomes was between 150-170 nm. Under SEM, the exosomes exhibited a round morphology. The flow cytometry analysis of N-exo and H-exo showed that CD63 and CD81 which are typical exosomal markers, were expressed on the surface of exosomes (Unpublished data).
Characterization of hESC-CMs
The cardiogenic differentiation efficiency was determined by counting the number of beating spheroids at day 10 after the onset of differentiation using an inverted cell culture microscope (Figure 1 A). Approximately 100% of spheroids were beating (see supplementary video online). The 14-day-old beating spheroids were subsequently collected and dissociated for immunostaining of cardiac-specific markers; Actinin, MLC2v and cTnT ( Figure 1B). hESC-CMs were approximately 90% positive for the tested cardiac specific markers ( Figure 1C).
CoCl 2 -induced apoptosis in hESC-CMs
To determine apoptogenic concentration of CoCl 2 in hESC-CMs, the caspase-3/7 activity was measured in the presence of different concentrations of CoCl 2 (1, 2 and 3 mM). The analysis showed that treatment of the cells with 3 mM CoCl 2 for 3 h resulted in 30.63 ± 2.66% cell apoptosis in terms of caspase-3/7 activation. As indicated in Figures. 2, 3 mM CoCl 2 resulted in significant higher caspase positive cells comparing to than 1 and 2 mM (3 mM vs. 2 mM p <0.01, 3 mM vs. 1 mM p <0.001).
The effect of exosomes-derived CDCs under hypoxic and normoxic conditions on induced apoptosis in hESC-CMs
The effect of hypoxic (H-exo) and normoxic exosomes (N-exo) on CoCl 2 -induced apoptosis was investigated. Accordingly, hESC-CMs were treated with N-exo and H-exo at different concentrations (10, 25 and 50 μg/mL) for 24 h and then the caspase-3/7 activity was measured after apoptosis induction wih CoCl 2 . We found that both N-exo and H-exo inhibited hESC-CMs CoCl 2 -induced apoptosis ( Figure.
Discussion
CDCs have shown to promote cardiac regeneration of the infracted human heart (2, 17). Exosomes generated by CDCs are beneficial paracrine signals that reproduce CDC-induced therapeutic regeneration. They are sufficient to mediate the entire effect of CDCs (3,5). Few studies have investigated the therapeutic potential of CDC-exosomes in animal MI models and some other cardiovascular diseases. These studies have shown that exosomes secreted by CDCs replicate the cardioprotective and regenerative effects of CDCs such as apoptosis inhibition of cardiomyocytes. Gallet and coworkers indicated that CDC-derived exosomes delivered by intramyocardial (IM) injection has the ability to decrease acute ischaemiareperfusion injury, halt adverse remodeling and to improve LVEF in pig models of acute (AMI) (2). In the study of Ibrahim et al., exosomes secreted by human CDCs inhibit apoptosis and promote proliferation of cardiomyocytes, while enhancing angiogenesis. Injection of exosomes into injured mouse hearts recapitulates the regenerative and functional effects produced by CDC transplantation, whereas inhibition of exosome production by CDCs blocks those benefits (3). All of these data confirmed in-vivo anti-apoptotic effect of CDC-exosomes (2, 3,18). In all of these studies, CDC-exosomes were obtained under normoxic conditions, which likely could not reflect the state of post-infarct tissue. While most in-vitro cultured cells maintained at oxygen levels of approximately 20%, natural cell micro-environments seem to have much lower oxygen tensions with considerable variation based on location. For instance the mean oxygen concentration of arterial blood is approximately 12%, and that of tissue is 3% (19). Adult stem cells similarly live under hypoxic conditions of 3-5% O 2 in-vivo and these hypoxic conditions are the physiological norms for a variety of stem cell niches (20). These studies have shown that the level of oxygen play a crucial role in the maintenance, differentiation, and function of stem cells. Nevertheless, hypoxia can also induce mitochondria-mediated apoptosis and subsequent caspases activation in bone marrowderived mesenchymal stem cells (21). In this study we have analyzed the anti-apoptotic activity of exosomes generated by CDCs under both normoxic and hypoxic conditions. Here, we showed that H-exo and N-exo significantly decrease CoCl 2 -induced apoptosis in hESC-CM.
Here, to isolate the CDC-exosomes from conditioned media, differential ultracentrifugation was used as described in the literatures with some modifications (12, 13). The size of H-exo and N-exo were almost similar, with mean hydrodynamic diameter of 150 to 170 nm. CDC-derived exosomes possessed highly positive expression for exosome surface markers, such as CD63 and CD81 (unpublished data).
CoCl 2 is a well-established hypoxiamimicking substance. CoCl 2 -treated cells share common features with cells incubated at 1% oxygen (22). CoCl 2 , as a substrate of the ferrochelatase enzyme, is thought to mimic the hypoxia by binding to the heme molecules (instead of Fe 2+ ). It was shown that the expression level of hypoxia-inducible factor-1α (HIF-1α), which is a major transcription factor and key regulator of adaptive responses to hypoxia, is markedly increased following treatment with CoCl 2 in a dose-dependent manner (23). In this study, CoCl 2 at concentration of 3 mM was used to induce apoptosis. In the study of Guo et al.,U937 and NB4 the cell lines were treated by CoCl 2 at different concentrations of 150, 200 and 300 μM. They found that at the concentration of 150, 200, and 300 μM the viability is reduced to 55, 20, and 7 % in U937 Figure 2. Apoptosis induction. The caspase-3/7 activity was measured to determine apoptogenic concentration of CoCl 2 on hESC-CMs. Each column represents the mean ± SEM of independent experiments. (**p <0.01, ***p <0.001 vs. 1mM, # # p <0.01). and to 60, 50, and 25% in NB4, respectively (15). Kim et al. also demonstrated that neural cells viability is reached to about 60% after 24 h CoCl 2 treatment at concentration of 1 mM (16). However, it should be taken into consideration that the method of apoptosis detection is not the same in all studies. For example, trypan-blue exclusion and MTT assay were used to evaluate cell viability by Guo (15) and Kim (16) et al., respectively. Totally, our data are in agreement with the results of Guo et al. (15) which indicate CoCl 2 at the concentration of greater than 50 μM induce apoptosis via mitochondria pathwaymediated caspase -3 activation. (15).
In this study, we found that exosomes secreted by human CDCs were cultured 48 h under hypoxic and normoxic conditions (1% O 2 ) inhibited apoptosis at both 25 and 50 μg/ mL concentrations. However, the anti-apoptotic effect of N-exo was significantly higher than that of H-exo at concentration of 25 and 50 μg/mL (p <0.0001). In the present study, higher oxygen percentage (1% O 2 ) was used for hypoxic preconditioning compared to hypoxic culture condition (0.5% O 2 ) that was used in the study of Chacko et al. (24). Their results indicated that exposure to sub-lethal hypoxia (0.5% O 2 ) for as long as 72 h by itself does not induce cell death by apoptosis in mesenchymal stem cells Totally, it can be concluded that different parameters in hypoxia preconditioning of
Discussion
CDCs have shown to promote cardiac regeneration of the infracted human heart (2, 17).
Exosomes generated by CDCs are beneficial paracrine signals that reproduce CDC-induced therapeutic regeneration. They are sufficient to mediate the entire effect of CDCs (3,5). Few cardiac stem cells including the percentage of oxygen and the duration of hypoxia play critical roles in their anti-apoptotic effect. Therefore, to obtain the optimum anti-apoptotic effect of cardiac stem cells (CSCs)-derived exosomes, further investigation is highly required to choose the proper manner of hypoxic preconditioning. Additionally, the way of apoptosis induction in cardiomyocyte might be important in the antiapoptotic effect of CSCs-derived exosomes. The severe hypoxic (0.1% O 2 ) and reduced serum conditions that led to decreasing cells apoptosis in the study of Chacko et al.,(24) may be different from the apoptosis condition that was induced by cobalt chloride (3 mM) in our study . In the study of Xiao et al.,H 2 O 2 was used to induce the oxidative stress that originates mainly in mitochondria from reactive oxygen species (ROS) (27,28). Their results demonstrated that H 2 O 2 -exosomes reduce H 2 O 2 induced apoptosis. Further investigations dealing with the kind of apoptosis induction will be helpful.
We can get to the conclusion that CDCsecreted exosomes have the potential to prevent apoptosis in cardiomyocytes and they will hopefully provide a promising therapeutic strategy for ischemic cardiac disease. Our results imply the need for further investigation of the effect of hypoxia-preconditioning method of cardiac stem cells on the anti-apoptotic activity of their secreting exosomes. | v3-fos-license |
2020-08-20T10:09:30.793Z | 2020-08-13T00:00:00.000 | 221838479 | {
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} | pes2o/s2orc | IdentIfIcatIon of the bIndIng sIte for plasmInogen krIngle 5 In the α-chaIn of fIbrIn(ogen) d-fragment
The interaction of the fifth kringle of Glu-plasminogen with fibrin triggers activation and initiation of fibrinolysis, yet the site on fibrin that binds kringle 5 remains unknown. The aim of our work was to determine an amino acid sequence in the D-fragment of fibrin(ogen) molecule, which is complementary to the lysine-binding site (LBS) in kringle 5. We studied the interaction between kringle 5 of plasminogen with polypeptide chains of the D-fragments of fibrin and cyanogen bromide fragments FCB-2 and t-NDSK and showed that kringle 5 bound specifically to αand γ-chains of the D-fragment and the α-chain of FCB-2. Tryptic peptides of D-fragment α-chain were obtained, separated by their ability to bind with the immobilized kringle 5, and then all studied peptides were characterized by MALDI-TOF analysis. The critical amino acid residues of the α-chain of D-fragment, which provide its interaction with kringle 5, turned out to be α171Arg and/or α176Lys. The binding site of Glu-plasminogen complementary to the LBS of kringle 5 is located within Аα168Ala−183Lys, a sequence in a weakly structured loop between two supercoils in the α-chain of the Dfragment of the fibrin(ogen) molecule.
Introduction
Fibrin is the main component of blood clots, which are formed to stop and prevent blood loss after vessel damage. Also, fibrin clot formation plays the primary role in the development of life-threatening thrombotic conditions and vascular pathologies, such as ischemic heart disease, atherosclerosis, myocardial infarction, stroke, etc. Therefore, investigation of the molecular mechanisms of protein interactions related to fibrin degradation has vital clinical implications. Proteolytic enzyme plasmin (EC 3.4.21.7) is the key fibrinolytic proteinase responsible for clot degradation in vivo. Plasmin is derived from circulating non-active zymogen plasminogen. The interaction between plasminogen and tissue activator with polymeric fibrin localizes plasmin generation on the surface of the fibrin clot, and therefore makes possible the selective fibrin hydrolysis and keeps plasmin protected from the plasma inhibitor, α-2-antiplasmin [1,2].
Glu-plasminogen is the native form of plasminogen, which has a glutamate residue at the NH2terminus. It consists of several domains: the N-terminal domain, five sequential homologous kringle domains (K1, K2, K3, K4, and K5) and the proteinase domain. Lysine-binding sites (LBSs) of kringle domains allow the interaction of plasminogen with various proteins and receptors [3][4][5][6]. Glu-plasminogen exists in the closed conformation in plasma, it is stabilized by intramolecular interactions of the Nterminal domain with the K5 [7]. When K5 binds to ligands, Glu-plasminogen acquires open conformation [8,9]. Limited proteolysis of plasminogen by plasmin can detach the N-terminal domain from Glu-plasminogen molecule resulting in the partially degraded Lys-plasminogen, which has an open conabbreviations: desAB fibrin -fibrin containing neither A nor B fibrinopeptides; t-NDSK -thrombin-treated N-terminal disulfide knot of fibrin; FCB-2 − cyanogen bromide fragment of fibrin(ogen) consisting of fibrinogen chain fragments Aα148-208, Bβ191-224, 225-242, 243-305 and γ95-265, linked by disulfide bonds; D-fragment − one of the two identical terminal regions of fibrinogen; DD-fragment − two identical terminal regions of adjacent fibrin molecules, covalently cross-linked by factor XIIIa.
formation. In healthy individuals, Lys-plasminogen is not detected in blood plasma. The transformation of Glu-plasminogen into the partially degra ded form in vivo may occur on the cell surface [10,11] or on the fibrin network during clot hydrolysis [12]. Plasminogen activation by endogenous activators (e. g. tissue plasminogen activator or urokinase) occurs by cleavage of the Arg561-Val562 peptide bond, resulting in the formation of the active center and two-chain molecule of plasmin. An activation loop, containing the Arg561-Val562 peptide bond, is located between K5 and the proteinase domain. In the closed conformation of Glu-plasminogen, it is shielded by a connector, which holds together K3 and K4, and is presented to the activator during the transition of zymogen into the open conformation.
Both Glu-and Lys-forms of plasminogen specifically bind to fibrin. Adsorption of Lys-plasminogen is inhibited by 6-aminohexanoic acid (6-AHA) at concentrations saturating the high-affinity ligand binding site located in the K1, while adsorption of Glu-plasminogen is suppressed at the saturation of the low-affinity ligand binding sites located in the K4 and K5. The fact that Glu-plasminogen interaction with fibrin is mediated by the K5 is confirmed by the data that the mini-plasminogen and the kringle fragment 1-5 inhibit, while kringle fragment 1-3 does not affect the binding of Glu-plasminogen to fibrin [13,14]. Isolated K5 exerts an inhibitory effect on desAB fibrin hydrolysis upon Glu-plasminogen activation by tissue activator [15]. The contribution of the LBS of K5 to interaction with fibrin leads to dissociation of the electrostatic bond between this site and the N-terminal domain, causing Glu-plasminogen to adapt open conformation, which provides its activation by the tissue activator and initiates fibrin hydrolysis.
Polymeric fibrin is formed from fibrinogen under the action of thrombin. Structural rearrangements occurring in the D-regions of fibrin(ogen) during polymerization lead to exposure of plasminogen and tissue activator binding sites [2]. It was shown that the binding site for plasminogen is localized within 148-160 amino acid residues sequence of the Аα-chain, but it is unclear with which kringle do-main it interacts. A synthetic peptide corresponding to the Аα148-160 sequence in fibrin(ogen) stimulates activation of plasminogen by tissue activator and does not affect activation of mini-plasminogen [16]. A thermostable region of the D fragment (TSD, 28 kDa), whose α-chain encompasses a 148-160 amino acid residues sequence, has one plasminogen-binding site of high affinity for Lys-plasminogen (K d = 0.44 µM), interacts with the kringle K1-3 and does not bind K4 and mini-plasminogen [17]. These data convincingly show that the plasminogenbinding site localized in the 148-160 sequence of the D-fragment Аα-chain is complementary to the LBS in kringle fragment 1-3 of plasminogen molecule. Given that the K5-mediated Glu-plasminogen interac tion with fibrin triggers the process of zymogen activation and fibrinolysis initiation, the determination of the binding site for K5 in the fibrin mole cule is of essential importance to understand the molecular mechanisms of fibrinolysis and to search ways to regulate this process. Thus, the aim of our study was to identify the binding site for K5 LBS of Glu-plasminogen in the D-fragment of fibrin(ogen) molecule.
materials and methods
Fibrinogen was isolated from donor blood plasma by fractional salting out with sodium sulfate [18]. Fibrin, covalently crosslinked by coagulation factor XIIIa, was obtained by thrombin-induced polymerization of fibrinogen in the presence of calcium ions (25 mM) [19]. Non-crosslinked fibrin was obtained from thrombin-activated fibrinogen in the presence of 6-AHA and para-hydroxymercuribenzoate [20]. DD-fragment was obtained by plasmin hydrolysis of the crosslinked fibrin in the presence of 5 mM Ca2+ [19]. D-fragment was produced by plasmin hydrolysis of non-crosslinked fibrin [21]. The hydrolysis reaction was stopped by 6-AHA in the presence of EDTA. FCB-2 and t-NDSK were obtained by cyanogen bromide cleavage of fibrin [22].
Purity of the obtained proteins was determined electrophoretically in 10% PAGE with 0.1% SDS [26] using molecular mass markers of 10-260 kDa (Spectra Multicolor Broad Range Protein Ladder, Fermentas, Lithuania). Protein concentration was calculated by the optical density of solutions at 280 and 320 nm using their E 280 1% and molecular masses . All reagents were at least chemically pure grade.
Determination of proteins able to specifically bind K5 was done by Western-blotting [27] using polyclonal antibodies to K5. Proteins and their polypeptide chains, reduced by β-mercaptoethanol, were separated by electrophoresis in 10% PAGE with 0.1% SDS and transferred from the gel onto nitrocellulose membrane with 0.45 µm pore size (GVS North America/Sigma, St. Louis, USA). Membrane was blocked by 3.5% solution of non-fat skim milk (BioRad, Hercules, USA) in phosphate buffer saline (PBS). K5 (20 µg/ml in PBS with 0.05% Tween-20, PBST) was bound to proteins, adsorbed on the nitrocellulose membrane, during 2 hours at 37 °С. In the next stage, the membrane was incubated with antibodies to K5 (5 µg/ml in PBSТ) for 1 hour at 37 °С. Then, the membrane was incubated with goat anti-rabbit IgG-HRP conjugate (Sigma, St. Louis, USA) in PBST for 1 hour at 37 °С. After every incubation stage, the membrane was thoroughly washed by PBST from non-specifically bound antibodies. Specific immunostaining was developed in 0.05% 4-chloro-1-naphthol (Sigma, USA) solution in 0.05 M potassium phosphate buffer (рН 6.0) with 0.06 % H 2 O 2 .
α-Сhains were obtained from the D-fragment of non-cross-linked fibrin by preparative electrophoresis in 10% PAGE with 0.1% SDS. To accomplish this, the disulfide bonds of the D-fragment were reduced by 5% β-mercaptoethanol in PBS with 8 М urea under argon atmosphere for 3 h at 37 °С. The SH-groups were alkylated by 4-vinylpyridine (Sigma, USA) under argon atmosphere for 2 h at 20 °С [28]. The proteins were precipitated by acetone and fractionated by electrophoresis. α-chains fractions were eluted from the respective bands of the gel with 0.1% trifluoroacetic acid (TFA) and precipitated by acetone.
Trypsin hydrolysis of the α-chains was performed as previously described [29]. The acetone pellet, which contained α-chains, was dissolved in 0.1 М ammonia-bicarbonate buffer, рН 9.5 and in- . The amount of protein in the fractions was measured spectrophotometrically using BCA TM Protein Assay Kit (Thermo Fisher Scientific, Pittsburgh, USA) at λ = 570 nm. Both fractions of tryptic peptides (bound to K5 and not bound) were dialyzed against 0.05 M Na-phosphate buffer (рН 7.4) to remove 6-AHA and NaCl and concentrated on Speed-Vac (Thermo Fisher Scientific, Waltham, USA). In total, 0.024 mg of protein in 0.01 ml solution bound to K5 was obtained by affinity chromatography, and 0.23 mg of protein in 0.1 ml solution appeared to be not bound.
The tryptic peptides were studied by MALDI-TOF using the Voyager DE PRO spectrometer (Applied Biosystems, Carlsbad, USA) in the positive ion mode, in the mass range МН+ of 500 to 5000 kDa. We used reflex mode of time-of-flight detector of the mass-spectrometer with the applied voltage of 20 kV. Internal calibration was performed using matrix peaks (МН+ 666.0293) and autolyse peptides of trypsin (МН+ 2163.0566, 2273.1599). Н+matrix ionization of tryptic peptides was done using 3,5-dimethoxy-4-hydroxycinnamic acid (DHCA, Sigma, USA) under laser emission. DHCA concentration in the matrix reagent was 1 mg/ml, reagent was dissolved in a solution of equal volumes of
Fig. 1. Electrophoregram of the D-fragment of noncross-linked fibrin and DD-fragment of fibrin (left from markers). Western blot image of fragments, to which K5 binds (right from markers). M − molecular mass markers
acetonitrile (Sigma, USA) and 0.1% TFA acid. For analy sis, 1 µl of each sample (fractions of affinity bound and not bound to K5 tryptic peptides with 2.4 mg/ml and 2.3 mg/ml concentrations respectively) was mixed with 1 µl of matrix reagent and transferred to the sample plate, then dried and analyzed. The mass spectrum data were treated using Data-Explorer 4.1 (Applied Biosystems, Carlsbad, USA). We measured monoisotopic values of the protonated molecules, using the average of 4 to 10 mass spectrum data. The amino acid sequence of the tryptic peptides was determined using an online resource Peptide Mass Calculator (Expasy.org). The presented electrophoregrams and blotograms are typical for repeated experiments. The results of experiments, the permissible error of which did not exceed 5% (p < 0.05) were included in the work. Graphic modeling was performed using the Internet resource Protein Data Bank (PDB) archive. The ribbon diagrams of D-fragment (PDB ID: 1FZC) and plasminogen K5 (PDB ID: 2KNF) are based on their crystal structure [30,31].
results and discussion
We have recently shown that plasminogen fragments of K1-3 and K5 bind to different sites that are adjacent in the DD fragment of fibrin [32]. The known binding site for plasminogen located in the Aα148-160 sequence of the D-region of fibrin(ogen), according to the literature, is complementary to the LBS of K1-3, while the binding site for K5 remains unknown. Binding of the isolated K5 to fibrin Dand DD-fragments and their polypeptide chains was investigated by means of immunoblotting to localize the region responsible for the K5-mediated interaction of Glu-plasminogen with fibrin. This approach reveals the binding sites on the linear fragments of the polypeptide chains. Fibrin fragments and their chains were separated by SDS-PAGE in the presence of β-mercaptoethanol for reducing disulfide bonds. Proteins were transferred from gel to a nitrocellulose membrane, which was then incubated with K5. The bound K5 was detected using monospecific polyclonal antibodies and visualized using anti-rabbit antibodies conjugated to horseradish peroxidase. The data presented in Fig. 1 show that K5 is specifically adsorbed to the DD-fragment of fibrin and the D-fragment obtained from non-cross-linked fibrin. K5 binds to α-, γ-and γγ-chains but is not adsorbed on the β-chains of the fragments (Fig. 2). The results suggest that the interaction of plasminogen, It is known that the cyanogen bromide fragment of fibrin(ogen) FCB-2 stimulates activation of Glu-plasminogen by tissue activator (e.g., it has binding sites for these proteins), while t-NDSK does not exhibit effector properties [33]. Subsequently, the interaction of K5 with the polypeptide chains of these fragments was investigated using an immunoblot, and t-NDSK was used as a negative control. The FCB-2 fragment consists of peptides of all three polypeptide chains of the D-fragment: α -5.9 kDa; β -5.8; 4.3; 2.2 kDa and γ -21 kDa, which are interconnected by disulfide bonds. The fragment of the central part of the molecule, t-NDSK, has structural similarities with the E-fragment and consists of the following chains: α -3.5 kDa; β -9 kDa; γ -10.5 kDa. Fig. 3 shows a scheme that illustrates location of the studied polypeptides in the structure of the fibrinogen molecule.
The SDS-PAGE of fragment FCB-2, reduced by β-mercaptoethanol, clearly shows two protein bands, one for the γ-chain (21 kDa ) and the other for a mix of α-and β-chains (5.9 and 5.8 kDa, respectively). Polypeptide chains of the t-NDSK frag-molecular and clinical studies of hemostasis (Fig. 4, А). The Western blot image (Fig. 4, B) shows the binding of K5 to polypeptide chains of the studied fragments. It is seen that K5 does not interact with the γ-chain of FCB-2, which consists of the amino acid residues γ95-265, i.e. there is no binding site for plasminogen K5 within this polypeptide fragment in the D-fragment of the fibrin(ogen) molecule. Meanwhile, K5 interacts with the protein band, which includes the α-and β-chains of the FCB-2 fragment. As we have shown, K5 does not bind to the β-chains of the DD-and D-fragments. Therefore, the detected band, corresponding to the α-and β-chains, indicates the interaction of kringle 5 with the α-chain of the FCB-2 fragment, represented by amino acid residues α148-207. Less intense staining of this band is due to the partial contribution of the β-chain. It should be noted that K5 does not bind to any of the presented chains of the t-NDSK fragment (Fig. 4, B).
Fig. 2. Electrophoregram of polypeptide chains of D-fragment of non-cross-linked fibrin and DD fragment of fibrin (left from markers). Western blot image of polypeptide chains of the fragments, to which K5 binds (right from markers). M − molecular mass markers
Absence of the cationic center is the key feature of the K5 ligand binding site, while the anionic center is represented by the canonical Asp516 and Asp518. During the study of K5 ligand specificity, it was shown that it has affinity to aminohexyl-, homoarginine-and guanidine-hexyl-Sepharose 4B and does not interact with arginine-Sepharose 4B, the ligand mimicking the C-terminal arginine in proteins [34]. Therefore, the structural requirements of K5 ligand binding site are met by the lysyl or arginyl residues in the polypeptide chains of proteins.
The obtained data showing the lack of K5 interaction with the polypeptide chains of t-NDSK fragment and β-chains of DD-and D-fragments, which contain a certain amount of lysyl and arginyl residues, indicate that not all positively charged amino acid residues, exposed on linear fragments of polypeptide chains under experimental conditions, may serve as ligands for K5. Obviously, the site within the fibrin(ogen) molecule, complementary to the K5 ligand binding site, has a structure, that provides the necessary presentation of the side radical of lysine or arginine, the positive charge of which is not compensated by interactions with surrounding amino acid residues.
Thus, as a result of the conducted researches, the new information about localization of plasminogen kringle 5 binding sites in the polypeptide chains of the fibrin(ogen) D-regions has been obtained. It is shown that K5 specifically interacts with the α-chains of D-and DD-fragments of fibrin, which are represented by Aα111-197 and Aα111-206 sequences, respectively, as well as with the α-chain of the cyanogen bromide fragment FCB-2, which is identical to the Аα148-207 fragment of fibrin(ogen) molecule. A comparison of these fragments of the polypeptide chains allows one to assume that the K5 binding site is located in the polypeptide sequence Аα148-197. Because the Aα148-160 section is responsible for interaction with the K1-3 plasminogen fragment, the K5 binding site probably is located within the Aα160-197 sequence. This section of the α-chain contains arginyl residues in the 162, 167, 171, and 197 positions and lysyl − in the 176, 183, and 191 positions, making them potential targets for K5 binding. It was shown that the γ-chain of the Dfragment also contains the K5 binding sites outside the sequence γ95-265.
Taking into consideration that the binding sites for K1-3 and K5 in the fibrin DD-fragment are adjacent [32], further studies were aimed to explore more detailed localization of the K5 binding site in the α-chain of D-fragments. To accomplish this, an isolated α-chain was obtained from the D-fragment of fibrin, tryptic peptides of the α-chain were produced, The isolated α-chain was obtained by preparative SDS-PAGE. Preliminarily, the disulfide bonds of the D fragment were reduced, then the SH-groups were alkylated. From each 2.0 mg of the D-fragment, we obtained 0.3 mg of α-chain. It was hydrolyzed by trypsin at a 1:25 mass ratio of enzyme : substrate at pH 9.5. The hydrolysate was applied to a 1.4 ml column with K5-Sepharose 4B, containing 0.6 mg of immobilized protein. Rechromatography was performed three times for depletion of the peptides affine to K5, after which the column was washed with 0.5 M NaCl in 0.05 M Na-phosphate buffer (рН 7.4), peptides specifically bound to K5 were eluted with 0.25 M 6-AHA. As a result, 24 µg of peptides with affinity for K5 and 230 µg of peptides that are not adsorbed on the affinity sorbent were obtained. The obtained tryptic peptides were analyzed by massspectrometry. The results are given in the Table and Fig. 5. The Table presents tryptic peptides of the α-chain of the D-fragment of fibrin, which do not show affinity for K5. There are 11 arginine residues and 9 lysine residues within the studied sequence of αVal111−Arg197 of the D-fragment. In the Table, the arginine and lysine residues located at the C-terminus or inside the tryptic peptides are marked in red.
As expected, peptides having positively charged amino acid residues at the C-terminus did not interact with K5. However, a number of peptides with lysyl and arginyl side radicals do not show affinity for K5 as well. Interestingly, the tryptic peptide 192Asp − 199Arg, which includes Arg197, was found among them. That is, the location of Arg197 in so-called "proline brackets" (195Pro-203Pro) does not give it the advantage to interact with the K5. Besides, the presence of peptides 192Asp−199Arg, 192Asp−197Arg and 177Asp−197Arg in trypsin hydrolysate suggests that plasmin hydrolysis of fibrin results in a D-fragment formation, whose С-terminal amino acid of the α-chain can be equally likely 197Arg or 199Arg. Only two peptides, specifically bound to K5-Sepharose, were eluted with 0.25 M 6-AHA, e.g. 168Ala−176Lys and 172Glu−183Lys. Their mass spectra are presented in Fig. 5. They are referred to as "peptides with a possible one missed cleavage", which corresponds to 171Arg and 176Lys.
It should be noted that the tryptic peptides 163Gly − 171Arg and 172Glu − 176Lys, which contain 171Arg and 176Lys on the C-terminus, respec-tively, do not bind to immobilized K5. Thus, we obtained data that directly indicate that 171Arg and/or 176Lys at polypeptide α-chains of fibrin D-fragments are involved in binding to Glu-plasminogen K5. It can be argued that the binding site for Glu-plasminogen, that is complementary to ligand binding site of K5, is located within sequence Аα168Ala−183Lys in a weakly structured loop between two supercoils in the α-chain of D-fragment of the fibrin(ogen) molecule. The relative positions of 516Asp and 518Asp in the ligand binding site of K5 and the 171Arg and 176Lys of the α-chain of the D-fragment are shown in Fig. 6. Fig. 7 presents a ribbon scheme of the fibrin(ogen) molecule D-fragment. All three polypeptide chains − α, β and γ, which are part of the D-fragment, in their N-terminus have the structure of the α-helix. Beyond the peripheral disulfide node, the β-and γ-polypeptide chains form С-terminal globular β-and γ-modules, while the α-chain turns in the opposite direction. After a short weakly structured section, it regains the α-helical structure and exits the D-fragment. The black color in the scheme indicates the Аα148-160 sequence, which has the known plasminogen binding site complementary to the LBS of K1-3. The arrow points the position of 171Arg and 176Lys responsible for K5 binding. It can be seen that the fragments, which have binding sites for plasminogen K1-3 and K5, are located in the D-fragment near to each other.
It is hypothesized that interaction of Glu-plasminogen kringle 5 with αArg171/Lys176 of fibrin leads to the zymogen molecule acquiring an open conformation, which allows K1-3 binding to nearby αLys148−Ser160 sequence, thus presenting the activation loop for tissue activator cleaving Arg161-Val162 peptide bond of plasminogen to form active plasmin. This two-center interaction of plasminogen with fibrin is a necessary condition for zymogen activation.
The obtained novel data on the localization of binding sites of plasminogen K5 in the polypeptide chains of the fibrinogen D-domains expand our understanding of the functional importance of conformational peculiarities of fibrinogen and plasminogen molecular interactions during fibrinolysis. Our findings are related to the fundamental haemostasis study, which has a potential applied significance in targeting pharmacorrection of fibrinolysis dysfunction and various cardiovascular diseases. | v3-fos-license |
2020-11-26T09:04:57.058Z | 2020-01-01T00:00:00.000 | 229504990 | {
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"year": 2020
} | pes2o/s2orc | Engineered Plant‐Based Nanocellulose Hydrogel for Small Intestinal Organoid Growth
Abstract Organoids are three‐dimensional self‐renewing and organizing clusters of cells that recapitulate the behavior and functionality of developed organs. Referred to as “organs in a dish,” organoids are invaluable biological models for disease modeling or drug screening. Currently, organoid culture commonly relies on an expensive and undefined tumor‐derived reconstituted basal membrane which hinders its application in high‐throughput screening, regenerative medicine, and diagnostics. Here, we introduce a novel engineered plant‐based nanocellulose hydrogel is introduced as a well‐defined and low‐cost matrix that supports organoid growth. Gels containing 0.1% nanocellulose fibers (99.9% water) are ionically crosslinked and present mechanical properties similar to the standard animal‐based matrix. The regulation of the osmotic pressure is performed by a salt‐free strategy, offering conditions for cell survival and proliferation. Cellulose nanofibers are functionalized with fibronectin‐derived adhesive sites to provide the required microenvironment for small intestinal organoid growth and budding. Comparative transcriptomic profiling reveals a good correlation with transcriptome‐wide gene expression pattern between organoids cultured in both materials, while differences are observed in stem cells‐specific marker genes. These hydrogels are tunable and can be combined with laminin‐1 and supplemented with insulin‐like growth factor (IGF‐1) to optimize the culture conditions. Nanocellulose hydrogel emerges as a promising matrix for the growth of organoids.
Introduction
Organoids are complex 3D cell culture systems able to self-renew and reorganize in vitro. These "mini-organs" mimic many of the physiological properties of tissues and organs including much of their behavior and functionality. [1,2] These attributes make organoids a robust and reliable biological model for many applications in biomedicine, especially studies on drug screening and design, disease modeling, and developmental biology. [3] Organoids can be generated from embryonic, adult, pluripotent or induced pluripotent stem cells (ESC, ASC, PSC, or iPSC, respectively), as well as from primary healthy or cancerous tissues. [1,4] For establishment and long-term culture, organoids are commonly embedded within an Engelbreth-Holm Swarm (EHS) matrix derived from the reconstituted basement membrane of mouse sarcoma. [5,6] This decellularized extracellular matrix (ECM)-based gel is known as Matrigel (Corning), Cultrex BME (Trevigen), or Geltrex (Gibco). In spite of its wide application in organoids culture, the EHS matrix has a poorly defined composition and an important batch to batch variability. Indeed, more than a thousand unique biomolecules are found in Matrigel and the batch to batch consistency of its proteic components is limited to 50%. [7] The lack of understanding of how these factors influence the experimental conditions, such as cells and its microenvironment, has delayed its translation to clinics. [8] Modifying the regular matrix stiffness as well as the laborious process to recover cells encapsulated within the gel stand as critical limitations in research and development. Lastly, to facilitate large-scale experimental studies and use of cultures in clinical environments, a cost-effective product is required. Therefore, alternative matrices able to reduce the dependency of Matrigel, or fully replace this animal-based material-without its critical drawbacks-are urgently needed.
In addition to decellularized tissues, natural and synthetic polymeric materials have been explored for organoid systems. [9] Cruz-Acuna et al. reported a four-armed maleimide-terminated poly(ethylene glycol) (PEG) hydrogel able to grow human intestinal organoid from ESC and iPSCs. [10] Gjoresvski et al. evaluated another PEG-based matrix with dynamic stiffness where intestinal stem cells expand and form organoids. [11,12] These two studies have demonstrated that mechanical properties play a key role in the generation and expansion of organoids in culture. The presence of adhesive sites, mainly Arg-Gly-Asp (RGD) peptides, also stands as a fundamental element in these matrices, inducing high cell attachment and differentiation. Similarly, laminin-1 is suggested to be a required additive to induce robust organoids formation in a fibrin-based hydrogel. [13] However, carbohydrate polymers and plant-based hydrogels remain poorly explored. Capeling et al. presented a non-adhesive alginate hydrogel for intestinal organoids growth which was unable to progress into enteroids. In contrast, Wilkinson et al. showed that alginate beads functionalized with type I collagen can support the generation of lung organoids. [14] To the best of our knowledge, functionalized cellulose-based hydrogels have not been rigorously investigated nor successful in growing organoids. Cancerous spheroids (non-structured clusters of proliferating transformed cells) were previously cultured in neat microfibrilated cellulose scaffolds. [15] Similarly, inert cellulose nanofibrillar hydrogel was shown to promote the differentiation of human liver organoids. [16] Here, we introduce an engineered plant-based nanocellulose hydrogel for the growth of mouse small intestinal organoid (Figure 1a).
Results and Discussion
In a step-wise approach, the design of the hydrogel is guided to match the biochemical and mechanical properties of the EHS matrix. Oxidized cellulose nanofibers (CNF) are functionalized with fibronectin-derived moieties (RGD peptides) to enhance cellular interaction between the organoids and the cellulosic surface, inducing adhesion. The temperature-responsive gellification of Matrigel is mimicked by the ionic crosslinking of CNF. Crypts are suspended in a physically crosslinked hydrogel that undergoes in situ Ca 2+ -mediated ionic crosslinking. The internal remodeling of the hydrogel 3D structure is enabled by the rearrangement of charges and fibers entanglement which are independent of the metalloproteinases expression and activity. This strategy allows the hydrogels not only to reach identical mechanical properties as Matrigel, but also to achieve any level of viscoelastic modulus, whether higher or lower ( Figure S1, Supporting Information). The rheological properties of Matrigel (Corning, protein concentration = 8.6 mg mL −1 ) were characterized by the viscosity-shear, showing a shear-thinning behavior ( Figure 1c). Oscillatory strain sweep of Matrigel measures a storage modulus (G') of 101 Pa and a loss modulus (G") of 6 Pa in the linear viscoelastic (LVE) region ( Figure 1d). The curves do not exhibit a crossover point, highlighting the dominance of the elastic regime. The Matrigel yielding point occurs at a shear stress of 56 Pa (Figure 1e). Similar to the EHS matrix, nanocellulose hydrogel-here referred to as RGD-GLY hydrogel (0.1 wt% solids content)-exhibits a characteristic shear-thinning curve. Measuring the dynamic strain shows RGD-GLY hydrogel to have an elastic modulus (G') of 98 Pa, a viscous modulus (G") of 8 Pa and a yield point of 15 Pa under shear stress. The absence of grafted RGD peptides on the cellulose surface has minimal effects on the material's rheological profile ( Figure S2, Supporting Information), whilst hydrogels at higher solids content (0.2-0.5 wt%) form considerably stiffer matrices ( Figure S3, Supporting Information) which do not degrade over time ( Figure S4, Supporting Information). The remarkable difference between the yielding point of both materials does not show any impact on the culture of organoids, allowing easy recovery of cells by mechanical disruption of the hydrogel, followed by the usual centrifugation when passaged. The material's structure was analyzed under small-angle neutron scattering (SANS), demonstrating that ionic crosslinking of cellulose nanofibers as well as CNF functionalization modify the hydrogel network organization (Figure 1f). The RGD-grafted hydrogel porosity is characterized by a correlation length of 730 Å, lower than the 750 Å measured for the non-functionalized gels (Figure 1g). The structural characterization of the porous hydrogel shows the nanocellulose dimensions to match those of the constitutive collagen fibers present in the EHS matrix. [17] These results summed to the rheological measurements show the engineered cellulose gel to have the required mechanical properties.
By opposition to scaffolds for tissue engineering, hydrogels for organoid systems must reconstitute the microenvironment of the basal membrane beyond its stiffness or elasticity. Indeed, cell proliferation as well as cell differentiation are directly dependent on the niche where they are confined. [18] For this reason, the osmolality and pH of the hydrogel are controlled to emulate the physiological conditions. Made essentially of water (99.9%), nanocellulose hydrogel has no osmolality (0 mOsmol kg −1 ). Glycine (250 mm), a non-polar amino acid, is dissolved in the hydrogel to increase its osmolality to 295 mOsmol kg −1 (Figure 1h), matching Matrigel (290 mOsmol kg −1 ). This strategy not only preserves the hydrogel colloidal stability before the ionic crosslinking takes place, but also conserves cell viability until the culture media is added to the wells. HEPES (25 mm), a widely used buffering agent in cell culture, is also added to buffer the matrix pH to the physiological range. A highly hypotonic material induces massive apoptosis within 24 h causing crypts to turn into an aggregate of debris. In addition, an acidic hydrogel also impacts the survival and growth of crypts encapsulated within the matrix ( Figure S5, Supporting Information). In this study, the combination of glycine and HEPES is referred to as GLY. The preliminary test of neat, GLY-and RGD-GLY hydrogels as matrices for organoid growth is shown in Figure 1i. Mus musculus small intestinal organoids were adopted as the biological model and initially established in Matrigel. After passaging, mouse crypts embedded in neat hydrogel turn into an agglomerated cluster of debris within 2 days. Once the osmolality of the matrix is balanced by GLY, small intestinal crypts progress into cystic organoids. Organoids are formed only in the presence of RGD-GLY hydrogel, showing morphology and size similar to the crypts established in Matrigel.
In order to assess the biocompatibility of engineered hydrogels, small intestinal crypts are embedded in different formulations of the material and cultured for 4 days. On the second and fourth day, calcein AM staining was used to identify the viable cells and propidium iodide to reveal the dead cells. Crypts encapsulated in the neat nanocellulose hydrogel exhibit a high level of apoptosis, with on average 80% of each unit containing dead cells, with only 20% viable cells after 2 days. Once the gel osmolality is balanced and the matrix is buffered by GLY, the viable area within cystic organoids is increased to 78% and the dead area reaches 22%. Grafting RGD peptides onto the cellulose nanofibers does not affect overall viability, preserving 80% and 20% of living and dead areas, respectively. Organoids embedded in Matrigel present viability of 88% and an apoptotic region of 18% after being cultivated for 48 h (Figure 2a). On the fourth day of culture, crypts in the neat hydrogel continue as a cluster of apoptotic debris, representing 97% of its area. Organoids encapsulated within the GLY hydrogel sustain 75% of viability with the remaining 25% of organoid area containing apoptotic cells. RGD-GLY hydrogel supports 81% viability, indicating the maintenance of cells in small intestinal organoids to be similar to those seeded in the EHS matrix (80% living and 20% dead areas) (Figure 2b).
The ability of nanocellulose hydrogels to establish organoids from freshly isolated mouse small intestine crypts was assessed. Small intestinal organoid viability. A) Crypts were seeded in neat, GLY, and RGD-GLY nanocellulose hydrogel. After 2 days, a major apoptotic area is detected in the samples cultured in the neat hydrogel. Once GLY is added, cystic organoids are formed, predominantly composed of living cells. However, morphology and topography strongly differ from the control (Matrigel). Crypts seeded in RGD-GLY hydrogel form cystic organoids, presenting a development similar to those in Matrigel with 80% viability. B) After 4 days, RGD-GLY hydrogel induces progression to budding organoids. In the absence of RGD peptides, the organoids remain cystic, growing in size only. Scale bars: 100 µm. Results shown represent independent experiments performed in triplicates (n = 3, error bars = SD). * = p < 0.05. **** = p < 0.0001. ns = non-significant.
Whilst organoids are easily established in Matrigel, dissected crypts directly seeded in RGD-GLY hydrogel do not progress into organoids. However, when the hydrogel is supplemented with a minor volume of Matrigel-20% (v/v)-, organoids are established from fresh intestinal crypts ( Figure S6, Supporting Information). The combination of nanocellulose and Matrigel at this condition does not alter the stiffness of the matrix (Figure S7, Supporting Information). Interestingly, organoids could also be formed from single cells seeded in RGD-GLY hydrogel, although the growth rate is considerably slower than those Once established and passaged, the growth of organoids embedded into RGD-GLY hydrogel (absent of any volume of Matrigel) is tracked over four days and compared to those within the EHS matrix. On day 1, small intestinal crypts embedded within both materials acquire spherical morphology with a lumen charac-teristic of the cystic phase. On the second day, the spheres become asymmetric with the initiation of budding. The following day, organoids in RGD-GLY hydrogel and Matrigel contain fully formed budding crypts, requiring passage on the fourth day (Figure 3a). Interestingly, all the organoids cultured in GLY hydrogel remain in their cystic phase without forming buds even after 7 days ( Figure S9, Supporting Information). The presence of adhesive sites plays a key role in the process of organoid development, and removal is known to inhibit the formation of buds. Indeed, the lack of integrin beta-1 ligand induces the reversion of polarization, with the external exposure of the apical layer and internalization of the basal region. [19] Despite the disruption of this process in GLY hydrogels, cells are still viable, and organoids grow in size. In contrast, RGD-GLY hydrogel promotes crypts to form cystic organoids that evolve to budding units in 2 or 3 days and require to be passaged after 4 days. However, some cystic organoids are still present in RGD-GLY hydrogel ( Figure S10, Supporting Information). The organoids cultured in the RGD-GLY hydrogel were passaged every 4 days for 2 weeks, preserving the expected development and morphology ( Figure S11, Supporting Information). Organoids passaged from nanocellulose back to Matrigel also present the usual morphology and normal growth for 7 days (Figure 3b). These results indicate that both the nanocellulose and the animal-based matrix are interchangeable, whilst engineered nanocellulose seems not to interrupt the self-renewing ability of the stem cell population and subsequent self-organizing processes present in the organoids.
We further examined cytoskeletal and proliferative cell markers to characterize organoid growth in nanocellulose hydrogels. The development of organoids within the nanocellulose hydrogel and Matrigel was analyzed by culturing organoids derived from fluorescence ubiquitination cell-cycle indicator (FUCCI) mice in both matrices. [20] F-actin filaments are observed by the presence of LifeAct-green fluorescent protein (GFP) in FUCCI organoids. Prominent apical staining of the well-known network of F-actin, which support microvilli, was observed indicating the preservation of cytoskeleton integrity and cellular polarity in both cystic and budded phases. A similar proportion of red fluorescent protein (RFP)-labeled cells in G1 phase of the cell cycle was also observed in organoids cultured in nanocellulose for 2 and 4 days (Figure 3c). Proliferative cells are found in small intestinal organoids revealed by staining for cells expressing the nuclear protein Ki67, after 3 days of culture in nanocellulose hydrogel and Matrigel (Figure 3e). Metabolic activity is increased by 75% during the first 2 days of culture in the hydrogel and remains stable on the fourth day ( Figure S12, Supporting Information). Interestingly, yes-associated protein 1 (YAP-1), a mechanosensing effector of the Hippo signaling pathway required for initiation of intestinal organoid budding, is shown to be inactive in both cystic and budded organoids (Figure 3d). [11,21] Recently, based on organoids formed from single stem cells, Serra et al. clarified that YAP-1 is active and localized within the nuclei during the first 24 h of culture, and then re-localized to the cytosol once inactivated over time. [21] However, since the organoids assessed here were not formed from single cells, the transient activation of YAP could not be detected. This suggests that the organoids could have been seeded after breaking of the initial symmetry has occurred in the early stages. Overall, the RGD-GLY hydrogel provides the biological conditions required for organoids to develop from crypts to cystic organoids which progress to mature organoids containing crypt domains.
To further assess the characteristics of organoids cultured in RGD-GLY hydrogel compared to Matrigel, comparative transcriptomic analysis was performed. RNA sequencing reveals that small intestinal organoids embedded in nanocellulose exhibit some differences in expression of specific groups of genes when compared to those in Matrigel. Biological variations are also detected between organoids derived from three different mice, and cultured in each material (Figure 4a). Overall, a good correlation in the whole transcriptome is observed, with the coefficient of determination as high as 0.93 (Figure 4b). One hundred ninety eight genes are identified to be significantly differentially expressed in organoids growing in nanocellulose with fold change ≥ 2 (FDR and p ≤ 0.05) (Figure 4c), notably those related to receptor ligand activity and calcium-dependent binding ( Figure S13, Supporting Information). This event is expected and understandable, considering the ionic crosslinking method explored to provide the mechanical properties of the hydrogel. Interestingly, the expression of crypt base columnar (CBC) stem cell markers, such as Lgr5 and Ascl2, decreases in RGD-GLY hydrogel (Figure 4e), whilst those related to revival stem cells present up to a 20-fold increase (Figure 4g). [22] CBC stem cells refer to a population of cells responsible for the self-renewability of the intestinal epithelium. [23] Recently, a population of quiescent stem cellsnamed revival stem cells-was characterized on its ability to drive the regeneration of damaged intestinal tissue and to reconstitute the eventual loss of CBC stem cells. [24] Both populations are of critical importance in reliable intestinal models. Despite these variations, the Spearman's correlation coefficient for CBC and revival stem cells genes remains as high as 0.89 (Figures 4d,f). The expression of proliferative markers is downregulated, in contrast to the YAP signaling and differentiation genes which appear to be enhanced ( Figure S14, Supporting Information). Indeed, markers related to Paneth (Oit3 and Ang4), tuft (Trpm5 and Gfi1b) and goblet cells (Muc2 and Tff3) as well as genes associated to enteroendocrine cells (Tph1 and Reg4) and enterocytes (Alpi and Apoa1) are found in higher levels for organoids in the cellulosic matrix ( Figure S15, Supporting Information). These differences can be justified by the high number of growth factors and other biomolecules existing in Matrigel but absent in RGD-GLY hydrogel. [5,7] Our simplified system is essentially made of 99.9% of water and only 0.1% solids content, functionalized with a single cell adhesive peptide. In this context, the ability to supplement the hydrogel with ECM-derived elements is welcome, allowing the design of a controlled microenvironment for specific needs. Overall, the culture of small intestinal organoids in standard RGD-GLY hydrogel may be a good model that supports studies focused on differentiated cell types and the process of regeneration following injury.
As a proof-of-concept of the tunability of nanocellulose hydrogel, this matrix was combined to insulin-like growth factor-1 (IGF-1) and laminin-1; these are two of the elements present in Matrigel. IGF-1 has the ability to induce the crypt expansion by activation of intestinal stem cells. [25] Laminin-1 is the major protein found in the EHS matrix and forms a network associated with collagen, fibronectin and other proteins. [26] More than reinforcing the mechanical properties, laminin also promotes epithelial cell adhesion and differentiation. [27] The concentration of IGF-1 in the EHS matrix ranges between 1.7 to 4.7 ng mL −1 , whilst laminin makes up to 60% of the protein content in Matrigel. The addition of IGF-1 at its minimal concentration to the hydrogel accelerates the expansion of early-stage cystic organoids in less than 24 h. Within 3 days of culture, organoids in IGFcontaining hydrogel expand substantially in size (Figure 5a). In contrast, organoids growing in IGF-free hydrogel require at least 5 days to achieve similar dimensions ( Figure S16, Supporting Information). This indicates that IGF-1 might contribute to the development of the organoids seen in the EHS matrix. On the other hand, the addition of laminin-1 (0.5 mg mL −1 ) has minimum effects on the nanocellulose rheological properties, preserving the storage and loss moduli at 94 and 6 Pa, respectively (Figure 5b). Organoids grown in hydrogels containing this protein present a similar morphology to those cultured in absence of laminin-1 (Figure 5c). Previous works reported that enriched laminin-materials (up to 3 mg mL −1 ) benefited the formation and growth of organoids. [11,13] However, the success of these gels might simply be due to the generous amount of laminin, also extracted from mouse sarcoma. In contrast, Rezakhani et al. recently showed that laminin-1 in gels at concentrations as low as 0.3 mg mL −1 can also improve organoid growth, whilst lamininfree hydrogels were found to support organoid derivation. [28] Yavitt et al. also demonstrated that hydrogels without laminin were restricted to expand intestinal stem cell colonies, but could not form organoids. [29] In this study, the effect of a higher concentration of laminin was not evaluated; this was to maintain functionalized nanocellulose fibers as the predominant element in a fully defined plant-based hydrogel. Inspired by these outcomes, the addition of the ECM-derived factors can be further explored to achieve the conditions required to sustain different organoids derived from other organs.
Conclusion
To the best of our knowledge, we introduce the first engineered plant-based nanocellulose hydrogel as a very low-cost but performant material for organoid growth. Organoids represent a robust model for key applications in biomedicine including drug screening and disease modelling. The EHS matrix has become the standard material for organoid culture and is widely used. However, Matrigel and similar matrices remain expensive, biochemically variable and undefined. These are major obstacles for fundamental research studies and the translation of organoids to clinics. Alternative matrices able to sustain organoid systems are required to drastically reduce costs and to eliminate the liability from unknown biomolecules. These materials must be compliant with good manufacturing practices (GMP) procedures required for re-producible and accredited pre-clinical tests. Synthetic ECM-like structures allow organoids to be cultivated for further use as tissue replacement in regenerative medicine. Our hydrogel has similar mechanical properties to those of the EHS matrix. Easy to functionalize, cellulose nanofibers are grafted with RGD peptides, inducing small intestinal organoids formation and growth. Both matrices are interchangeable, showing the presence of proliferative cells and regular cell cycle. In this study, mouse small intestinal organoids were adopted as biological model. Despite the positive outcomes reported, nanocellulose is limited by not establishing organoids from dissected tissues. In addition, other types of organoids, including those derived from human tissues, must be individually evaluated to determine the ideal mechanical and biochemical conditions of specific cultures. Overall, nanocellulose hydrogel stands as a promising material for its tunability and compatibility with ECM-components. Indeed, based on recent reports, the specific combination of nanocellulose and laminin may support the long-term culture of organoids, allowing multiple passages. Engineered nanocellulose hydrogel represents a performant and sustainable alternative for the growth of organoids, contributing to significantly reducing the costs in studies against diseases of global concern such as cancer.
Experimental Section
Synthesis of Nanocellulose Hydrogel: A 4 wt% never dried bleached Eucalyptus Kraft (BEK) pulp suspension (Australian Paper, Maryvale, Australia) containing proportional amounts of TEMPO (Sigma-Aldrich 214000) and NaBr (Sigma-Aldrich 71329) was prepared according to Saito et al. [30] To initiate the oxidation, 12 w/v% NaClO (Sigma-Aldrich 425044) (pH adjusted to 10) was added drop-wise at a ratio of 5 mmol NaClO g −1 fiber. The reaction was maintained at pH 10 by adding 0.5 m NaOH. The oxidation process was proceeded until reaction termination-corresponding to the stabilization of pH. Oxidized fibers were recovered and washed through filtration with a Buchner funnel and stored refrigerated (4°C). The oxidized pulp was dispersed in deionized water to achieve a concentration of 0.1 wt% and converted into gels via mechanical fibrillation through a high-pressure homogeniser (GEA Niro Soavi Homogenizer Panda) at 1000 bar. The carboxylate group content was determined by conductometric titration as previously reported. [30,31] Freeze-dried oxidized pulp samples (≈30 mg) were suspended into 40 mL deionised water. Then, 40 µL 1% NaCl was added to the suspended sample. The pH of the suspended sample was adjusted between 2.5 and 3 prior to titration with 0.01 m NaOH using a Mettler Toledo T5 titrator. The conductivity of the sample was monitored throughout the titration progress. The carboxyl group content CC (mmol COO − Na + g −1 fiber) was determined as: Where V 1 and V 2 are the amount of titrant (start and end of conductivity titration curve) required to neutralize the carboxylic groups (in L), c is the NaOH concentration (mol L −1 ), and w is the sample weight (g).
Hydrogel Functionalization: Cellulose nanofibers were functionalized with RGD peptides via EDC/NHS coupling. EDC (Sigma-Aldrich E6383) and NHS (Sigma-Aldrich 130672) were dissolved in 2-(Nmorpholino)ethanesulfonic acid (MES, Sigma Aldrich M0164) buffer containing NaCl (Merk 1064040500) 0.9% at pH 6. To activate the carboxylate groups, equimolar solutions of EDC and NHS were added to the gel in a mole ratio 1:1:1 to the existing carboxylate concentration and stirred 15 min at room temperature. A volume of (GRGDSPC) RGD peptides (GenScript) (final concentration = 2 mm) was added to the hydrogel and stirred for 2 h at room temperature. The pH of the hydrogel was adjusted to 7 by adding NaOH (Sigma-Aldrich S8045) (1 m) measured by pH strips. Glycine (Sigma-Aldrich 50046) (250 mm) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Sigma-Aldrich H7523) (25 mm) were dissolved in the hydrogel. A volume of 100 µL was used to measure the osmolality in a K-7400S osmometer (Knauer). The pH was adjusted to 7 by addition of NaOH (1 m), measured with pH strips. The hydrogel was sterilized under ultraviolet radiation exposure during 20 min prior to use. For functionalization, hydrogels were mixed with IGF-1 and laminin-1, at 5 ng mL −1 and 0.5 mg mL −1 final concentration, respectively.
Rheology: Nanocellulose hydrogels and Matrigel were characterized by rheology using an Anton Paar MCR302 rheometer with a parallel plate. All testing was performed at 37°C and a solvent trap was used to ensure temperature stability. Two types of rheological tests were performed: viscosity measurements and oscillatory strain sweep. Viscosity measurements were performed between shear rates of 0.5 to 100 s −1 . Oscillatory strain sweep was measured between 0.01% and 100% strain at constant 1 Hz frequency. A volume of 1 mL of nanocellulose hydrogels was crosslinked with 100 µL CaCl 2 (100 mm) and incubated at room temperature for 5 min. A volume of 1 mL of Matrigel was incubated at 37°C, 5% CO 2 for 15 min. Samples were covered with organoid culture media and incubated at 37°C for 3 h. After incubation, the culture media was removed and the hydrogels or Matrigel transferred to the rheometer surface for measurements. For stability test, RGD-GLY hydrogel was prepared in a similar manner and then incubated at 37°C, 5% CO 2 during 1 to 4 days before measurement. All measurements were performed in triplicates to ensure repeatability.
Small-Angle Neutron Scattering: Small-angle neutron scattering (SANS) experiments were made at the Time-of-Flight BILBY beamline at the Australian Nuclear Science and Technology Organization (ANSTO), NSW, Australia. The D 2 O-hydrated samples were placed in the demountable cells of wall thickness 2 mm. For measurements, neutrons of wavelength of 6 Å was selected by neutron-velocity selector. Two sample to detector lengths were applied which cover the Q-range from 0.00254 to 0.31Å −1 . The raw collected data was reduced by the Mantid software with the BILBY package. During data reduction, the background of the D 2 O was subtracted from the hydrated samples. The reduced data was normalized to the absolute scattering values by the pre-calibrated scattering curve of D 2 O. Data analysis was performed by SASview.
Small Intestinal Organoids Culture: All animal procedures were approved by the Monash Animal Ethics Research Platform ethics committee in strict accordance with good animal practice as defined by the National Health and Medical Research Council (Australia) Code of Practice for the Care and Use of Animals for Experimental Purposes. Mice (M. musculus) were culled by cervical dislocation. The small intestine tube was dissected and flushed with PBS to remove feces. Small intestinal tissues were opened longitudinally, scraped with a glass coverslip to remove villi, cut into 5-mm pieces and washed with PBS five times to remove unattached epithelial fragments, mucus and feces. Following incubation for 30 min at 4°C in 3 mm EDTA-PBS solution, intestinal crypts were released from small intestine tissue fragments by mechanically pipetting them with a 10 mL pipette in PBS and repeating this step three times. Isolated intestinal crypts were strained (70-µm cell strainer, Falcon) and pelleted by centrifugation three times at 1500 rpm for 2 min at 4°C. The supernatant was discarded and the pellet containing the crypts was re-suspended in Growth Factor Reduced (GFR) Matrigel (Corning 356 231), nanocellulose hydrogels or in hydrogels supplemented with Matrigel (20% v/v). A volume of Matrigel containing crypts (50 µL) was seeded into a 24-well plate (Nunc) and incubated for 15 min at 37°C until solidified. For nanocellulose hydrogels, the 24-well plate was previously coated with 5 µL of calcium chloride (100 mm), then followed by the seeding of 50 µL of hydrogels containing intestinal crypts. Hydrogels were incubated for 5 min at room temperature. Each well containing each matrix received 500 µL of crypt culture media (DMEM/F12 (Gibco), B27 (Gibco), Glutamax (Gibco), N2 (Gibco), 10 mm HEPES (Gibco), Penicillin-Streptomycin (Gibco), 0.5 µg mL −1 Amphotericin B (Gibco), 50 ng mL −1 EGF (Peprotech), 2% Noggin conditioned media, and 10% R-spondin 1 conditioned media). Crypt culture media was replaced every 2 days, and organoids were passaged every 3-4 days. For passaging, Matrigel or nanocellulose hydrogels were mechanically disrupted and the organoids recovered by centrifugation (3 min at 1500 rpm). Residual volumes of Matrigel or hydrogel were removed, and the organoids resuspended in DMEM/F12 media followed by mechanical disruption or dissociation achieved by manual pipetting for at least 30×. For single cell experiments, the organoids were resuspended in TrypLE Express (Invitrogen, 12604021) and incubated 5 min at 37°C. Crypts or cells were washed with 5 mL of DMEM/F12 media during 3 min at 1500 rpm and resuspended in Matrigel or nanocellulose hydrogels for seeding as previously described.
Viability Test: Small intestine organoids encapsulated in the hydrogels or Matrigel were cultured during 2 or 4 days and stained with Calcein-AM (Sigma-Aldrich C1359) and Propidium Iodide (Sigma-Aldrich P4170). The crypt culture media was replaced by 500 µL of DMEM/F12 media containing 1 µm calcein-AM and 3 µm propidium iodide. The plate was incubated at 37°C 5% CO 2 during 30 min in absence of light. Organoids were imaged under EVOS XL Core microscope. Quantification of living and dead area was performed by image processing using FIJI software and data analysis with OriginPro 9.
Metabolic Activity: Organoids in Matrigel or RGD-GLY hydrogel had their metabolic activity measured by the Prestoblue (Invitrogen A13261) assay. Prestoblue solution was diluted in DMEM/F12 media (10% v/v) and incubated at 37°C for 5 min; 200 µL was added to each well (matrix and organoids and matrices only). Samples were transferred to the incubator (37°C, 5% CO 2 ) for 40 min. Fluorescence was measured by a Pherastar FSX plate reader (540 nm excitation; 590 nm emission). Values from days 2 and 4 were normalized to those from day 1 (100%).
RNA Isolation and Sequencing:
Organoids isolated from three different mice were cultivated for 4 days in both Matrigel and hydrogel. The complete media was removed and wells washed three times with filtered PBS. A volume of 1 mL TRIZol (Invitrogen) was used to dissolved Matrigel and hydrogels and release the cellular content. Chloroform (200 µL) was added to TRIzol and tubes vigorously mixed for 15 s. After 3 min incubation at room temperature, samples were centrifuged at 14 000 rpm for 15 min at 4°C. The aqueous phase was transferred to empty tubes and combined to 500 µL of isopropanol. Samples were incubated at room temperature for 15 min and then centrifuged at 14 000 rpm for 10 min at 4°C. The supernatant was removed and the pellet washed with cold ethanol (75% v/v). Sample were mixed and centrifuged as before. The supernatant was removed and pellet was air dried for 5 min at room temperature. A volume of 15 µL of RNase-free water was added to the tubes. Sample were dried in RNA stabilization tubes (GENEWIZ) by centrifugal vacuum concentrator (Eppendorf) for 30 min at 1500 rpm in 30°C. RNA sequencing was performed and analyzed by GENEWIZ. Briefly, total RNA of each sample was quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies). 1 µg total RNA with RIN value above 6.5 was used for library preparation by poly(A) mRNA Magnetic Isolation (NEBNext) for poly(A) mRNA isolation, then priming with First Strand Synthesis Reaction Buffer and Random Primers followed by cDNA synthesis using ProtoScript II Reverse Transcriptase and Second Strand Synthesis Enzyme Mix. After T-A ligation, size selection (≈420 base pairs) and PCR amplification, the PCR products were cleaned up and quantified by Qubit3.0 Fluorometer (Invitrogen). Multiplexed RNA sequencing was performed on Illumina HiSeq instrument using a 2 × 150 bp paired-end configuration which revealed more than 50 m reads. Quality control and assessment were performed using Cutadapt (V1.9.1) and FastQC (V0.10.1) respectively. [32] Sequence reads were then aligned to M. musculus genome (ENSEMBL:GRCm38.97) using Hisat2 (v2.0.1). [33] Gene expression level was determined using HT-Seq (V0.6.1). [34] Differential gene expression of mouse small intestinal organoids grown in Hydrogel relative to in Matrigel was calculated using the Bioconductor package DESeq2 (V1.6.3) [35] and reported in fold change.
Statistical Analysis: The statistically significant differences in live and dead areas of organoids were assessed by one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test in Graphpad Prism 8. For RNAseq, genes with fold change ≥ 2 with False Discovery Rate (FDR) and adjusted p value < 0.05 were considered significantly differentially expressed. Principal component analysis (PCA) was performed using R to visualize sample-to-sample distances. Gene ontology (biological process) analyses was performed using Metascape. [36] Correlation analyses were performed using liner regression and Spearman's Rank-Order in Prism 7 to compare the expression profiles of whole transcriptome and cell-types specific marker genes between organoid from different matrices.
Supporting Information
Supporting Information is available from the Wiley Online Library or from the author. | v3-fos-license |
2019-09-20T13:04:59.213Z | 2019-01-01T00:00:00.000 | 202688048 | {
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} | pes2o/s2orc | Thermal field formation during wIRA-hyperthermia: temperature measurements in skin and subcutis of piglets as a basis for thermotherapy of superficial tumors and local skin infections caused by thermosensitive microbial pathogens
Thermal field formation during wIRA-hyperthermia: temperature measurements in skin and subcutis of piglets as a basis of caused ABSTRACT Purpose: The temporal and spatial formation of the temperature field and its changes during/upon water-filtered infrared-A (wIRA)-irradiation in porcine skin and subcutis were investigated in vivo in order to get a detailed physical basis for thermotherapy of superficial tumors and infections caused by thermosensitive microbial pathogens (e.g., Mycobacterium ulcerans causing Buruli ulcer). Methods: Local wIRA-hyperthermia was performed in 11 anesthetized piglets using 85.0mW cm (cid:1) 2 , 103.2mW cm (cid:1) 2 and 126.5mW cm (cid:1) 2 , respectively. Invasive temperature measurements were carried out simultaneously in 1-min intervals using eight fiber-optical probes at different tissue depths between 2 and 20mm, and by an IR thermometer at the skin surface. Results: Tissue temperature distribution depended on incident irradiance, exposure time, tissue depths and individual ‘ physiologies ’ of the animals. Temperature maxima were found at depths between 4 and 7mm, exceeding skin surface temperatures by about 1 – 2K. Tissue temperatures above 37 (cid:3) C, necessary to eradicate M. ulcerans at depths < 20mm, were reached reliably. Conclusions: wIRA-hyperthermia may be considered as a novel therapeutic option for treatment of local skin infections caused by thermosensitive pathogens (e.g., in Buruli ulcer). To ensure tempera- tures required for heat treatment of superficial tumors deeper than 4mm, the incident irradiance needed can be controlled either by (a) invasive temperature measurements or (b) control of skin surface temperature and considering possible temperature increases up to 1 – 2K in underlying tissue.
Thermal field formation during wIRA-hyperthermia: temperature measurements in skin and subcutis of piglets as a basis for thermotherapy of superficial tumors and local skin infections caused by thermosensitive microbial pathogens 1
. Introduction
Water-filtered infrared-A (wIRA) radiation (780-1400 nm) [1] shows a significantly deeper penetration into skin and subcutaneous tissue compared to unfiltered IR-A radiation and both, the middle (IR-B, 1400-3000 nm) and the long-wavelength (IR-C, 3000 nm-1 mm) infrared radiation [2,3]. This property causes basic differences concerning tissue heating as compared to conventional heat sources used in thermotherapy such as unfiltered IR-A, IR-B, IR-C, heating packs, hotwater baths and heated air/vapor flows reported by various authors. Advantages include (a) significant heat generation by absorption of radiation not only in the uppermost skin layers or at the skin surface, but also in deeper tissues layers [3]; (b) threshold skin surface temperatures for induction of heat pain [4,5] are reached at significantly higher incident irradiances as compared to short-wavelength IR (by a factor of about 2.5) and IR-C (by a factor of about 3.6) [2,6] and (c) significantly smaller temperature decrease within deeper tissue layers as compared to short-wavelength IR or hot packs [7].
New perspectives for thermal therapy to treat infectious diseases and to eliminate a number of thermosensitive pathogens in vivo were recently discussed by Gazel and Yilmaz [28]. In this context, wIRA-hyperthermia could be a useful alternative/adjuvant ('thermal-microbiology concept' [28]) to antibiotic (e.g., Rifamycine and Streptomycine) and surgical measures [29,30] as well as to the application of jackets filled with heated water [31] or of chemical (exothermal) phase change material packs [32] for local treatment of, for example, Buruli ulcers, frequent infections in tropical and subtropical countries [33].
Respective therapeutic interventions use skin surface temperatures of about 40 C in the ulcerated area for several hours per day for about 2 months. Temperatures above 37 C are needed to inactivate and eliminate the pathogen in the infected tissue [31,32,34]. According to Ruf et al. [35] the causative thermosensitive pathogen Mycobacterium ulcerans is located at depths in the tissue up to about 13 mm. In contrast to the sole thermally based treatment [28][29][30][31][32][33][34][35], Kuratli et al. [36] have described nonthermal reduction of chlamydial infectivity by wIRA in combination with visible radiation. This thermotherapeutic concept, that is, eradication of thermosensitive bacteria by hyperthermia, has been described in detail recently [37][38][39][40][41][42][43].
Whereas thermotherapy should be effective to ensure the therapeutic goal, it should also avoid unintentional thermal injury of the exposed tissue due to overheating using temperatures above about 43 C for too long exposure times [4,[44][45][46]. In most cases it is inconvenient to directly assess the tissue temperatures therapeutically required during the treatment by invasive measurements. Instead, heat pain response by the patient or temperature measurements on the skin surface are commonly used to estimate the actual heat impact within the target region. However, it is well known that tissue burns may occur during IR therapy even if the skin surface temperature remains below the heat pain threshold, an effect which depends on the individual ability for heat dissipation in the irradiated tissues.
Heat dissipation is determined by the thickness of the subcutaneous fat layer, the body core temperature and the effectiveness of both, conductive (by molecular movements) and convective (by blood flow) heat transport. Thus, only in the case of defined and known relationships between tissue temperature and skin surface temperature, assessment of the latter is sufficient. Therefore, using a porcine model, an excellent animal model for the profiling of this new therapeutic intervention [47], as a first step before starting systematic investigations in humans, two goals of this preclinical in vivo study were defined: (a) determination of the interrelation between the skin surface temperature and the tissue temperature upon wIRA-irradiation and its individual variability, and (b) assessment of the relationship between the temperature depth profile and the kinetics of tissue heating as a function of irradiance, exposure time and individual factors affecting heating (e.g., thickness of the skin and of the fat layer, body core temperature and efficacy of thermoregulation). Key parameters of the current evaluation were (a) heating-up times to reach target temperatures between 39 C and 43 C up to depths of 20 mm within the tissue, (b) maximum and mean temperatures and their deviations as a function of tissue depth and (c) persistence of tissue temperatures and thermal skin erythema after wIRA-heating. Experiments were performed using a diameter of the exposed skin area !10 cm for skin and subcutis heating which is representative for both, large-size ('loco-regional') skin IR-irradiation [48] as used in localized hyperthermia and whole body IR-irradiation before the onset of systemic heat regulation (e.g., by sweating).
Animals
Eleven piglets were incorporated in the study (body weights 15-25 kg). Before treatment, skin hairs were sheared within the target area, then the skin was cleaned, and the thicknesses of skin and fat layers were measured with an ultrasound scanner (type: HS-166V with probe No. HCS-8712M/ 7.5 MHz, Honda Electronics, Toyohashi, Japan).
Animals were divided by random sampling into three groups for three different incident irradiances (see Table 1) and were anesthetized using the following protocol: after sedation with ketamine (10 mg kg À1 i.m.) and medetomidine (50 mg kg À1 i.m.), general anesthesia was induced with propofol i.v. until anesthetic depth allowed intubation. Anesthesia was maintained with sevoflurane (end-tidal 3%). The piglets were ventilated to keep respiratory parameters constant (inspiratory O 2 concentration: 40 ± 5%, and end-tidal CO 2 partial pressure: 45 ± 5 mmHg), and were infused with a balanced infusion solution (Ringer's lactate: 5 ml kg À1 h À1 ) in order to keep mean arterial blood pressure >50 mm Hg. Before treatment the body core temperatures of the animals varied between 37.7 C and 39.0 C, and ranged between 38.3 C and 39.0 C upon treatment.
wIRA-irradiation
As shown in Table 1, three different incident irradiances were achieved by distance variation between the exit window of the irradiator and the skin surface (37, 42 and 47 cm) in order to exclude changes of the incident wIRA-spectrum. Verification measurements were performed using a double- Table 1. Individual thicknesses of skin and fat layers, and body core temperature before and immediately after wIRA-irradiation of piglets p2-p12, divided into three groups according to the different irradiances (IR-A) applied. monochromator spectroradiometer (type SPECTRO 320D, Instrument Systems, Munich, Germany). Before starting the study, the spectroradiometer was calibrated by the manufacturer, whereas offset correction was performed before each measurement. Measured spectra of irradiance are shown in Figure 1. Irradiance data were calculated by integrating the spectral irradiance data over the wavelength within the respective spectral subranges (shown in Table 2). According to these data, about 73% of the total irradiance came from the IR-A range. During the measurements, both the exit window of the irradiator and the Ulbricht sphere used as entrance window of the spectroradiometer were parallel and centered to each other.
Temperature measurements
Tissue temperatures were measured simultaneously in 1-min intervals within the treated area before, during and after wIRA-irradiation with an IR thermometer at the skin surface . The sheathing at the end of the probes was stripped according to the desired depths (tip diameter: 0.5 mm). Before measurements, the probes were calibrated in ice water. In this way, the relative error of the probes was limited to ±0.3 K. According to the manufacturer's information, resolution of the probes is 0.03 K, and the response time 10 ms. The accuracy of the infrared thermometer was verified by comparison to the readings of a precision mercury thermometer immersed into homogeneously tempered water. Temperatures of the infrared thermometer measured at the water surface differed by less than ±1.5% within the range of 30-50 C. For this test, the infrared thermometer was used with an emission coefficient of e % 0.93, and was set to e % 0.98 for skin temperature measurements.
Assessment of skin redness
Skin redness was recorded to assess changes in peripheral blood flow/blood volume before and after exposure with a spectrophotometer type SPECTROPEN (Dr. Lange, D€ usseldorf, Germany) which differentiates object colors into the components red-green (a à ), blue-yellow (b à ) and light-dark (L à ) according to CIE [49]. The spectrophotometer was calibrated before each experiment with a white reflectance standard (type: SRS-99-010 AQS-01160-060, S/N OC55C-1537, Labsphere, North Sutton, NH, USA).
General procedures
Each piglet was exposed only once choosing an incident irradiance according to Table 1. Treatments were performed on a special table in an operating room of the Animal Hospital of the University of Z€ urich, Switzerland. Body-core temperatures (in the esophagus) and vital functions of the animals were monitored continuously during wIRA-irradiation. Room air temperature was between 22 C and 24 C (no air flow, monitoring of humidity).
wIRA-irradiation was focused on a circular area (10 cm diameter) at the upper left thigh, which was nearly planar (deviations from ideal planarity 1 cm) and centric to the radiation exit window of the irradiator. According to Figure 2, there was only a deviation of 0.47% from an ideal homogeneity of irradiance within the target area. The angle between the beam and the skin surface was orthogonal ( Figure 3).
wIRA-treatments were carried out for a maximum of 60 min, or until a thermal steady state was reached. The irradiation was terminated when temperatures reached 45 C, that is, the threshold level for possible burns according to DIN 33403/2 [5]. After completion of wIRA-exposure, temperature measurements were continued until the baseline values before irradiation were reached again. When symptoms of the end of anesthesia became obvious, temperature measurements were also stopped. Table 2. Incident irradiance in the spectral range of IR-A (780-1400 nm), in the 'total' spectral range of spectroradiometric measurement (380-1700 nm) and in the ranges of VIS 1 (380-590 nm), of VIS 2 þ IR-A (590-1400 nm) and of IR-B Ã (1400-1700 nm) as part of the spectral range of IR-B (1400-3000 nm) in the center of the irradiated skin area as a function of longitudinal distances between this area and the exit window of the irradiator.
Longitudinal distance (cm) Irradiance (mW cm À2 ) in the spectral range of
Individual thermal responses to wIRA-irradiation
The complete documentation of surface and tissue temperature data sets measured as a function of time and depth before, during and after wIRA-irradiation of each piglet is presented in the Supplemental Material. Measured temperatures showed significant individual differences during wIRA-irradiation. This is exemplarily shown for four piglets in Figures 4a-d and 5a-d. Table 3 provides related relevant temperatures and a characterization of thermal responses for different layers of the heated tissue.
Two of these piglets (p2 and p3) were exposed to the same wIRA-irradiance (126 mW cm À2 ). However, in p2 there was a continuous temperature rise (heat accumulation) in all tissue layers, exceeding the threshold for burns in the skin and in the upper fat layer (Figure 4d). In contrast, p3 reacted with an effective thermoregulation which resulted in a temperature maximum of 43.6 C at the skin surface and in lower temperatures within the underlying tissue (see Figure 4a).
Piglets p6 and p10 could regulate their tissue temperatures; however, with different kinetics and extents compared to p3 (Figure 4b-c). Temperature maxima upon wIRA-irradiation were observed within the tissue for both piglets, but not at the skin surface (Figure 4b-c). Moreover, minor irradiance in p10 resulted in a more homogeneous but lower tissue heating (as compared to p2 and p3) and in an earlier final thermal steady state (as compared to p3 and p6).
More details are illustrated in Figures 4a-d and 5a-d showing individual responses depending on incident irradiance, exposure time, thicknesses of skin and fat layer, tissue depths and individual ability for heat dissipation within the tissue by conduction and convection. The body core temperature increased only marginally from 38.0 to 38.5 C for p3, p6 and p10, or remained almost constant at 39.0 C for p2 (curves 10). Because of this complexity, the interrelation between all these influencing factors needs to be analyzed in more detail as outlined in the following.
Effects of wIRA-irradiation within the different groups
From Figures 4d and 5d it is obvious that piglet p2 most probably is an outlier due to higher body core temperature and inability for effective thermoregulation. Therefore, analyses of mean thermal responses in group 1 were performed without considering the temperature data of p2 in Table 4 provides a summary of means and standard deviations of the maximum temperatures and the thermal steady-state temperatures (SST) for different tissue depths. Temperature maxima during the wIRAirradiation differed in the tissue on average by 2.1 K in group 1 (exposed to 126.5 mW cm À2 ) and by only 1.1 K in group 2 (103.2 mW cm À2 ) and group 3 (85.0 mW cm À2 ) which indicates a more homogeneous heating of the tissue in the latter groups as compared to group 1. SST was observed only in groups 2 and 3 at all depths, whereas it was not observed at the skin surface and in the deeper part of the fat layer in group 1.
Initial temperature increase and the onset of local thermoregulation in the skin
Immediately after starting wIRA-irradiation, a rapid temperature rise was observed depending on the incident irradiance and the tissue depth. All piglets reacted by initiating local thermoregulation in the skin. Thermal steady states were reached after about 20-30 min. However, intermediate thermal responses showed two main characteristics: (a) striking deviances of the standard deviations of the temperatures from their respective means as depicted in Figures 6a-d and 7a-d. This was observed even for the same irradiance applied within the groups indicating significant differences in the individual processes of heat dissipation in terms of onset time and extent of increased peripheral perfusion, and (b) occurrence of temporary temperature maxima at the skin surface and in the skin before the onset of an effective temperature down-regulation in the case of higher irradiances. This phenomenon was observed in groups 1 and 2 (exposed to IR-A of 126.5 and 103.2 mW cm À2 , respectively; see Figures 6a-c, curves 1 and 2) whereas the skin surface temperature and the temperature within the skin increased continuously up to thermal steady states in group 3 (exposed to 85 mW cm À2 , see Figures 6a-c, curve 3).
Impact of the fat layer
Due to radiation absorption mainly in the skin [2,3,6,7] and due to the insulating properties of the fat layer, rapid temperature increases, heat accumulation and temperature decrease by local thermoregulation occurred mainly above the fat layer. Moreover, the predominant conductive heat transport from the skin surface to deeper tissues and the ineffective heat dissipation in the fat layer caused (with varying delays) temperature rises with increasing depth in tissues underlaying the skin. Therefore, temperature profiles in the upper part of the fat layer (at a depth of 7 mm) were similar, but less pronounced than in the skin (see Figure 6d),
Temperature differences between skin surface and tissue
According to the data shown in Table 4, temperatures in the upper layers of the tissue exceeded the temperatures at the skin surface in all animals. This most probably is caused by (a) heat accumulation in the tissue above the fat layer, (b) decreased heat transfer into the subcutis due to the insulating fat layer and (c) heat transfer from the skin surface to the environment.
Vertical temperature profiles
The baseline tissue temperatures before wIRA-irradiation increased continuously from the skin surface to deeper tissue layers (curves 0 and black symbols in Figures 8a-d). wIRAirradiation resulted in the following characteristic effects (curves 1-3 in Figures 8a-d): (a) temperature maxima within in the skin at a depth of 4 mm were observed immediately after starting wIRA-irradiation, persisting during the whole irradiation period; (b) mean temperatures increased with higher irradiance and longer exposure time and (c) enlarging differences between the maximum temperature in the tissue and at the skin surface with increasing exposure time.
Notably, the depth profile of group 2 (medium irradiance) was similar to the profile of group 1 (high irradiance) after 5 min of exposure, whereas it resembled the profile of group 3 (low irradiance) after 30 min, obviously initiated by effective thermoregulation in the animals of group 2 (see Figures 8a-d). This phenomenon could be of interest for scheduling of therapeutic hyperthermia when mild tissue heating with low irradiances is needed.
Impact of individual deviations from mean hyperthermia temperatures
wIRA-irradiation can individually result in (unintentional) lower or higher tissue temperatures compared to the mean data in Table 4, and as documented in Figures 6-8. The temperature at the lower limit of the standard deviation: The temperature at the lower confidence limit of the mean using a significance level of 5%: (T mean : mean temperature, r: standard deviation, n: number of piglets, t: critical value of Student's t-distribution (t ¼ 4.30 for n ¼ 3, groups 1 and 3 and t ¼ 3.18 for n ¼ 4, group 2) [50].
According to these conditions, tissue temperatures T rl may be expected in about 16%, and temperatures T lcl in about 2.5% of all cases. This results in decreased depths of tissue layers which can be heated up to therapeutically required temperature levels as shown in Table 5 for irradiation times of 30 min. Irradiation times <20-30 min are too short to generate thermal steady states. (For more details see Figures S13a-d and S14a-d in the Supplemental Material.) As shown in Figures 4d, 5d, 6c, 8d and 11, unintentional hot spots in the tissue with temperatures up to about 45 C were observed in single cases after exposure times of 20-30 min. As stated earlier [44][45][46], these temperature maxima may be sufficient to cause irreversible epidermal injury and skin burning in both porcine and human skin if critical exposure times were exceeded. Therefore, hyperthermia using wIRA and treatment times !30 min requires additional measures of heating control to ensure both, the therapeutic success and the prevention of possible thermal injury and tissue burns.
Correlation between surface and tissue temperature during wIRA-irradiation
Skin surface temperatures are often used to control hyperthermia instead of (more complex) invasive temperature measurements. For safety reasons, when surface temperature measurements are used, it is mandatory to consider the formation of temperature maxima that may occur within the tissue. Therefore, a comparison between synchronous maximum temperatures at the skin surface and in the tissue is shown for each piglet in Figure 11a. According to these data, the maximum tissue temperature can exceed the skin surface temperature by about 1 K in most cases and can reach about 2 K in some piglets. The differences of the temperature maxima at the skin surface and in the tissue were up to about 1.8 K and were negligible in two cases (Figure 11b).
wIRA irradiation times to reach therapeutically relevant tissue temperatures
Depending on the indication, therapeutic tissue temperatures between 39 C and 43 C are needed. As marked by solid symbols in Figure 9a and b, temperatures of 39 C and 40 C were achieved in all piglets between the skin surface and depths of 20 mm for all irradiances applied. In all animals, temperatures between 41 C and 43 C were observed, but in certain layers only (solid symbols), or in single piglets (open symbols) (Figure 9c-e).wIRA-irradiation times to reach certain target temperatures in the tissue depended on irradiance and heat transport from the skin to deeper tissue layers. Shortest times were recorded for skin heating due to heat accumulation. The latter is caused by (a) decreased heat transport from the skin to the fat layer (as an insulating barrier), and (b) by heat loss in the upper skin layer due to heat transition from the skin surface to the environment (by thermal radiation and conductive heat exchange with the air) reducing the temperature gradient between the skin surface and deeper skin layers. Both effects prolonged the wIRA-irradiation times to reach the required tissue temperatures or even prevented therapeutically relevant temperatures in some piglets (see Figure 9a-e). Table 6 provides respective data of the exposure times to generate temperatures between 39 C and 43 C at the skin surface as well as in the skin at a depth of 4 mm, at the lower part of the fat layer at a depth of 10 mm, and in the muscle at depths of 16 and 20 mm.
Persistence of increased tissue temperatures after termination of wIRA-irradiation
Effective tissue heating can be achieved by pulsed infrared irradiation controlled by simultaneous monitoring of the skin surface temperature [27]. In combined therapeutic concepts, infrared heating of the tissue is used to increase the efficacy of subsequent therapeutic measures, as described for recurrent breast cancer by Notter et al. [23,24]. In both cases, information about the kinetics of the temperature decline in the tissue after finishing wIRA-irradiation is needed (a) for safety reasons to prevent tissue overheating that may be caused by too fast changes between irradiation and break (pulse rate), and (b) to retain a required hyperthermia status of the tissue between preheating and the following treatment (e.g., re-RT [23,24]). Thus, considering a stop of wIRAirradiation as soon as skin surface temperature reaches 43 C and continuation of irradiation after reaching 42 C again, model calculations performed by Dombrovsky et al. [51] for human skin resulted in a temperature increase in the skin of about 0.2 K (as compared to skin surface temperature under the condition of thermal steady state and before the onset of thermal regulation processes). Some examples of the temperature decreases measured at the skin surface and in the tissue after wIRA-irradiation are presented in Figure 4a-d. If a certain post-irradiation hyperthermia level (PIHL) is required, decay times and tissue depths have to be considered. Considering a minimal hyperthermia level of 39 C, decay times to reach the baseline values again were dominated by individual heat dissipation which resulted in an extreme variation of the data. However, mean decay times increased with increasing depth as expected, and were up to about 8 min at the skin surface, up to about 15 min in the skin, 5-28 min in the fat layer, and about 42 min in the muscle at a depth of 20 mm (see Figure 10).
For higher PIHL values the decay times decreased significantly and showed means of less than 10 min in the tissue for PIHL ¼ 40 C and less than 5 min for PIHL ¼ 43 C. However, in only a few piglets, tissues were heated up to temperatures >41 C at depths below the fat layer (see Figures 6-9). The mean decay times to reach reliable PIHLs between 40 C and 42 C increased slightly with increasing irradiance after wIRA-irradiation. This was caused by higher mean skin surface and tissue temperatures at the end of irradiation. (For more details see related Figures S12a-d in the Supplemental Material)
Skin reddening upon wIRA-irradiation
Skin reddening may indicate an intensification of the peripheral blood flow rate for thermoregulation, blood pooling and/or early thermal skin damage. It was observed in five piglets after wIRA-irradiation. To quantify the induced skin reddening, the colorimetric erythema index (CEI) was calculated according to COLIPA [52] using the equation: where a à (t 0 ) and a à (t e ) denote the red-green components in the CIE color space before (t ¼ t 0 ) and immediately after wIRA-irradiation (t ¼ t e ). As shown in Table 7, calculated data of the symptomatic animals were limited to values of CIE 2.9, indicating only a small degree of redness. In two cases the redness was homogeneous, in one piglet reticular-dendritically speckled. In two additional cases it changed from a reticular-dendritic to a homogeneous distribution and disappeared completely within the cooling-down period after exposure for all five piglets (see Table 7). Thus, these results can be interpreted as a hint for (a) a probably minor role of an increase in peripheral blood flow for thermoregulation in piglets compared to humans, and (b) the absence of thermal skin damages upon wIRA-treatment [53].
Conclusions
4.1. Temperature at the skin surface and in the tissue during and following wIRA-irradiation (a) The temperature rise in porcine skin and subcutis depends on irradiance, exposure time, tissue depth and thermally effective factors such as body core temperature, thicknesses of skin and fat layer and effectiveness of the local thermoregulation.
(b) Abnormal high body core temperature and a thick subcutaneous fat layer may promote overheating of the tissue including burns during exposure even with irradiances well-tolerated by normal individuals.
(c) Due to the limitation of the differences between the temperature maxima in the tissue and the corresponding skin surface temperature up to 1-2 K, skin surface temperature measurements considering these differences can be used in almost all cases for an estimation of the maximum tissue temperature during hyperthermia in order to prevent tissue overheating and acute thermal damages of the tissue as discussed in Section 3.2.6. Further recommendations to prevent thermal injury and burning are: (i) reduction of irradiance, (ii) reduction of irradiation time to a therapeutically needed minimum and (iii) the use of pulsed exposure controlled by simultaneous skin surface temperature measurement.
(d) Since temperature decay times after wIRA-treatment depend on tissue depth and decrease with increasing PIHL, they exhibit significant inter-individual changes. The time between heating and the start of the following treatment should be shorter than the shortest decay time measured to retain the heating state of the tissue needed for therapeutic efficacy of a combination therapy. Shortest decay times for PIHL ¼ 39 C were up to about 5 min, for PIHL ¼ 40-42 C up to about 1 min, and can only be determined in single cases for PIHL ¼ 43 C. Therefore, the risk of overheating tissues during pulsed wIRA-irradiation is low, but cannot be excluded and requires further experimental investigations.
4.2. Therapeutic options using wIRA-irradiation times to reach thermal steady state (a) Within mean irradiation times of 30 min, temperatures !40 C at depths up to 20 mm were reliably reached even with an incident irradiance of only 85.0 mW cm À2 (IR-A). Considering this fact, an effective thermal inactivation of the M. ulcerans and other thermosensitive pathogens as noted by Gazel and Yilmaz [28] can be assured. Therefore, wIRAhyperthermia is suitable for the treatment of Buruli ulcer infections as an effective and more convenient alternative (contact free heating) compared to conventional methods. It is also recommended to determine the efficacy of wIRAhyperthermia for thermal inactivation of other microbial pathogens by exceeding their minimum inactivation temperature in the infected tissue (e.g., Chlamydia trachomatis, M. leprae, periodontal pathogens). This could be a promising new therapeutic approach to prevent possible problems due to antibiotic resistances and to surgical inaccessibility.
(b) Target temperatures !42 C can be generated with an irradiance of 126.5 mW cm À2 up to a depth of 8 mm (see Figure S13d Data are presented for piglets p3-p5 [group 1, exposed to 126.5 mW cm À2 (IR-A)], for piglets p6-p9 [group 2, exposed to 103.2 mW cm À2 (IR-A)] and for piglets p10-p12 [group 3, exposed to 85.0 mW cm À2 (IR-A)]. Single values for piglet p2 are shown for comparison. Table 5. Depth of heated tissue after 30 min of wIRA-irradiation as a function of (i) irradiance (IR-A) for different mean temperatures (T mean ) according to Figure 8(d), of (ii) the temperatures at the lower limit of standard deviation (T rl ) according to Equation (1) and Figure S13d, and of (iii) the temperatures at the lower confidence limit of the mean using a significance level of 5% (T lcl ) according to Equation (2) and Figure S14d ( Figures S13 and S14 Figure 11. Maximum tissue temperature as a function of synchronously measured skin surface temperature (a) and maximum temperature at the skin surface during wIRA-irradiation (b). Diamonds: piglets 2-5, exposed to 126.5 mW cm À2 (IR-A); triangles: piglets 6-9, exposed to 103.2 mW cm À2 ; dots: piglets 10-12, exposed to 85.0 mW cm À2 . Piglets p2 and p5 showed maximum tissue temperatures above 45 C. Values are means and standard deviations (r) for all piglets of the group reaching the target temperature. Individual data: only one piglet reached the target temperature. Table 7. a à -data before (t ¼ t 0 ), immediately after wIRA-irradiation (t ¼ t e ) and after the cooling-down period (t c ), duration of the cooling-down period (Dt c ¼ t c À t e ). adjusted measures to compensate heat dissipation are needed. These include (i) higher incident irradiances and (ii) extended exposure times. In any case, heating has to be controlled by invasive temperature measurements or temperature estimation as described in Section 4.1.c.
4.3.
Relevance of the experimental data for wIRA-hyperthermia of humans (a) In order to assess the transferability of the results described to humans, the following facts must be taken into consideration: (i) in contrast to humans, pigs are unable to cool their skin by sweating; (ii) normal values of the body core and the blood temperature are about 1 K higher than in humans; (iii) skin and fat layers in pigs are thicker than in humans and (iv) an increase in peripheral blood flow is an essential part of the thermoregulation in healthy humans, which results in skin reddening to intensify heat dissipation. This phenomenon was observed in five piglets only immediately after the end of wIRA-treatment. Moreover, it is known that general anesthesia can cause hypothermia in patients, typically by 1-2 K [55]. However, as verified by the continuous body core temperature recordings (see Table 1, Figures 4a-d, curves 10, and in the Supplemental Figures S1a-S11a, curves 10), this phenomenon did not occur in the piglets during the experiments. Therefore, further investigations based on direct in vivo measurements in humans are needed to adapt the data described above to the requirements in the human situation. | v3-fos-license |
2019-06-12T14:56:11.278Z | 2019-05-06T00:00:00.000 | 184488166 | {
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} | pes2o/s2orc | Consumption of Minerals, Toxic Metals and Hydroxymethylfurfural: Analysis of Infant Foods and Formulae
Infant foods and formulae may contain toxic substances and elements which can be neo-formed contaminants or derived from raw materials or processing. The content of minerals, toxic elements, and hydroxymethylfurfural (HMF) in infant foods and formulae were evaluated. The effect of storage temperature on HMF formation in infant formulae and its potential as a quality parameter was also evaluated. Prune-based foods contained the highest HMF content. HMF significantly increased when the storage temperature was elevated to 30 °C for 21 days. All trace elements were present in adequate amounts, while the concentration of nickel was higher when compared to those of other studies. The study indicates that HMF can be used as a quality indicator for product shelf-life and that the concentrations of minerals and toxic elements vary greatly due to the diverse compositions of foods and formulae. Such contaminants need to be monitored as infants represent a vulnerable group compared to adults.
Introduction
Infants are more sensitive than adults to food contaminants due to a higher rate of uptake by the gastrointestinal tract, an incompletely developed blood-brain barrier, an undeveloped detoxification system, and high food consumption relative to body mass [1]. Heavy metals are contaminants which can accumulate in infant foods through the food chain, during food processing or leakage from packaging materials [2]. Their effect on living organisms depends on the nature and concentration of the element concerned. Some elements are an essential part of the human diet, while others can be xenobiotic and highly toxic [3]. Maximum levels for heavy metals in infant foods and formulae are only defined for cadmium, lead, and tin through Regulation (CE) No. 1881/2006 and subsequent updates [1]. Contaminants can also be formed during the heating or preservation of foods and can pose harm to human health. These are termed neo-formed contaminants. Hydroxymethylfurfural (HMF) is a neo-formed contaminant in food, being an intermediate in the Maillard reaction which consists of a series of reactions, starting with a reaction between the carbonyl group of a reducing sugar with a free amino group, or it can result from the direct dehydration of sugars [4]. It is practically not present in fresh food but it is found in variable amounts in processed foods, such as jams, fruit juices, and syrups, as its synthesis depends on the temperature, pH, concentration of saccharides, presence of organic acids, and presence of divalent ions [5]. The aim of the study was to assess the content of minerals, toxic metals (Cr, Cu, Hg, Ni, Zn, Mn and Fe), and HMF in infant foods and formulae. This would provide an insight into the potential effects of undesirable substances within a vulnerable group.
Sample Collection
Thirty-two infant foods from four different manufacturers were randomly selected via convenience sampling from local pharmacies and supermarkets, and categorized as apple, pear, prune, fish, poultry, and ruminant-based foods. Six infant formulae from 3 different manufacturers were randomly collected from local pharmacies and were categorized as beginner infant formulae (0-6 months) and follow-on formulae (6-12 months).
Determination of pH
The pH of samples was measured with a Thermo scientific Orion Star A215 pH meter (Life Technologies Ltd., Paisley, UK). For infant foods, the pH was measured directly using a probe for viscous samples while for the powdered infant formulae, a reconstitution in de-ionized water at a ratio of 1:10 was carried out.
Determination of HMF
HMF content was determined according to a spectrophotometric method after White [6]. The determination of HMF content was based on the determination of UV absorbance of HMF at 284 nm (SpectroStar-Nano, BMG, Labtech, Ortenberg, Germany). The difference between the absorbance of a clear sample solution and the sample solution after the addition of 0.2% NaHSO 3 was determined to avoid the interference of other compounds at this wavelength. Five grams of each of the baby foods and infant formulae were tested for HMF content at a temperature of 18 • C. Furthermore, the infant formulae were incubated and maintained at 30 • C for 21 days in a water bath. The same HMF test procedure was used to determine the effect of temperature on HMF levels. Limits of detection (LOD) and limits of quantification (LOQ) for HMF were calculated as 3 s/m and 10 s/m, respectively, where s refers to the standard deviation of the intensity of blank samples and m refers to the slope of the calibration curve for HMF (Table 1).
Determination of Trace Elements
For mineral and toxic metal analysis, the samples were mineralized by digesting 1 g of the sample using 5 mL of 5% HNO 3 at 80 • C, followed by 2 mL of 34.5-36.5% H 2 O 2 after the acid evaporated. Further mineralization of the sample was carried out by ashing at 500 • C in a muffle furnace (Wisetherm, Wisd, Laboratory Instruments, Germany) for 6 h. The ash was reconstituted in 5 mL of 5% HNO 3 and filtered. Deionized water was added up to 50 mL and the samples were quantitatively analyzed using a Microwave Plasma-Atomic Emission Spectrometer (MP-AES 4100, Agilent Technologies Inc., Santa Clara, CA, USA). The method was validated according to Berg [7]. The LOD and LOQ for each heavy metal were calculated as 3 s/m and 10 s/m, respectively, with respect to the calibration curve for each element (Table 1).
Statistical Analysis
All measurements were conducted in triplicate and average results were reported. The statistical program Prism 5 (GraphPad Software Inc., San Diego, CA, USA) was used for data analysis. The results for the heavy metal elements and hydroxymethylfurfural contents were analyzed by one-way ANOVA with the Bonferroni post hoc test to compare the statistical difference between means of the data sets and their mean difference. The same statistical test was carried out to compare the mean content of hydroxymethylfurfural between infant formulae stored at room temperature and infant formulae stored at 30 • C for 21 days. Principal component analysis and Pearson correlations were conducted on all samples, using XLSTAT v.2014.4.04 (Microsoft, version 19.4.46756, SAS Institute Inc., Marlow, Buckinghamshire, UK) to determine any clustering of minerals and toxic metals. A P value less than 0.05 was considered as statistically significant.
Results
A total of 38 samples were assessed for HMF content and selected heavy metal elements. The infant foods (n = 32) exhibited variable amounts of HMF, ranging from 0.89 mg/kg to 144 mg/kg, with the lowest content being present in poultry-based infant foods, while the highest content was present in prune-based products ( Table 2). The HMF content in infant formulae (n = 6) ranged from 0.29 mg/kg to 7.87 mg/kg when examined at room temperature. The HMF content in all types of infant formulae significantly increased (p ≤ 0.05) after being stored at 30 • C for 21 days and ranged from 1.80 mg/kg to 9.43 mg/kg ( Figure 1). The mean heavy metal content of Cr, Cu, Hg, Ni, Fe, Mn, and Zn is shown in Table 3. The trace elements were detected in all infant food and formulae samples analyzed except for Hg, which was detected only in one sample from the pear-based infant food category (n = 6).
Toxics 2019, 7, x FOR PEER REVIEW 3 of 8 in 5 ml of 5% HNO3 and filtered. Deionized water was added up to 50 ml and the samples were quantitatively analyzed using a Microwave Plasma-Atomic Emission Spectrometer (MP-AES 4100, Agilent Technologies Inc., Santa Clara, CA, USA). The method was validated according to Berg [7]. The LOD and LOQ for each heavy metal were calculated as 3 s/m and 10 s/m, respectively, with respect to the calibration curve for each element (Table 1).
Statistical Analysis
All measurements were conducted in triplicate and average results were reported. The statistical program Prism 5 (GraphPad Software Inc., San Diego, CA, USA) was used for data analysis. The results for the heavy metal elements and hydroxymethylfurfural contents were analyzed by one-way ANOVA with the Bonferroni post hoc test to compare the statistical difference between means of the data sets and their mean difference. The same statistical test was carried out to compare the mean content of hydroxymethylfurfural between infant formulae stored at room temperature and infant formulae stored at 30 °C for 21 days. Principal component analysis and Pearson correlations were conducted on all samples, using XLSTAT v.2014.4.04 (Microsoft, version 19.4.46756, SAS Institute Inc., Marlow, Buckinghamshire, UK) to determine any clustering of minerals and toxic metals. A P value less than 0.05 was considered as statistically significant.
Results
A total of 38 samples were assessed for HMF content and selected heavy metal elements. The infant foods (n = 32) exhibited variable amounts of HMF, ranging from 0.89 mg/kg to 144 mg/kg, with the lowest content being present in poultry-based infant foods, while the highest content was present in prune-based products ( Table 2). The HMF content in infant formulae (n = 6) ranged from 0.29 mg/kg to 7.87 mg/kg when examined at room temperature. The HMF content in all types of infant formulae significantly increased (p ≤ 0.05) after being stored at 30 °C for 21 days and ranged from 1.80 mg/kg to 9.43 mg/kg ( Figure 1). The mean heavy metal content of Cr, Cu, Hg, Ni, Fe, Mn, and Zn is shown in Table 3. The trace elements were detected in all infant food and formulae samples analyzed except for Hg, which was detected only in one sample from the pear-based infant food category (n = 6).
Discussion
Toxic substances may be either present in the raw materials or evolve during the processing of the raw materials into the finished products. Although the assurance of food quality is the responsibility of the producer and manufacturer, authorities worldwide do not control food products for safety. Several reports have shown that baby foods may contain contaminants, some of which include microorganisms [8,9], mycotoxins [10,11], aromatic compounds [12,13], furans [14,15], and metals [16][17][18].
The HMF content was determined at a temperature of 18 • C for the baby foods, and at two temperatures (18 and 30 • C) for the infant formulae. Since baby foods in individual jars are consumed within one meal and the foods have undergone extensive processing in industry, the baby foods were not tested at a temperature of 30 • C for a 21-day period. It is more likely that for infant formulae, repeated quantities are consumed from the same can over a period of time. There is no limit for the HMF content in foods, apart for honey at 40 mg/kg in general environmental conditions, 80 mg/kg for honey produced in tropical climates, and 15 mg/kg for honey with low enzymatic activity [19]. This makes it difficult to ascertain whether acceptable or excessive levels of HMF are found in the studied foods. The results from studies carried out by Kalábová and Večerek [20], andČížková and coworkers [21], for the determination of the HMF content in infant foods, reported ranges from 2.10 mg/kg to 9.80 mg/kg and 4.10 mg to 28.90 mg/kg, respectively. The current study showed a larger spread of values nearly fifteen times the upper limit, observed by Kalábová and Večerek [20], and seven times the upper limit, observed byČížková and coworkers [21]. This variability could be related to the type of food tested, since this varied in the different studies. A significant difference in the HMF content of prune-based infant foods compared to other infant foods (p ≤ 0.05) was observed and these were identified as a potential source of high HMF consumption in children. Products processed from prunes, such as pitted prunes and prune juices, have been reported to have an HMF content as high as 291 mg/kg and 528 mg/L, respectively. The higher HMF value in fruit-based foods is due to greater carbohydrate degradation as a consequence of the Maillard reaction, which is favored by a lower pH (Table 2). On the other hand, a higher furan content is present in vegetable-based foods compared to fruit-based foods. This is related to either a greater furfural content or a greater ascorbic acid degradation [14].
The HMF content in infant formulae observed in the study, ranging from 0.29 mg/kg to 7.87 mg/kg, was comparable with other studies, such as that by Michalak and coworkers [22], reporting an HMF content between 1.22 mg/kg and 8.20 mg/kg. With respect to the changes of HMF content during storage at 30 • C for 21 days, the HMF content in all formulae increased significantly after storage (p ≤ 0.05). This temperature-dependent effect was shown in various studies, such as that by Chávez-Servín and coworkers [23], where they demonstrated a similar significance and proportional increase after 70 days of storage at 25 • C. However, the relationship between HMF concentration and pH in infant formulae was not significant (p > 0.05). Therefore, HMF synthesis was not dependent on the pH of infant formulae. In a study conducted earlier by Chávez-Servín and coworkers [24], it was observed that infant formulae at a neutral pH for a period of 12 months of storage exhibited insignificant formation of HMF.
There was a variation in the absorbance value with respect to the concentration of the heavy metal element, and, therefore, a strong positive linear relationship was present between the two parameters (r = 0.9986). The low LOD and LOQ values demonstrate that the MP-AES method for the analysis of heavy metal elements was highly sensitive ( Table 1). The heavy metal content varied widely due to many factors, such as differences between food types, the characteristics of the manufacturing practices and processes, and possible contamination during processing. The present study demonstrated wide variations in the concentration of the most essential and toxic elements in infant formulae and foods (Table 3). In the infant formulae, the manufacturer's fortification of essential elements resulted in concentrations many times higher than those found in foods, especially Fe, Zn, and Cu. The concentration of nickel in the samples, ranging from 0.63 mg/kg to 1.07 mg/kg, exceeded the reference value of 5 µg/kg bw/day set by the Food and Agriculture Organization/World Health Organization [25], as the daily intake of Ni through infant formulae ranged from 7 µg/kg bw day to 19 µg/kg bw day. Mehrnia and Bashti [26] reported daily intake values of nickel through infant formula more than tenfold the reference value set by the JECFA. Nickel toxicity is associated with immediate and delayed hypersensitivity reactions. It has the potential to cause immunological disturbances and act as an immunotoxic agent in humans [27]. Only one sample was contaminated with Hg at a concentration of 0.7 mg/kg. Since Hg was detected in a pear-based food, the presence of methylmercury is excluded, as this bioaccumulates in fish. Therefore, this value cannot be compared to the EFSA [28], which establishes a TWI reference value of 1.3 µg/kg bw for methylmercury. Cruz and coworkers [29] reported infant formulae testing positive for mercury, with levels of 0.63 mg/kg and 0.83 mg/kg.
Factor analysis using principal components was used to identify latent traits within the data. Pearson correlation (Table 4) Figure 2b demonstrates the factor scores of the two latent factors. Factor 1, on the horizontal axis, demonstrates the clustering of baby foods on the left hand side of the scatter plot, while the infant formulae scattered more on the right hand side. This demonstrates the distinction of the foods and formulae characteristics with respect to mineral and toxic metal values. [25], as the daily intake of Ni through infant formulae ranged from 7 µg/kg bw day to 19 µg/kg bw day. Mehrnia and Bashti [26] reported daily intake values of nickel through infant formula more than tenfold the reference value set by the JECFA. Nickel toxicity is associated with immediate and delayed hypersensitivity reactions. It has the potential to cause immunological disturbances and act as an immunotoxic agent in humans [27]. Only one sample was contaminated with Hg at a concentration of 0.7 mg/kg. Since Hg was detected in a pear-based food, the presence of methylmercury is excluded, as this bioaccumulates in fish. Therefore, this value cannot be compared to the EFSA [28], which establishes a TWI reference value of 1.3 µg/kg bw for methylmercury. Cruz and coworkers [29] reported infant formulae testing positive for mercury, with levels of 0.63 mg/kg and 0.83 mg/kg.
Factor analysis using principal components was used to identify latent traits within the data. Pearson correlation (Table 4) revealed that there were several correlations between the minerals and toxic metals. There were positive correlations between Cr and Cu, Fe, Zn (r = 0.718, 0.725 and 0.631), Cu and Fe, Zn (r = 0.996 and 0.984), and Fe with Zn (r = 0.974). There were negative correlations between Cu and Mn (r = −0.636), Mn with Fe, and Zn (r = −0.654 and −0.641). Two latent factors had an eigenvalue greater than 1, which together explained 80.04% of the total variance ( Figure 2a). The factor loadings demonstrated the different groups of variables. For the first factor, the factor loadings of Cr, Cu, Fe, and Zn, and the second factor, weighed heavily on Hg, Ni, and Mn. Figure 2b demonstrates the factor scores of the two latent factors. Factor 1, on the horizontal axis, demonstrates the clustering of baby foods on the left hand side of the scatter plot, while the infant formulae scattered more on the right hand side. This demonstrates the distinction of the foods and formulae characteristics with respect to mineral and toxic metal values.
Conclusions
Opinions on the cytotoxicity, carcinogenic, and genotoxic potential of hydroxymethylfurfural vary, while certain minerals and toxic metals are known to be deleterious if consumed in large quantities. However, the concentrations of such metals vary depending on the food type used. Infant foods and formulae contained varying amounts of HMF and metals, thus, the total daily intake of these contaminants is affected by individual feeding patterns. Notably, a high HMF content was observed in prune-based infant foods. On the other hand, with regard to the metal contents, it was observed that infant foods contained Mn, Zn, Fe, Cu, and Cr, while infant formulae contained Zn, Fe, Cu, Mn, and Cr in decreasing order. There was a low presence of Ni and negligible quantities of Hg. Infants are within a vulnerable age group and have a restricted diet compared to other age groups, therefore, it is recommended that foods are monitored to ensure safe use. The setting up of limits with respect to this vulnerable group should be considered through further studies, using a greater diversification of samples that are subjected under varying conditions. Author Contributions: Conceptualization, writing-review, editing and supervision: E.A.; methodology and investigation: C.V.
Funding: This research received no external funding. | v3-fos-license |
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} | pes2o/s2orc | Supercritical Fluid Extraction of Eucalyptus globulus Bark—A Promising Approach for Triterpenoid Production
Eucalyptus bark contains significant amounts of triterpenoids with demonstrated bioactivity, namely triterpenic acids and their acetyl derivatives (ursolic, betulinic, oleanolic, betulonic, 3-acetylursolic, and 3-acetyloleanolic acids). In this work, the supercritical fluid extraction (SFE) of Eucalyptus globulus deciduous bark was carried out with pure and modified carbon dioxide to recover this fraction, and the results were compared with those obtained by Soxhlet extraction with dichloromethane. The effects of pressure (100–200 bar), co-solvent (ethanol) content (0, 5 and 8% wt), and multistep operation were studied in order to evaluate the applicability of SFE for their selective and efficient production. The individual extraction curves of the main families of compounds were measured, and the extracts analyzed by GC-MS. Results pointed out the influence of pressure and the important role played by the co-solvent. Ethanol can be used with advantage, since its effect is more important than increasing pressure by several tens of bar. At 160 bar and 40 °C, the introduction of 8% (wt) of ethanol greatly improves the yield of triterpenoids more than threefold.
Introduction
Presently, technologies based on the use of fossil fuels for energy and chemical production are still predominant. Nonetheless, because of dwindling feedstocks, growing concerns about global climate change and pollution, and stricter emission laws, new approaches are being sought to produce novel and better quality products to meet the new sustainability targets of present politic and industrial thinking [1][2][3][4]. At this point, forest and mill residues, agriculture crops and wastes, wood and wood wastes, animal wastes, livestock operation residues, aquatic plants, fast-growing trees and plants, and municipal and industrial wastes can play an important role. These renewable resources can be transformed into a variety of products, including chemicals, energy, transportation fuels, and materials, giving rise of the concept of biorefinery as an integral unit which accepts, converts and processes such feedstocks [5][6][7][8].
For the sustainability of biorefineries, low environmental impact technologies must be used throughout, which may be accomplished by integrating that target into a performance criterion of the design. It is vital to avoid the unacceptable aspects of petrochemical production and avoid hazardous and environmentally harmful process auxiliaries, such as toxic reagents and volatile organic solvents. In this scenario, green chemical technologies, such as supercritical fluid extraction (SFE) [5,9], receive particular attention, and novel solvents become highly desirable, such as supercritical carbon dioxide (SC-CO 2 ) and ionic liquids [1,4,10,11].
The interests of agro-forest industries, particularly pulp and paper mills, in the integrated exploitation of plants biomass is growing enormously, since they produce considerable amounts of by-products, such as bark and general logging wastes (e.g., leaves, branches, fruits, sawdust), which are either left in the forest for soil nutrition or ultimately burned in the biomass boiler for energy production. Some of these residues contain high value components whose extraction does not affect the current pulp and power outputs of existing mills. For instance, the exploitation of valuable extractives, such as phytosterols, namely β-sitosterol [12][13][14], lignans [15][16][17], and betulin [18,19] from by-products of the industrial processing (e.g., bark, wood knots, pulping liquors) is a strategy implemented in some pulp industries.
In this paper, the supercritical fluid extraction of triterpenoids from eucalyptus deciduous bark is presented. The effects of pressure, co-solvent (ethanol) content, and multistep operation are studied in order to enlighten subsequent investigations on the applicability of SFE for their selective and efficient production. The extracts obtained from one step and multistep operation with SC-CO 2 and SC-CO 2 modified with ethanol are analyzed. The individual cumulative extraction curves of the main families of compounds are also discussed in detail.
Soxhlet Extraction of Eucalyptus globulus Deciduous Bark
The yield of the lipophilic extractives of deciduous bark extracted with dichloromethane was 2.1% (wt), which is in good agreement with previous results for this biomass fraction of E. globulus [21]. A GC-MS chromatogram of this extract (as TMS derivatives) is shown in Figure 2. In Table 1 the corresponding retention times, composition and concentrations are listed. It may be observed that the extract is mainly composed of several triterpenic acids with lupane, oleanane and ursane skeletons (Figure 1), mostly ursolic acid and its acetyl derivative, 3-acetylursolic acid, accounting for 2.77 g/kg and 2.64 g/kg, respectively, in a total of 10.74 g/kg quantified compounds. Betulonic (0.80 g/kg), oleanolic (0.71 g/kg), 3-acetyloleanolic (0.69 g/kg) and betulinic (0.62 g/kg) acids are also abundant in this extract, being the main components of the triterpenoid family of compounds, which also includes minor amounts of β-amyrin. Several fatty acids (C 14 to C 28 , accounting globally for 0.48 g/kg), mainly hexadecanoic, tetracosanoic and hexacosanoic acids, some long chain aliphatic alcohols (C 16 to C 28 , accounting globally for 0.44 g/kg), as hexacosan-1-ol, and β-sitosterol (0.24 g/kg) are also present in considerable amounts in the extract.
One-Step Supercritical Fluid Extraction of Eucalyptus globulus Deciduous Bark
In this section, the results obtained for the five supercritical extractions carried out in one step are presented and discussed. On the whole, the extraction yields ranged from 0.48% to 1.76% (wt), a slight increase from 100 bar to 160 bar being observed (Figure 3), but a considerable jump of 103% was obtained when passing from 160 bar to 220 bar. This behavior is due to the direct effect of pressure upon the density of CO 2 , which determines solubility at constant temperature. The densities of CO 2 , calculated with the Pitzer and Schreiber equation of state [31], for pressures of 100, 160, and 220 bar, are 633.1, 796.8 and 858.7 kg/m 3 , respectively, which shows the significant impact of density in the region around 800 kg/m 3 , a value typical of current organic solvents. An analogous trend was found for the extractions carried out at constant temperature and pressure (40 °C, 160 bar) when the co-solvent (ethanol) introduced was increased from 0% to 5%, and then to 8% (wt) (Figure 3). In this sequence, the densities of the CO 2 and ethanol mixtures are very similar: 796.8, 794.2 and 795.0 [32]-but the extraction yields show two large increments (154% and 240%). Concerning the entrainer effect, which is defined as an increase in both solubility and selectivity [33], the polarity modification imparted by ethanol to the non-polar CO 2 , with final positive effect on solvent power, reveals the intermolecular interactions between ethanol and the extract components. It is worth noting that the yield achieved at 160 bar/40 °C/8% (wt) ethanol is close to that obtained by Soxhlet extraction with dichloromethane (1.8% versus 2.1%).
Regarding the composition of those extracts, they exhibit considerable differences, both quantitatively and qualitatively. In Figure 4, the abundances of the most important chemical families detected are plotted together with the Soxhlet extraction results for comparison. The numerical values are listed in Table 2, where the individual concentrations of triterpenoids are specified, given their interest in this work. Table 2. Main components and families (mg/kg of dry bark) of Eucalyptus globulus deciduous bark extracts obtained with SC-CO 2 and SC-CO 2 /ethanol. Comparison with Soxhlet results using dichloromethane (see Table 1). In the assays without co-solvent addition, the extracts show similar compositions. The main components are triterpenoids, particularly 3-acetylursolic, 3-acetyloleanolic and betulonic acids, also as β-amyrin and β-sitosterol, followed by fatty acids, (from which palmitic and oleic are the most representative), and minor fractions of long chain aliphatic alcohols. β-sitosterol was included in the triterpenoids group. Besides, considering the recognized biological activities of this molecule [34,35], its eventual exploitation can be of relevant interest for the upgrade of E. globulus biomass residues. In comparison to the dichloromethane extract, it is noteworthy that the three assays without co-solvent have low content levels (or even an absence of content) of ursolic and oleanolic acids ( Table 2), two of the main components of E. globulus deciduous bark (see Table 1). This is due to the polarity gap between them and CO 2 , which implies that higher pressure or, alternatively, a modifier is necessary to improve their solubility. This is clear from the successful removal of their acetylated forms (i.e., 3-acetylursolic, 3-acetyloleanolic), since after esterification the polarity imparted by the hydroxyl group is much attenuated. Table 2). Comparison with Soxhlet extraction with dichloromethane. (FA, fatty acids; LCAA, long chain aliphatic alcohols; TT, triterpenoids; EtOH, ethanol).
In experiments with co-solvent addition, a significant increase (151%) of the amount of triterpenoids extracted with 5% (wt) ethanol (5.17 g/kg at 160 bar) was observed in comparison to the extract obtained with pure carbon dioxide at the same pressure and temperature (2.06 g/kg)-see Table 2. The recovery of triterpenoids was once again enhanced (in this case, 26%) after raising the ethanol content to 8% (wt), attaining 6.53 g/kg of bark. These results emphasize the chief role played by solvent polarity, since such increments can be attributed mainly to the extraction of non-acetylated triterpenic acids. In fact, at 160 bar and 40 °C, their evolution along with ethanol percentage in the supercritical solvent may be taken from Table 2: ursolic acid, 0.073, 1.74, 1.78 g/kg, and oleanolic acid, 0.050, 0.66, 0.69 g/kg. In conjunction, these variations account for 52.5% of the global increment of triterpenoids. In contrast, the two acetylated acids (acetylursolic and acetyloleanolic), justify only 18.6% of triterpenoid extraction enhancement.
Compared with the dichloromethane extract, the quantities of triterpenoids obtained by SC-CO 2 /ethanol extraction reached about 70.7% of its total potential. Nonetheless, such yields may/should be optimized by adjusting operating conditions, such as extraction time, pressure, temperature, and co-solvent percentage in the SC-CO 2 stream.
On the whole, the increased amount of triterpenoids extracted is, essentially, the main difference between the extracts obtained with and without co-solvent, the remaining composition being similar.
Stepwise SC-CO 2 Extraction of Eucalyptus globulus Deciduous Bark
The individual cumulative curves for each family of compounds obtained in the stepwise extraction assay are plotted in Figures 5 and 6 as functions of the mass of the CO 2 spent per unit mass of bark. In Figure 5, they are plotted as absolute weight (mg) of solute in the extract, while in Figure 6 they are normalized by their maximum extractable values (taken to be equal to that of the Soxhlet dichloromethane extract). In the first step (120 bar, 40 °C), one extracts mostly long chain aliphatic alcohols and other compounds (41% and 66% of its total potential in the bark, respectively) and low quantities of triterpenoids and fatty acids. Of the triterpenoids, 3-acetylursolic acid is the predominant component due to its higher lipophilic character, at least in comparison with parent ursolic acid. Moreover, it should be noted that it represents ca. 30% of the triterpenoids extracted by Soxhlet with dichloromethane (see Table 1).
In the second step (180 bar, 40 °C), the improved solvent power of SC-CO 2 explains the extraction of higher amounts of triterpenoids, which increased from ca. 55 to 72 mg (see Figure 5). It is worth noting that the extraction rate of triterpenoids and fatty acids increases instantaneously at the beginning of this step (see Figure 6 also), and then diminishes continuously with time, as in the first step. The remaining families of components do not exhibit this trend. That jump is essentially justified by the effect of pressure upon solubility, as already observed and discussed in section 2.2, whereas the deceleration is due to the fact that the driving force to mass transfer attenuates along the extraction. At higher pressures the proximity between CO 2 and solute molecules is shortened, making possible interactions with CO 2 quadrupole that are almost absent at low densities. Increasing density leads to the creation of substantial dipole (induced or not)-quadrupole interactions that favor the solubility of non-acetylated triterpenic acids, from which the ursolic acid must be detached because it is the most abundant (ca. 68%; see Table 1). As has been mentioned above, this enhancement can be largely incremented using ethanol as co-solvent, which has been accomplished in the following step. Figure 6 illustrates that the introduction of ethanol (5%, wt) in step 3 contributed to the removal of higher amounts of triterpenoids and fatty acids, which doubled from 72 to 142 mg and from 10.8 to 22.2 mg, respectively, the variation of other families being much smaller. Nevertheless, this figure points out that we are still far from exhausting the total amount of triterpenoids present in eucalyptus deciduous bark; therefore an optimization work on this topic is still needed to improve the extraction yield. This will be the main target of future researches, in order to achieve selective and quantitative triterpenoid extraction using supercritical CO 2 (modified or not) at economically tractable pressures.
Considering the results of this essay, one may expect that a multistep process based on different operating conditions may generate extracts enriched in triterpenoids by firstly removing part of the aliphatic matrix (fatty acids, long chain aliphatic alcohols and other compounds) with supercritical CO 2 , since numerous substances are CO 2 -philic.
Bark Samples
Deciduous bark of E. globulus was randomly harvested from a 20-year-old clone plantation cultivated in the Eixo (40°37′13.56″N, 8°34′08.43″W) region of Aveiro, Portugal, air dried until a constant weight was achieved, and milled to granulometry lower than 2 mm prior to extraction. Deciduous bark was selected as substrate, since it is mostly outer bark (very similar to the last one in terms of triterpenic acids composition) and avoids felling a large number of trees to ensure a continuous supply of raw material of controlled origin for a long-term study.
Soxhlet Extraction
Samples of E. globulus deciduous bark (15 g) were Soxhlet-extracted with dichloromethane for 7 h. The solvent was evaporated to dryness, the extracts were weighed and the results were expressed as a percent of dry bark. Dichloromethane was chosen because it is a fairly specific solvent for lipophilic extractives and was used as a reference to evaluate the efficiency of the SFE extractions.
SFE Apparatus
The SFE apparatus used to carry out the extraction assays is schematically shown in Figure 7. In this diagram the CO 2 taken from a cylinder is compressed to the desired extraction pressure by means of a Nova Swiss gas compressor (model 5542121), and then heated to the desired temperature by passing through a high pressure tubing coil immersed in a temperature-controlled (up to ±0.5 °C) water bath. SC-CO 2 flows at the desired pressure and temperature conditions upwards through a packed bed of E. globulus deciduous bark contained in the extraction vessel (316SS; internal diameter of 24 mm; total length of 572 mm). The extractor is heated by passing hot water through a heating jacket surrounding the outer surface of the vessel. The extraction pressure was controlled by means of a back pressure regulator, BPR (Tescom 27-1700), where depressurization of the extract flow stream took place. The extracts were solubilized in n-hexane and collected in a glass trap, T1. To ensure total recovery of compounds, the gas flow passes through a second glass trap, T2. Both traps are kept under 0 °C immersed in an ethylene glycol bath. The gas flow rate and total mass of carbon dioxide used in the assays are measured with a coriolis-type gas flow meter (Danfoss, Mass 6000). The extraction pressure is measured at the exit of the extraction vessel with an accuracy of ±0.1 MPa (Wika, model 881.14.600). Spent CO 2 is vented to the atmosphere. The addition of co-solvent to the system was made by a liquid pump (LDC Analytical miniPump) coupled to the gas line between the high pressure tubing coil immersed in the water bath and the extraction vessel. SC-CO 2 and the liquid co-solvent are mixed in a static mixer before entering in the extraction vessel. The co-solvent container is placed on a balance being the flow rate measured by weight difference and controlled by the liquid pump.
SFE Procedure
In each run, about 30 g of milled bark were introduced in the extraction vessel. A first set of extractions were carried out at 100, 160 and 220 bar without the addition of co-solvent, and a second one at 160 bar with 5% and 8% (wt) ethanol during 3 h. The average SC-CO 2 flow rate was 12.5 g/min, and the temperature was kept at 40 °C. The extracts were collected in both traps at the end of each run and combined.
A second sequence of extractions in series was performed at 40 °C using about 70 g of milled bark and 6 g/min of solvent. The following steps of 5 h were carried out: (1) extraction at 120 bar with pure CO 2 ; (2) extraction at 180 bar with pure CO 2 ; and (3) extraction at 180 bar with CO 2 modified with 5% ethanol. The extracts were collected within 1 h interval in the first two steps, while in step 3 they were collected after 2 and 5 h of extraction time. All extracts were collected from both traps, combined and analyzed individually. The solvent was evaporated to dryness. The extracts were weighed and the results expressed as a percentage of dry bark.
GC-MS Analyses
Before each GC-MS analysis, nearly 20 mg of dried sample were converted into trimethylsilyl (TMS) derivatives according to the literature [23]. GC-MS analyses were performed using a Trace Gas Chromatograph 2000 Series equipped with a Thermo Scientific DSQ II mass spectrometer, using helium as carrier gas (35 cm·s −1 ), equipped with a DB-1 J&W capillary column (30 m × 0.32 mm i.d., 0.25 µm film thickness). The chromatographic conditions were as follows: initial temperature: 80 °C for 5 min; temperature rate of 4 °C·min −1 up to 260 °C and 2 °C min −1 until the final temperature of 285 °C; maintained at 285 °C for 10 min; injector temperature: 250 °C; transfer-line temperature: 290 °C; split ratio: 1:50. The MS was operated in the electron impact mode with electron impact energy of 70 eV and data collected at a rate of 1 scan·s −1 over a range of m/z 33-700. The ion source was maintained at 250 °C.
For quantitative analysis, the GC-MS instrument was calibrated with pure reference compounds, representative of the major lipophilic extractives components (namely, palmitic acid, nonacosan-1-ol, β-sitosterol, betulinic acid, ursolic acid and oleanolic acid), relative to tetracosane, the internal standard used. The respective multiplication factors needed to obtain correct quantification of the peak areas were calculated as an average of six GC-MS runs. Compounds were identified, as TMS derivatives, by comparing their mass spectra with the GC-MS spectral library, with data from the literature [23,[36][37][38][39][40][41] and, in some cases, by injection of standards. Two aliquots of each extract were analyzed. Each aliquot was injected in triplicate. The results presented are the average of the concordant values obtained for each part (less than 5% variation between injections of the same aliquot and between aliquots of the same sample).
Conclusions
In this work, eucalyptus deciduous bark was investigated as a source of triterpenoids due to their interest as new bioactive agents. The main components are ursolic acid and its acetyl derivative, 3-acetylursolic acid, which accounted for 2.77 and 2.64 g/kg, respectively, in a total of 10.74 g/kg of quantified compounds. The supercritical fluid extraction with pure and modified carbon dioxide was evaluated by carrying out experiments at 40 °C at pressures from 100 to 220 bar. Pressure has a large influence upon the extraction yield and on the concentrations of the extracts. Furthermore, the introduction of 8% (wt) of ethanol at 160 bar and 40 °C more than trebles the yield of triterpenoids, which highlights the important role played by co-solvent in this extraction. Hence, ethanol may be used with advantage, since its effect is more important than increasing pressure by several tens of bar. Taking into account the additional multistep extraction performed in series in this work, the results showed that an appropriate combination of operating conditions may generate extracts enriched in triterpenoids. | v3-fos-license |
2018-06-13T09:30:58.183Z | 2018-06-13T00:00:00.000 | 48353628 | {
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} | pes2o/s2orc | Hypoxia with Wharton’s jelly mesenchymal stem cell coculture maintains stemness of umbilical cord blood-derived CD34+ cells
Background The physiological approach suggests that an environment associating mesenchymal stromal cells with low O2 concentration would be most favorable for the maintenance of hematopoietic stem/progenitor cells (HSPCs). To test this hypothesis, we performed a coculture of cord blood CD34+ cells with Wharton’s jelly mesenchymal stem cells (WJ-MSCs) under different O2 concentration to simulate the growth of HSPCs in vivo, and assessed the impacts on stemness maintenance and proliferation of cord blood HSPCs in vitro. Methods CD34+ cells derived from cord blood were isolated and cocultured under 1%, 3%, or 20% O2 concentrations with irradiated WJ-MSCs without adding exogenous cytokines for 7 days. The cultured cells were harvested and analyzed for phenotype and functionality, including total nuclear cells (TNC), CD34+Lin− cells, colony forming unit (CFU) for committed progenitors, and long-term culture initiating cells (LTC-ICs) for HSPCs. The cytokine levels in the medium were detected with Luminex liquid chips, and the mRNA expression of hypoxia inducible factor (HIF) genes and stem cell signal pathway (Notch, Hedgehog, and Wnt/β-catenin) downstream genes in cord blood HSPCs were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Results Our results showed that the number of TNC cells, CD34+Lin− cells, and CFU were higher or similar with 20% O2 (normoxia) in coculture and compared with 1% O2 (hypoxia). Interestingly, a 1% O2 concentration ensured better percentages of CD34+Lin− cells and LTC-IC cells. The hypoxia tension (1% O2) significantly increased vascular endothelial growth factor (VEGF) secretion and decreased interleukin (IL)-6, IL-7, stem cell factor (SCF), and thrombopoietin (TPO) secretion of WJ-MSCs, and selectively activated the Notch, Wnt/β-catenin, and Hedgehog signaling pathway of cord blood HSPCs by HIF-related factors, which may play an important role in stemness preservation and for sustaining HSPC quiescence. Conclusions Our data demonstrate that cord blood HSPCs maintain stemness better under hypoxia than normoxia with WJ-MSC coculture, partially due to the increased secretion of VEGF, decreased secretion of IL-6 by WJ-MSCs, and selective activation of stem cell signal pathways in HSPCs. This suggests that the oxygenation may not only be a physiological regulatory factor but also a cell engineering tool in HSPC research, and this may have important translational and clinical implications.
Background
Umbilical cord blood (UCB) is an alternative source of hematopoietic stem/progenitor cells (HSPCs) for transplantation in malignant and nonmalignant hematologic diseases. However, since the amount of HSPCs in a single unit of cord blood is insufficient for transplantation in most adult patients, the application of cord blood HSPCs remains with a major limitation [1]. The ex-vivo expansion of cord blood HSPCs is one feasible method to increase the HSPC number and has recently become a focus for research. HSPCs maintain their stemness by interacting with stromal cells and the extracellular matrix through cell-to-cell contact and paracrine factor secretion [2]. The microenvironment of the placenta or umbilical cord, where the UCB-HSPCs reside in, is different to that in bone marrow, with the stromal cells including Wharton's jelly mesenchymal stem cells (WJ-MSCs) and vascular endothelial cells being dissimilar to bone marrow stromal cells (e.g., no osteoblasts). Compared with MSCs from the bone marrow, WJ-MSCs not only express cell markers of BM-MSCs, but also additionally express many molecules involved in HSPC expansion and interaction, such as granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and CD117 [3]. These advantages make WJ-MSCs a preferable feeder layer choice for UCB-HSPC expansion in vitro. Furthermore, in physiological situations, HSPCs are distributed in the tissues with low oxygen tension, and it is now widely accepted that gradients of oxygen from below 1% in hypoxic niches to 6% in the sinusoidal cavity exist within the human bone marrow [4,5]. Like all other established cell lines, stem cells were typically cultured under ambient oxygen tension with very little attention paid to the metabolic milieu of the niche in which they were grown or normally resided. In this study, we compared the effects of combining WJ-MSCs at different O 2 concentrations in coculture on stemness maintenance and proliferation of HSPCs in vitro without adding exogenous cytokines. Our results show that stemness of HPSCs can be better maintained at 1% O 2 with WJ-MSC coculture. Under hypoxia, the levels of secretion of different cytokines via WJ-MSCs and selective activation of stem cell signal pathways may impact a few mechanisms.
Isolation and characterization of WJ-MSCs from umbilical cord
Umbilical cord samples were collected from healthy full-term deliveries after obtaining informed consent as a donation for research. The preparation of the umbilical cord tissue was completed within 12 h. The umbilical cord sample was rinsed with normal saline to remove the residual blood. It was then cut into 1.0-cm long segments, and each segment was cut into a 1.0 mm 3 tissue mass and evenly plated in a 10.0-cm dish. The dishes were incubated at 37°C under 5% CO 2 for 24 h, and then 10 ml culture medium contained 45% low-glucose Dulbecco's modified Eagle's medium (DMEM), 45% Ham's F-12, and 10% fetal bovine serum (FBS) (Life Technologies, Grand Island, NY, USA) was added. Half of the medium was replaced 7 days later, and then half of the medium was replaced every 3 days until the primary cells reached subconfluence. The primary cells were detached with trypsin-EDTA (0.25%) (HyClone Laboratories, Logan, Utah, USA) and the reaction was terminated with FBS to passage. Cells harvested from every three dishes were plated in a T-75 flask, and daughter cells were passaged at 1:4 ratios in T-75 flasks. The phenotypic characterization on second-to fourth-passage WJ-MSCs was assayed using a FACScan flow cytometer (Beckman Coulter, USA) for CD90-FITC, CD45-PECY7 (eBioscience, San Diego, CA, USA), CD105-PE, HLA-DR-PECY5, CD34-PE, and CD166-PE (Biolegend, San Diego, CA, USA) according to the manufacturer's instructions. Appropriate isotype controls for nonspecific binding were set for each antibody. For each sample, the assayed cells were analyzed for at least 10,000 events. The WJ-MSCs expressed CD90, CD105, and CD166, and were negative for CD45, CD34, and HLA-DR (Fig. 1).
Isolation and purification of CD34 + cells from umbilical cord blood
Umbilical cord blood was collected from normal full-term delivery after obtaining informed consent from the mothers as a donation for banking, and only cord blood samples not appropriate for banking (< 100 ml) were used in our experiments. We mixed cord blood with 6% hetastarch in 0.9% sodium chloride (Hospira, USA) at a ratio of 4:1 and let it stand for approximately 30 min to allow most of the red cells to form a sediment. Cells in the supernatant was laid onto Ficoll-Paque PLUS (GE Healthcare Bio-Sciences, Pittsburgh, USA) and centrifuged to collect mononuclear cells (MNCs) by depleting the platelets, plasma, and residual red cells. Enrichment of CD34 + cells was performed with two runs of immunomagnetic selection on MiniMACS columns (Miltenyi Biotec, Gladbach, Germany) in accordance with the manufacturer's instructions.
Coculture of WJ-MSCs and CD34 + cells under hypoxic or normoxic conditions
WJ-MSCs at passages 2 to 4 were harvested and radiated (25 Gy) to prevent overgrowth. We then plated them into 12-well plates (8 × 10 4 /well) with H5100 medium contained 10 −6 M hydrocortisone (StemCell Technologies, Vancouver, BC, Canada). At least 24 h later, we removed the medium and seeded purified CD34 + cells (suspended in H5100 medium with 10 −6 M hydrocortisone) into the plate (4 × 10 4 /well). WJ-MSCs and CD34 + cells were cocultured at 37°C and 5% CO 2 under normoxic (20% O 2 ) and hypoxic (3% and 1% O 2 ) conditions for 7 days without adding exogenous cytokines, replacing half of the medium every 3 days. Hypoxic cultures were made in a two-gas incubator (Thermo Scientific, Forma™ Steri-Cycle i160 STERI-cycle, USA) equipped with an O 2 probe to regulate N 2 levels. We also set a control group without WJ-MSCs as feeder layers, with the medium and culture conditions as described above.
Colony-forming cell assay
On day 7, the harvested cells from each group were plated in semisolid culture (H4434, Stem Cell Technologies, Vancouver, BC, Canada) following the manufacturer's instructions for the colony-forming unit (CFU) assay. After incubation at 37°C under 5% CO 2 at 100% humidity for 14 days, the total colony-forming unit (T-CFU), burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte/macrophage (CFU-GM) levels were scored under an inverted microscope. Each CFU is equivalent to a colony-forming cell (CFC).
Long-term culture-initiating cell assay M2-10B4 (ATCC), a murine fibroblast cell line, was used as a feeder layer. At least 24 h before assay, M2-10B4 cells were radiated (80 Gy) and plated in six-well plates (2.5 × 10 5 /well). The plates were coated with collagen solution (StemCell Technologies, Vancouver, BC, Canada). Cells harvested from coculture systems at different oxygen concentrations on day 7 were resuspended with H5100 containing 10 −6 M hydrocortisone (StemCell Technologies, Vancouver, BC, Canada) and then seeded into the plate (2 × 10 5 /well) with the feeder layers. At weekly intervals we replaced half of the medium. Both nonadherent and adherent cells were harvested at week 5 and plated in semisolid culture (H4434, StemCell Technologies, Vancouver, BC, Canada) for CFC assay. After 18 days, colonies were scored under an inverted microscope. The
Cytokine concentration analysis
WJ-MSCs were radiated (25 Gy) and plated into 12-well plates (8 × 10 4 /well) suspended with H5100 medium containing 10 −6 M hydrocortisone (StemCell Technologies, Vancouver, BC, Canada). Twenty-four hours later, we replaced the medium and cultured the WJ-MSCs at 20% O 2 or 1% O 2 , replacing half of the medium twice a week. On day 7, we collected the supernatant of the medium and analyzed the concentration of the following cytokines in each group by Luminex assays Kit (R&D Systems, USA) on Luminex 200: tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-3, vascular endothelial growth factor (VEGF), stem cell factor (SCF), IL-7, GM-CSF, macrophage colony stimulating factor (M-CSF), G-CSF, and thrombopoietin (TPO). H5100 culture medium with 10 −6 M hydrocortisone incubated under the same conditions for each group was set as a blank control.
Signal pathway assays in MSC-HSPC low O 2 cocultures
We harvested the nonadherent cells of each coculture system on day 7. Total RNA was isolated using the MicroElute Total RNA Kit (OMEGA Bio-tek, Norcross, Georgia). Single-strand cDNA was synthesized using the HiFiScript cDNA Synthesis Kit (CW-Bio, Jiangsu, China). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the SYBR Green PCR Master Mix (Fermentas, Vilnius, Lithuania) on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad).
Statistical analysis
The statistical differences between each group were analyzed using the SPSS 20.0 statistical software for all the experiment data. The comparison was analyzed between two groups with an independent sample t test, and among three groups with single-factor analysis of variance (ANOVA). The values were plotted as mean ± standard deviation. Probability values P < 0.05 were considered statistically significant.
Results
The influence of different oxygen concentrations on cord blood HSPC coculture In this study, we defined CD34 + CD10 − CD14 − CD19 − phenotypic cells as the CD34 + lin − HSPC cells (Fig. 2). First, we compared the effects of 3% O 2 with 20% O 2 on the WJ-MSC-HSPC coculture in vitro for 7 days. The results showed that there was no significant difference between these two groups in the percentage or cell numbers of CD34 + Lin − cells, as well as the number of TNC (P > 0.05). We then cocultured UCB-HSPCs with WJ-MSCs at 1% O 2 and 20% O 2 concentrations. We 4 , respectively, and the percentage of CD34 + Lin − was 53.87 ± 5.02% (P < 0.05) (Fig. 3).
Differentiation and long-term reconstruction capacity maintenance
The TNCs were harvested on day 7 from the 1% O 2 and 20% O 2 culture systems for CFU and LTC-IC assay. Methylcellulose colony-forming assay was performed to evaluate the differentiation potential (CFU), and the LTC-IC was evaluated for the maintenance capacity of HSPCs in vitro (Fig. 4)
Concentration of cytokines secreted by WJ-MSCs
To explore the cytokine levels secreted by WJ-MSCs at different oxygen concentration, WJ-MSCs were cultured for 7 days under conditions of 1% O 2 and 20% O 2 and the medium was collected and the cytokines measured by Luminex liquid-phase chip. We assayed 10 cytokines related to hematopoiesis: TNF-alpha, IL-6, IL-3, VEGF, SCF, IL-7, GM-CSF, M-CSF, G-CSF, and TPO. The results show that the culture medium contained much higher levels of VEGF in the 1% O 2 group than the 20% O 2 group (P < 0.01), and more IL-6, IL-7, SCF, and TPO in the 20% O 2 group than in the 1% O 2 group (P < 0.05) ( Table 2 and Fig. 5).
Stem cell pathway activation under hypoxia
To investigate whether stem cell pathways were activated under hypoxic conditions, we analyzed the mRNA expression levels of the suspended cells for three important stem cell activation pathways (Notch, Wnt/β-catenin, and Hedgehog) downstream genes and hypoxia inducible genes (HIF1, HIF2, and ARNT).
Higher mRNA expressions were observed in HIF1, HIF2, and ARNT genes in the 1% O 2 group than in the 20% O 2 group (Fig. 6). Furthermore, some downstream gene expression upregulation was seen: HES1 on the Notch pathway; MMP7 on the Wnt/β-catenin downstream; and PTCH1 and SMO on the Hedgehog pathway (P < 0.05) (Fig. 6). However, for other genes such as HES3, HES5, HEY1, HEY2, TCF-1, Gli1, and PTCH2 after qRT-PCR 50 cycles, the fluorescence amplification curves of cDNA did not reach the plateau stage. This indicates that these genes were not activated in the hematopoietic cells in our low O 2 coculture system.
Discussion
The physiological approach suggests that a microenvironment associating MSCs with a low O 2 concentration would be most favorable for the maintenance of HSPCs in the course of ex-vivo expansion. The microenvironment of UCB-HSPCs in placenta or the umbilical cord is different from that in the bone marrow, and the stromal cells are dissimilar to those in bone marrow being composed of other stromal cells including WJ-MSCs and vascular endothelial cells. The microenvironment of bone marrow or cord blood is extremely hypoxic compared with ambient air [6]. [9]. These results confirmed the major role of the microenvironment for stromal cells and low O 2 concentrations for stem cell maintenance. The mechanisms of how low O 2 can maintain the stemness of HSPCs is still obscure. Some publications have already evaluated the effects of hypoxia on UCB-HSPCs. The reduction in cell division kinetics and a higher percentage of G 0 cells in hypoxic O 2 compared with 20% O 2 conditions have been reported [6,10]. Low O 2 tension increases the expression of hypoxia-inducible factor (HIF)-1 which mediates an active switch from oxidative to glycolytic metabolism, limiting reactive oxygen species (ROS) production and promoting its degradation. ROS terminates the quiescent state of HSPCs and promotes their differentiation [11]. Recently, Mantel et al. reported that, in ambient air, HSPCs were compromised through the activation of the mitochondrial permeability transition pore; this process can be inhibited by setting a hypoxia condition during harvesting and transplantation of donor bone marrow or by using cyclosporin A, which can protect HSPCs from extraphysiologic oxygen shock/stress (EPHOSS) [12]. G-CSF granulocyte colony stimulating factor, GM-CSF granulocyte macrophage colony stimulating factor, IL interleukin, M-CSF macrophage colony stimulating factor, SCF stem cell factor, TNF Tumor necrosis factor, TPO thrombopoietin, VEGF vascular endothelial growth factor*P < 0.05, **P < 0.01, versus 1% O 2 Fig. 5 Comparison of cytokine levels in 20% and 1% O 2 groups (n = 4). *P < 0.05, **P < 0.01. G-CSF granulocyte colony stimulating factor, GM-CSF granulocyte macrophage colony stimulating factor, IL interleukin, M-CSF macrophage colony stimulating factor, SCF stem cell factor, TNF tumor necrosis factor, TPO thrombopoietin, VEGF vascular endothelial growth factor Fig. 6 The expression levels of stem cell pathway target genes at 20% and 1% O 2 (n = 4). Detected genes included hypoxia inducible genes HIF1-α, HIF2-α, and ARNT, Notch pathway downstream genes HES1, HES3, HEY1, and HEY2, Wnt/β-catenin downstream genes AXIN2, MMP7, and TCF-1, and Hedgehog downstream genes GLI1, PTCH1, PTCH2, and SMO. *P < 0.05, **P < 0.01 The mechanisms behind MSC hematopoiesis support are still elusive, whether cell-to-cell direct contact and/ or soluble factor secretion [13,14]. Proteomic analysis of the WJ-MSCs revealed high levels of interleukins (IL-1a, IL-6, IL-7, IL-8), as well as SCF, hepatocyte growth factor (HGF), and ICAM-1, suggesting once again that they may be the agents involved in the expansion of UCB-HPSCs [15]. To investigate the combination roles of WJ-MSCs with low O 2 , we assayed 10 cytokines involved in hematopoiesis: TNF-alpha, IL-6, IL-3, VEGF, SCF, IL-7, GM-CSF, M-CSF, G-CSF, and TPO in the supernatant of WJ-MSC coculture medium at 1% and 20% O 2 . The results show that the culture medium contained much higher VEGF levels in the 1% O 2 group, and higher levels of IL-6, IL-7, SCF, and TPO in the 20% O 2 group. It is surprising that VEGF level was so high in our coculture system at 1% O 2 , and this indicates that the hypoxic response of WJ-MSCs is characterized by a rapid increase in VEGF secretion and glycolytic activity. This rapid increase in VEGF secretion may play a critical role in the survival and expansion of human UCB-HPSCs under hypoxia. VEGF is a principal regulator of hematopoiesis, which provides for quiescence and self-renewal as well as restraining the differentiation of HSPCs [16,17]. This may explain the reason why the TNCs harvested at 1% O 2 contained more long-term reconstruction cells and less lineage-committed progenitor cells. The function of IL-6 and IL-7 is to induce the differentiation of HSPCs [18,19], and our results showed that the levels of IL-6 and IL-7 were significantly higher at 20% O 2 . This may indicate a differentiation role under normoxia stimulation. Other cytokines, such as TNF-α, IL-3, M-CSF, and G-CSF which promote differentiation [20,21], tended to decrease at 1% O 2 . This may contribute to maintaining the stemness of HSPCs. In a recent research, Paquet et al. cultured human bone marrow MSCs at different oxygen concentration (21%, 5%, and 0.1%), and showed that hypoxic conditions increased the MSC paracrine secretion of angiogenic mediators such as VEGF, IL-8, RANTES, and monocyte chemoattractant protein 1, and significantly decreased the expression of several inflammatory/immunomodulatory mediators, such as IL-6, IL-15, and IL-1Rap [22]. Majumdar et al. compared the effects of normoxia (20% O 2 ) with hypoxia (2% O 2 ) on the paracrine secretion of WJ-MSCs; their results showed there was significantly increased secretion of VEGF and HGF under hypoxia [23]. These studies are consistent with our findings. Taken together, MSCs exposed to a hypoxic culture increase the expression of VEGF, promote the phosphorylation of focal adhesion kinase [24], and increase the expression of chemokine receptors such as CXCR4 and CX3CR1 [25].
To investigate whether stem cell pathways were activated under hypoxic condition, we analyzed the mRNA expression levels of the harvested HSPCs on hypoxia-related genes (HIF1, HIF2, and ARNT) and Notch, Wnt/β-catenin, and Hedgehog pathway downstream genes. Our findings showed that 1% O 2 induced higher mRNA expressions of HIF1, HIF2, and ARNT genes, and upregulated some downstream gene expression, such as HES1 on the Notch pathway, MMP7 on the Wnt/β-catenin downstream, and PTCH1 and SMO on the Hedgehog pathway. Hypoxic tension promotes the expression of the hypoxia inducible factor-related genes HIF-1α, HIF-2α, and ARNT. These genes encode HIF-α subunits that are stabilized under low oxygen tensions and exhibit tissue-restricted expression. Upon stabilization, these subunits dimerize with the β-subunit, HIF-β (ARNT), and translocate to the nucleus to regulate a spectrum of genes to maintain oxygen homeostasis, glucose metabolism, angiogenesis, erythropoiesis, and iron metabolism [26]. Hypoxia has been shown to activate molecular pathways in multiple stem cell systems. Notch signaling is widely appreciated to be critical for the maintenance of undifferentiated stem and progenitor cell populations. Ezashi et al. found that oxygen tensions as low as 1% appeared to decrease proliferation and maintain pluripotency of stem cells, while higher oxygen tensions (3-5%) appeared to maintain pluripotency with no effect on proliferation [27]. Our experiments showed that the activation of the three important signal pathways in HSPCs under hypoxia was significantly enhanced. However, the downstream genes of the three pathways were selectively activated and, thus far, their functions are not able to be shown. Some researchers have tried to confirm a correlation between HIF-related genes and stem cell pathways under hypoxia. Mukherjee et al. reported that, in drosophila blood cells, HIF-α binds to the Notch ligand intracellular segment to promote Notch downstream gene expression [28]. Bijlsma et al. found that HIF1-α induced hedgehog pathways activated by PTCH1 in the mouse [29]. Mazumdar et al. verified the relationship between HIF1 and TCF-1 in mouse embryonic stem cells [30], and Liu et al. confirmed the relationship between HIF1-α and MMP7 protein content in gastric cancer cell lines [31]. The increased expression of HIF-related factors, HES1, PTCH1, and MMP7 under hypoxia in our study can partially explain the positive effect of a hypoxic coculture system on stemness maintenance of UCB-HSPCs, but how the stem cell pathways are activated remains to be further explored.
Conclusions
In this study, we used WJ-MSCs as a feeder layer under low O 2 tension to simulate the physiological microenvironment in which UCB-HSPCs reside to explore the effects of the coculture system on stemness maintenance and proliferation of HSPCs in vitro without adding exogenous cytokines. The results showed that the populations of CD34 + Lin − cells and LTC-ICs could be preserved better at 1% O 2 than at 20% O 2 . Hypoxia increased VEGF secretion and decreased IL-6 secretion and selectively activated the Notch/Wnt/Hedgehog signaling pathway in UCB-HSPCs via HIF-related factors, which plays an important role in preserving stemness and sustaining HSPC quiescence. However, our findings are only a starting point for pursuing optimizing protocols aimed at expanding UCB-HSPCs ex vivo, such as adding some key cytokines and growth factors, using optimal low O 2 tension to protect HSPCs from EPHOSS to preserve the capacity of stemness of HSPCs, and developing bioreactor systems for in-vitro cultures, which may have important translational and clinical implications. | v3-fos-license |
2014-10-01T00:00:00.000Z | 2012-02-27T00:00:00.000 | 6507049 | {
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"Chemistry",
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} | pes2o/s2orc | Motion and Flexibility in Human Cytochrome P450 Aromatase
The crystal structures of human placental aromatase in complex with the substrate androstenedione and exemestane have revealed an androgen-specific active site and the structural basis for higher order organization. However, X-ray structures do not provide accounts of movements due to short-range fluctuations, ligand binding and protein-protein association. In this work, we conduct normal mode analysis (NMA) revealing the intrinsic fluctuations of aromatase, deduce the internal modes in membrane-free and membrane-integrated monomers as well as the intermolecular modes in oligomers, and propose a quaternary organization for the endoplasmic reticulum (ER) membrane integration. Dynamics of the crystallographic oligomers from NMA is found to be in agreement with the isotropic thermal factors from the X-ray analysis. Calculations of the root mean square fluctuations of the C-alpha atoms from their equilibrium positions confirm that the rigid-core structure of aromatase is intrinsic regardless of the changes in steroid binding interactions, and that aromatase self-association does not deteriorate the rigidity of the catalytic cleft. Furthermore, NMA on membrane-integrated aromatase shows that the internal modes in all likelihood contribute to breathing of the active site access channel. The collective intermolecular hinge bending and twisting modes provide the flexibility in the quaternary association necessary for membrane integration of the aromatase oligomers. Taken together, fluctuations of the active site, the access channel, and the heme-proximal cavity, and a dynamic quaternary organization could all be essential components of the functional aromatase in its role as an ER membrane-embedded steroidogenic enzyme.
Introduction
Cytochrome P450 aromatase catalyzes the biosynthesis of estrogens from their androgenic precursors by converting the partially unsaturated A-ring to an aromatic A-ring. Structurefunction relationships of aromatase have been studied for more than thirty years, but many issues remain unresolved. The recent crystal structure of human placental aromatase showing a compact active site cleft [1] has shed new light on the decades old problems. In the crystal, aromatase molecules are found to form head-to-tail oligomers [2]. This association of monomers is probably driven by electrostatic interactions between the ''head'' and ''tail'' segments of two adjacent molecules. Mutagenesis results demonstrate the functional implications of oligomerization of aromatase. Recently, Praporski et al. also reported a high order organization of aromatase in living cells using atomic force microscopy (AFM) and fluorescence resonance energy transfer [3]. The high-resolution AFM images support the formation of aromatase homodimer and oligomers that are stabilized in the lipid bilayer membrane.
However, the dynamical properties of aromatase that may play critical functional roles, such as membrane integration and active site access channel opening, have not yet been addressed. Availability of the crystal structure of aromatase has opened the door for investigating the dynamics by high resolution atomic/ coarse-grained simulated models, such as molecular dynamics (MD) simulations and normal mode analysis (NMA). NMA proves to be a very powerful tool to gain insights into the protein dynamics at a reasonable resolution (heavy atoms or Ca) at much less computational costs [4]. NMA in combination with elastic network (EN) model [5] has been developed for studying protein flexibility and dynamics [6,7,8,9,10,11,12,13,14,15,16,17]. Due to the simple harmonic nature of the potential, the methodology is valid only in proximity to equilibrium and unable to model energy barriers and multiple energy minima. Nevertheless, it has been proven to yield the slow normal modes just as effectively as those from complicated forcefields with specific non-linear terms [12,13]. The collective motions of a protein at the low-frequency spectrum are correctly correlated with the observed protein conformational changes upon ligand binding or protein-partner association [17].
In this paper, we present the results from EN-NMA on the membrane-free and membrane-integrated monomers and the crystallographic dimer and trimer of aromatase. We show that two major intermolecular modes of motion are responsible for alternations in the observed quaternary association of aromatase that could be utilized for its endoplasmic reticulum (ER) membrane integration. The two major intramolecular normal modes in the monomer are likely to be responsible for the active site access channel ''breathing''. The root mean square fluctuation (RMSF) from EN-NMA provides a measure for the intrinsic molecular flexibility and the analysis elucidates the rigid core structure of aromatase, regardless of its self-association and membrane integration.
EN-NMA of crystallographic aromatase oligomers
A tetramer is built using the crystallographic symmetry (Fig. 1A) and then subjected to normal mode analysis. Within the tetramer, the central green monomer is found to have the smallest amplitude of displacement in the first two slowest modes, indicating that its global mobility is constrained by the head-to-tail association and crystal packing. Other three monomers display higher mobility because they are devoid of the crystallographic constraints, or the periodic boundary conditions. Taking mode 7 as an example, two regions with distinctly different mobility are clearly visible. The inner region of the central monomer and its vicinity are much less mobile than the outer region of the blue, gold and gray monomers (Fig. S1A, Supporting Information).
The B-factors of Ca (B-C a ) are computed from the mean square fluctuation (MSF), for the green monomer and compared with the X-ray B-factors (Fig. 1B). The two agree with each other for a wide range of residues except for the termini. When compared with the X-ray B-factors, the B-C a factor profiles of the three outer monomers are significantly larger and exhibit substantial variations, unlike the inner green monomer (Fig. S1B, Supporting Information). The fluctuation patterns for the regions responsible for crystal packing and head-to-tail binding for these three monomers are dramatically different from the X-ray B-factors. The variations in global mobility and change in residue-fluctuation patterns are correlated well with the crystal contact interactions (see details in Supporting Information).
A monomer with the N-terminal helix is shown in Fig. 2A and the formation of large voids, the regions of lowest electron density, is observed in the crystal where the N-terminal helices reside (Fig. 2B). The monomers of the crystallographic tetramer are packed in the same way as those in the absence of the N-terminal helices (Fig. S2, Supporting Information). Interestingly, the motion of the N-terminal helix is found to be consistent with the crystallographic symmetry of the molecules in the crystal (Movie Figure 1. The crystallographic tetramer of aromatase and validation of the C a normal mode analysis against crystallographic B factors. A, three aromatase monomers from one oligomer chain (ribbon diagram colored blue, green and orange) in contact through the H-I loops with another monomer (gray) from the neighboring chain. B, the computed B-factors of C a of the central monomer (green line), simulating the closely packed aromatase in crystals, are compared well with those from X-ray data (black line). The substrate and heme group are represented by stick drawings. The former is colored in magenta while the latter is rendered in element colors: cyan, C; red, O; blue, N; brown, Fe. The coloring code and the atoms and bonds representations are the same in all figures unless otherwise noted. doi:10.1371/journal.pone.0032565.g001 S1, Supporting Information). The motion of the central monomer is highlighted in Fig. 2C, showing that its N-terminal helix has the largest eigenvectors among the entire monomer.
Collective motion in membrane-free and membraneintegrated aromatase monomers
The EN-NMA for a membrane-free monomer reveals interesting internal motions from the two slowest normal modes (Figs. 3A and B). In mode 7, three moving parts of the structure are identifiable: two in the lower half of the molecule librating in the opposite directions and the third in the upper half rotating against the lower half ( Fig. 3A and Movie S2A, Supporting Information). The membrane-integrating N terminus and its vicinity form the first part, the C-terminal loop regions the second, and the segments above the active site access channel the third. The access channel residues are at the borders of these three moving parts. The movements of each pair produce the so-called ''hingebending'' motion [18,19] with the common hinge being at the access channel. In mode 8, the front half of the molecule librates against the back, forming an intramolecular twisting motion with, again, the access channel at the interface ( Fig. 3B and Movie S2B, Supporting Information).
When the monomer is embedded in the lipid bilayer, motions similar to those in the membrane-free monomer are observed within the monomer but at a higher frequency. Due to interactions at the membrane-protein interfaces, the hinge-bending motion (the mode 19) has reduced amplitudes for the membrane integrating regions (the helices A9 and A, and the C-terminal b7-b8 and b9-b10 loops) ( Fig. 3C and Movie S3A, Supporting Information). Instead, the F-G loop and its vicinity have enhanced amplitudes and the C-terminal loop regions librate against the membrane, different from the movement in a membrane-free monomer. The twisting motion (the mode 17; Fig. 3D and Movie S3B, Supporting Information) exhibited is similar to that in the membrane-free molecule. The N-terminal helix is associated with the motions in the rear half of the molecule in Fig. 3D. It is noteworthy that the C-terminal loops are relatively stationary in both modes.
However, the three slowest modes, modes 7, 8 and 9, for the membrane-embedded monomer are unique and different from the above hinge bending and twisting motions. The former two are back-forth and left-right bending oscillations, respectively, and the latter is a twisting motion (Movie S4A, B, C, Supporting Information). The two bending modes result in rocking of the cytoplasmic domain of aromatase at the lipid interface in two directions. Twisting appears to be a counterclockwise, winding motion of the entire cytoplasmic domain about a vertical axis while keeping the N-terminal trans-membrane segments relatively stationary. As a result, the heme/active site region moves in and out of the lipid interior.
Slow modes of crystallographic oligomers
The two slowest normal modes 7 and 8 are basically rigid body rotations against each other when only the green-gold dimmer is considered (Fig. S3, Movie S5A and B, Supporting Information). In the process, the region above the D-E loop including helix J, b7, b10, and the b7-b8 loop of the green monomer, and the hemeproximal cavity region constituted by helices B9, C, H, H9 and J-K loop of the gold monomer move back and forth to each other. These movements lead to the simultaneous opening/closure of two head-to-tail extended regions formed by the neighboring monomer pairs.
These slowest normal modes are maintained within the crystallographic blue-green-gold trimer (Fig. 4A). In mode 7, the blue and gold monomers move away from/toward each other, while the green monomer undergoes a small back and forth translation (Movie S6A, Supporting Information). This movement consists of two asymmetrical hinge-bending motions, one between the blue and the green monomers and the other between the gold and the green. In mode 8, two twisting motions are formed through the rotation of either blue or gold monomer against the nearly stationary green monomer. The rotation axes are roughly the lines linking the centers of the mass with their respective headto-tail binding sites (a cross-section view in Movie S6BI and a plan view in Movie S6BII, Supporting Information). Interestingly, these two intermolecular motions are preserved in a trimeric aromatase even in the presence of the fourth gray monomer, simulating the crystal-packing environment (Movie S7A, BI and BII, Supporting Information). Nevertheless, examination of the higher frequency modes confirms the presence of bending and twisting modes similar to the ones in a membrane-free monomer, only muffled due to intermolecular association.
The electrostatic potentials of a dimer and a trimer are calculated and mapped on their van der Waals surfaces (Fig. 4B). In a dimer, two major groove sites form an electrostatic potential gradient near the head-to-tail binding site, site ''E'' with negative electrostatic potentials on the upper monomer and site ''P'' with positive electrostatic potentials on the lower. Nine negatively charged side chain residues, Asp 186, Asp197, Asp209, Asp222, Asp482, Glu177, Glu210, Glu483 and Glu489, contribute to the negative electrostatic potentials at the E site, and about six positive charges from Lys 142, Lys352, Lys440, Lys448, Arg 145, Arg375 and the heme group form the positive electrostatic potentials at the P site. In a trimer, a pair of such E and P sites is present at each head-to-tail binding site (Fig. 4B). The electrostatic potential . Intermolecular motions of the aromatase trimers from normal mode analysis and their complementarity with electrostatic interactions. A, two slowest normal modes in aromatase trimer. The dotted lines designate the rotational axes. B, electrostatic potentials mapped on the van der Waals surfaces of a trimer in a color scale red to blue representing a potential scale from 27kT/e to 7kT/e. The P and E sites, adjacent to the head-to-tail binding interfaces of the oligomers, correspond to the positively and negatively charged cavities, respectively. The arrow points roughly along the electrostatic potential gradient from negative to positive potentials. The orientation of oligomer is roughly the same in both panels. The inset shows the second dimer interface hidden from view. doi:10.1371/journal.pone.0032565.g004 gradient could be of interest here and could influence the intermolecular motions. The direction of intermolecular motions would be favorable along the gradient, but unfavorable against it.
We also probed by computational approaches other possible oligomeric interfaces that aromatase monomers may utilize in solution. An overwhelming majority of the models thus obtained showed the crystallographically observed interface as the intermonomer interaction surface. Furthermore, the results also suggested considerable flexibility in the D-E loop-to-heme proximal cavity association within the interface (Text S1 and Fig. S4, Supporting Information).
Fluctuation in aromatase
The Ca-RMSF of an aromatase monomer (Ca-RMSF) is calculated and visualized in a rendered ribbon diagram where red represents the lowest RMSF (at the heme group), and blue the highest RMSF (at the H-I loop) (Fig. 5). The H-I, D-E (not shown), G-H9 and F-G loops (including the short helix G9 and its connecting loops to helixes G and F) are quite flexible, but the inner core, defined as a spherical region within a radius of 15 Å from the center of substrate, is very rigid. The catalytic cleft is at the center of the core. The average RMSFs, either in the absence or the presence of the substrate, are calculated over four distinct regions: heme, the catalytic cleft, the heme-proximal cavity and the active site access channel, and also over three layers of interest within the aromatase molecule: the inner core (radius#15 Å ), middle-layer (15 Å ,radius#20 Å ) and outer layer (radius.20 Å ) (Fig. S5A, Supporting Information). Heme has the lowest RMSF, 0.47, followed by the catalytic site 0.52 and the inner core 0.65 in the presence of the substrate. They are all well below the average fluctuation (RMSF = 1) of the molecule. The putative access channel has a higher RMSF of 0.74 when compared with heme, the catalytic cleft and the inner core, probably due to some of its constitutive residues, such as Pro481, Asp482, Glu483 and Thr484 from the b9-b10 loop, are either lining the channel or bordering the lipid interface. A modest fluctuation, an RMSF of 0.92, has been found in the proximal cavity, most of whose constitutive residues are from the 21-residue long K0-L loop but stabilized by the heme group through coordination with Arg435 and the Cys437 ligation. The fluctuation of the middle layer is about 10% below the average fluctuation of the molecule and that of the outer layer is the largest (,35% above the average fluctuation).
Although the substrate is in direct contact with the catalytic cleft residues [1], only a marginal increase of 0.04 in the RMSF is found in the absence of substrate (Fig. S5A, B, Supporting Information). It appears that removal of the substrate does not significantly affect the rigidity of the catalytic cleft. Interestingly, however, similar small but consistent increases in RMSF in the heme-proximal cavity, the access channel and the inner core are observed on substrate removal, but not in the middle or outer layers. Therefore, the stabilizing effect of substrate binding on the protein rigidity is rather small due to the compact nature of the aromatase molecule, and is limited to the inner core, not exceeding a 15 Å radius. Furthermore, the heme moiety could primarily be responsible for the overall rigidity of the catalytic cleft resulting from stabilization of the side chains, such that the integrity of the functionally active enzyme is maintained even in the absence of the substrate.
The Ca-RMSFs calculated for a membrane-integrated aromatase monomer (Fig. S6A, Supporting Information) show that the catalytic cleft has similar low fluctuations as the heme, followed by the access channel and the proximal site. Notably, the fluctuations of these four segments have an order similar to those of a membrane-free monomer. The N-terminal helix has relatively higher fluctuations due to its location away from the body of the molecule. Thus, the rigid core structure of aromatase is intrinsic and independent of its membrane integration.
To evaluate possible impact of the side chains on these results, we compare the RMSFs of the catalytic cleft, the inner core, the middle layer and the outer layer with those obtained from allheavy atom NMA (Fig. S6B, Supporting Information). The results agree with each other within 0.09 RMSF, implicating that the side chain mobility is correlated with the main chain flexibility in the monomeric aromatase assuming that it does not undergo any large structural transition.
In addition, calculations of the fluctuations of aromatase oligomers show that the oligomerization does not deteriorate the rigidity of the active site ( Fig. S7 and Text S2, Supporting Information).
Model validation in crystal environment
The EN-NMA is attractive because it has the capability to identify the slowest internal modes of protein that are important for biological functions [12,17]. Our calculations on a tetramer validate the applicability of NMA. The computed B factors of the crystallographic central monomer agree well with the experimental X-ray B factor data. Moreover, the results show that the method is sensitive to inter-monomer association and crystal packing interactions. The RMSF peaks disappear at the tail (the D-E loop and vicinity) and the head (the K helix, J9-K and K0-L loops) regions upon head-to-tail association in which the helix, loops and strands embed into the protein interior (Fig. S1, Supporting Information). The D-E loop vicinity includes the b8 strand from sheet 3, and b7 and b10 from sheet 4. These results also confirm that the shape of the tail of one monomer compliments the proximal cavity of the next in an aromatase oligomer and the self-association is stabilized by intermolecular interactions. It is conceivable that the heme moiety plays a major role in the stability of the proximal cavity and hence influences the oligomerization. The peaks at the H-I loop interface disappear due to the crystal packing constraints.
Moreover, the NMA of crystallographic tetramer shows that the N-terminal helices have large mobilities. These motions, however, appear not to break the crystallographic symmetry or interfere with intermolecular packing. This could explain why the Nterminal region of the molecule appears dynamically disordered in electron density maps.
Complexation-induced rigidity
Self-association decreases the flexibility of a monomer in the oligomeric aromatase at the head-to-tail binding site and its vicinity. The result is similar to the phenomenon reported in the analysis of Ras-Raf using a molecular framework approach and MD simulation [20]. As we have also observed in the aromatase trimer, the regions distant from the binding sites become more flexible upon aromatase self association, the perturbation generated from aromatase self association can propagate from a binding site to remote regions by alternating the dynamic network of interactions in proteins. The translational and rotational degrees of freedom of the monomers in an aromatase oligomer are reduced due to monomer-monomer binding. The ''freezing-out'' of possible multiple structures of an oligomer upon binding results in loss of configurational entropy, but it could be compensated by the entropy gain from the increase in flexibility of the distant regions away from the binding site (Fig. S6A, Supporting Information) as proposed by Steinberg et al. [21]. Entropic contribution from the increased flexibility is believed to be a dominant factor in the free energy of protein-protein association [22].
The fluctuations of both the access channel cavity and proximal site relative to heme reduce on integration into the membrane. The RMSF ratio decreases from 1.77 to 1.33 for the proximal site and from 1.45 to 1.14 for the channel cavity, while the active cleft remains roughly the same. This could be due to the fact that the active site residues are located away from the membrane surface, whereas some of the access channel resides and the loop residues of the proximal site interact with the lipid bilayer. However, these predictions need further validation by site-directed mutagenesis in reconstituted membrane and/or cell-based activity assay on the mutant enzymes.
Furthermore, the observed reduction in the mobility of the membrane-associating C-terminal loops could result in enhanced stability and optimal alignment of the active site access channel for steroidal passage through the lipid bilayer.
Possible pathway for a crystallographic oligomer to integrate into membrane
A valid aromatase oligomer topology should be amenable to integration into the ER membrane. We have used two linear trimer units (Fig. 6A) to model a membrane-integrated circular hexamer (Figs. 6B and C) by a process described in the Materials and Methods section (see below). A combination of the twisting and hinge-bending motions shown in Fig. 4A could adjust the quaternary association along the lowest energy landscape [18]. Electrostatic interaction between the ''E'' site of the ''tail'' monomer and the ''P'' site of the ''head'' is presumed to play a role in driving the movements for the quaternary structural changes. The modeling suggests that a circular oligomer thus formed would use a similar loop-to-proximal cavity link as that used by the polymeric chain in the crystal. The N-terminal helix of each monomer penetrates into and across the lipid bilayer with its end in the lumen side.
The size of a circular oligomer may vary depending on aromatase concentration. An open passage in the membrane is just created for each monomer after membrane insertion and each passage is connected to the access channel of each molecule (same as for a monomer in Fig. S6A, Supporting Information). Organizations such as cyclic hexamers (size ,14 nm), octamers (,18 nm) and even higher orders could be modeled in this way. In the resting state, oligomerization could be a means of protection of integrity of the proximal site and/or from undesirable effects at the site, such as non-specific actions of redox agents, and phophorylation of Tyr 361 [23]. A likely scenario is that the monomers are replaced by the CPR molecules for the electron transfer reaction and aromatization to proceed.
Flexibility and dynamical motion: relevance to biological function
The N-terminal helix, novel to the P450 structures elucidated thus far, is the most mobile and flexible structural element identified. The F-G loop is the next most flexible region in the aromatase structure (Fig. 5) that is not significantly influenced by self-association and membrane integration. The F-G loop flexibility was previously reported to be one of the common features of cytochrome P450s 2B4 and BM-3 with functional relevance to enzymatic reactions [24]. The flexible loop undergoes an open/close motion that allows the steroids to enter into or leave from the active site through the access channel [1,25]. Our results provide new support to this notion. Furthermore, the NMA of a monomer reveals that the access channel could serve as a hinge for intramolecular bending and an interface for twisting motions. These motions, together with the intrinsic flexibility of the access channel, are likely to contribute to channel ''breathing'', opening and closing of the channel mouth and the cavity, perceived necessary for entry and exit of steroids to and from the active site.
The hinge bending and twisting motions at the access channel hinge/interface are also present in the lipid-embedded aromatase, but at a higher frequency. The membrane penetrating areas, such as helices A9 and A, have reduced amplitudes, owing perhaps to dampening of the oscillation by surrounding lipid molecules. However, the twisting motion is similar to the membrane free molecule, which suggests that twisting could be more closely related to a functional aromatase in vivo. Interestingly, the Nterminal helix motion does not coordinate with either of these two movements; instead, it is associated with the rear half of the molecule, suggesting that membrane integration of the N-terminal helix may have roles different from ''breathing'' or steroid passage, perhaps in intramembrane stabilization or CPR coupling. One of the slowest modes of the membrane-embedded aromatase suggests a periodic movement of the active site region deeper toward the lipid interior. Such a motion could be associated with the enzyme's substrate sequestration and/or product release phases of the catalytic cycle.
Two slowest modes at the interface of the head-to-tail association are intermolecular rigid-body hinge bending and twisting motions. They provide the flexibility for the aromatase molecules to reorganize themselves retaining the interface in order to form an oligomeric structure. Our data suggests that such reorganization and reorientation are necessary to position the trans-membrane helices and regions on the same side of each monomer for the oligomer as a whole to penetrate the lipid bilayer. The driving force for this interfacial motion could be drawn from the electrostatic potential gradient between the electronegative ''E'' site of the D-E loop region of one monomer and the electropositive ''P'' site of the heme-proximal region of the other. The heme-proximal electropositive ''P'' site of aromatase has been proposed to be critical for electron transfer by the FMN moiety from CPR [26]. The observed flexibility of the intermolecular interaction from this work suggests that the FMN moiety of CPR could bind at the interface, either by flexing the head-to-tail organization for a three-way binding or by competitively replacing an aromatase monomer.
One of the most important biological implications of our computational results is the corroboration that intermolecular contacts and flexibility observed in the crystal structure could be utilized into a higher order organization of aromatase that has the correct topology for membrane integration. Aromatase molecules function in the ER membrane and recent results suggest that the enzyme is multimeric when embedded in the lipid bilayer [3]. Our data derived from the crystal structure and flexibility calculations show a mechanism by which this could be done, maintaining the heme-proximal site orientation accessible for CPR coupling. Furthermore, the computational result on free monomer to monomer docking suggest that head-to-tail organization observed in the crystalline aromatase is the most favored interface albeit with a good deal of flexibility. Taken together, these results provide new atomic level insights into the form, function and flexibility of an oligomeric aromatase previously envisioned in the literature.
Lastly, the present aromatase atomic model for the first time shows the N-terminal trans-membrane helix, based partly on weak experimental electron density map not previously modeled, and partly on the crystal packing constraints. Although other microsomal P450's are known to have similar trans-membrane segments, aromatase in particular has longer and more pronounced membrane-integrating regions, as the crystal structure and sequence comparison suggest [1,26]. Modeling of this helix, and its juxtaposition in relation to other membrane integrating A9 and A helices, as well the C-terminal membrane associating areas all reaffirm the previously proposed notion that the opening to active site access channel rests just inside the lipid bilayer enabling easy passage of the highly hydrophobic steroidal substrate and product. The N-terminal helix appears to project out into the lipids via an extended peptide segment with residues Tyr41 to Gly49 away from the main enzyme structure. This is suggestive that the trans-membrane segment probably plays roles not directly associated with the enzyme catalysis, but in the aromatase-CPR interaction through the CPR's transmembrane segments. Indirectly, however, such ''tethered'' membrane anchoring may be crucial for the added flexibility of the business end of the molecule, as the slowest modes suggest. The observed dynamical disorder of the N-terminus in the X-ray data is simply a reflection of the fact that once separated from the membrane it dangles harmlessly away from the main structure and without any interference with the stability of the functional enzyme. The current modeling of the N-terminal helix and its vibrational modes within the apparent ''void'' of the aromatase crystal (see ''Calculations under crystal packing conditions'' in MATERIALS AND METHODS) is a confirmation that its enhanced mobility persists and accommodated in the crystalline state.
We show that the major normal modes in aromatase oligomers are inter-monomeric rigid body motions with the D-E loop to proximal site association as the interface and that this interface is directly linked to catalytic function of the enzyme. It is, therefore, likely that suitable small molecules binding at the interface would interfere with the CPR coupling, oligomer formation and/or its membrane integration. Such non-active site directed compounds could constitute a new class of aromatase inhibitors.
EN-NMA
The detailed description and recent reviews of the Ca-NMA method can be found in literature [7,12,13,15]. In this work, a single parameter potential was used as proposed by Tirion [5]. The building block approximation so called rotation-translation-block (RTB) [8] method is employed to speed up our calculations and reduce the computational limitation of a large system. As mentioned by Bahar [10,12] and Tama [8], this approximation has very little influence on slow modes, particularly for a large protein complex where the functional domains are expected to be large.
The oligomer coordinates were generated by the crystallographic symmetry operations using Coot [27] and the crystal structure of the human placental aromatase monomer [1], PDB code: 3EQM. NMA was implemented using the elNémo webserver [28]. The smallest system of study consisted of one monomer (452 residues) and there were 1940 residues (including the N-terminal helices) in the largest system consisting of a tetramer. The connectivity cutoff distance, used in pairwise Hookean potential between nodes, was tested in a range of 8,16 Å . A cutoff of 10 Å was selected for monomer and the default of 8 Å for oligomers after scaling with the experimental B factors. The block size was chosen by default, but could be varied with the system size. For convenience of visualization, the eigenvectors were scaled by a factor of 200. The eigenvector arrows in Fig. 2(C) represent the relative amplitude and direction of the associated C a atoms of the central monomer. The same eigenvector representation has been followed in Figs. 3 (A) to (D), Fig. 4 (A), and in Supporting Information (Fig. S1 and Fig. S3). The normal mode models were computed with a given perturbation amplitude in the direction of a single normal mode. Here the perturbation range was from 2100 to 100 with a step size of 20 [28]. The motion in supporting movies (Movies S1, S2, S3, S4, S5, S6, S7) was generated under perturbation; it could, therefore, be exaggerated when compared with equilibrium fluctuation.
The N-terminal helix was not included in the calculations except for the protein in complex with the membrane or wherever noted. It is seen that the movement of the N-terminal helix are predominant in the absence of membrane among the collective motion modes that have a low frequency and large amplitude. The usage of a truncated aromatase model is found more efficient than that with the N-terminal helix in the dynamics study of oligomers.
The frequency was normalized relative to the lowest mode frequency in all our calculations. The frequencies of modes 7 and 8 of a dimer were 1.00 and 1.09, and those of a free monomer were 1.00 and 1.04. Taking the slowest frequency to be 2.5 cm 21 , the frequencies of the first 20 slowest modes in the system of this study are in the range 2.5-15 cm 21 . Therefore, the time scale for the slowest modes range from a few picosecond to the order of 10 picosecond, in agreement with reported collective motion in proteins [18].
Calculations under crystal packing conditions
In the space group P3 2 21, the head-to-tail oligomers are formed about the crystallographic three-fold screw axis and packed in the crystal about a crystallographic 2-fold rotation axis perpendicular to the 3 2 screw axis (Fig. 1A). A head-to-tail intermolecular interaction among aromatase molecules is mediated via a surface loop between helix D and helix E of one aromatase molecule penetrating into the heme-proximal cavity of the next, thus forming a polymeric aromatase chain (Fig. 1A). Two oligomer chains form crystal contact through hydrogen bonding and salt bridge interactions via the H-I loops. For the system of four interconnected crystallographic monomers in Fig. 1A employed in the calculation, the crystal-packing environment was preserved for the central green monomer. However, the adjacent blue and gold monomers from the same polymer chain and the gray from the neighboring chain each had only one association, unlike the crystal environment.
The calculations were repeated for the crystallographic tetramer with the N-terminal helices (Fig. S2, Supporting Information). A putative atomic model, consisting primarily of an a-helix, for the N-terminal missing residues Asn12 to Thr44 is built using the partially visible weak experimental electron density [1] (Fig. S8, Supporting Information), and restrictions of the crystallographic 2fold rotation axis, which the two symmetry-related helices approach. The modeling was also guided by the fact that a helix between Ile13 and Tyr40 would traverse the lipid bilayer, positioning Asn12, a potential glycosylation site, in the ER lumen. Interestingly, N-terminal helices line up about the 3 2 symmetry axis within the crystal in the space that constitutes the largest void (a region of lowest electron density in the crystal), a channel of dynamically disordered solvent and detergent, thereby providing some rationale as to why the N terminus is disordered.
Modeling of aromatase monomer in lipid bilayer
It is known that phosphatidylcholine is the major lipid composition in ER membrane [29,30,31]. For simplicity, a bilayer model of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) is employed to represent the ER membrane. The coordinates of the phospholipids were generated with the builder module in the VMD package [32] and the membrane has a size of 80 Å |80 Å . The aromatase molecule was then aligned against the membrane according to the hydrophobic property of the protein and their topology, as described earlier [1]. The N-terminal helices, up to helix A, traverse into the bilayer with the Asn12 in the lumen side. The C-terminal loops, such as b7-b8 and b9-b10 loops are embedded into the lipids. The structures of protein and membrane were then merged with the VMD package by eliminating the lipid molecules that overlap with the protein. The complex were finally subjected to energy minimization at the protein-membrane interface with fixed backbone of the protein and lipid molecules in Molecular Operating Environment (MOE, 2009.10), Chemical Computing Group, Montreal Canada [33]. There are 160 lipid molecules and one aromatase molecule in a total of about 12,000 heavy atoms in the system.
Characterization method of aromatase flexibility
Root mean square fluctuation (RMSF) was used to characterize protein flexibility. The mean square fluctuation (MSF) of the ith node, Sx i T 2 , could be determined from the normal modes [13,16] where k B is the Boltzmann constant, T the system temperature, m i the mass of the ith node, U ij the eigenvector of the ith node with the frequency v j , and n v is the number of modes considered. An accurate evaluation of MSF was achieved from the average of the 100 slowest normal modes. The B factor of each node was calculated using the relationship of and further rescaled by an origin shift and a scale factor multiplier. The former is necessary to account for the contribution from rigid body motion implicit in the X-ray B-factors [14] and the latter is used to match the X-ray data. NMA was carried out with variables such as temperature, atomic mass and potential energy, in reduced units, so that the unit of MSF was also reduced. Here, the flexibility of a node was characterized by the relative RMSF of the node to the mean value of the system, i.e. the computed RMSF was in reference to system of study. For simplification, RMSF used in the paper refers to the relative RMSF unless otherwise noted. The residue RMSF was given as the average over the backbone atoms in all heavy-atom NMA, and as the value for the C a atoms in C a -NMA. Flexibility of a region of interest was depicted by the average of residue-RMSFs over this region. Because MSF is in reduced unit, the calculated B factors (from Eq. 2) were scaled to the experimental B factor data. Prior to scaling, the calculated MSF of each node was reasonably up shifted away from the origin to account for the translational and rotational rigid body motions in the lattice cell.
Calculation of the electrostatic potentials of aromatase oligomer
The software Adaptive Poisson-Boltzmann Solver (APBS) [34] as a plug-in to Pymol [35], was used to calculate the electrostatic potentials of the aromatase dimer and trimer. The coordinate files of these crystallographic oligomers were prepared in the same way as those in NMA. The electrostatic interactions between solutes in solvent media were evaluated by solving the Poisson-Boltzmann equation (PBE) [36], a popular continuum model. The parame-ters, such as grid dimension, length and spacing, etc. were setup in default values as suggested by the program. The calculated electrostatics were visualized with Pymol by mapping them on the van der Waals surface of protein molecules rendered in a color spectrum from red to blue representing the scale from 27kT/e to 7kT/e.
Modeling a hexamer on membrane
The atomic model of aromatase with the N-terminal helix was used to build by collapsing a linear chain of two units of crystallographically related trimers (Fig. 6A) into a circle in which all N-terminal helices have similar orientations (Figs. 6B and C). This was achieved primarily by rotating the each molecule pair roughly 6120u with D-E loop-in-proximal-cavity as the fulcrum as indicated in Fig. 7, such that the N-terminal helices all align on the same side of the hexamer. Some hinge bending and translational adjustments were made as well in order to form a symmetrical hexagon to avoid any steric violation. At the end, however, similar D-E-loop to proximal site contacts as observed in the crystal structure was maintained in all six monomer-to-monomer interfaces. Finally, the hexamer was built on a membrane (20nm|20nm) by the process described above (modeling of aromatase monomer in lipid bilayer). | v3-fos-license |
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} | pes2o/s2orc | Nickel-catalyzed N-terminal Oxidative Deamination in Peptides Containing Histidine at Position 2 Coupled with Sulfite Oxidation*
Peptides containing histidine at position 2 were observed to undergo spontaneous N-terminal oxidative deamination in aqueous solution in the presence of Ni(II), sulfite, and ambient oxygen. The reaction resulted in the formation of a free carbonyl on the N-terminal α-carbon (α-ketoamide) and was catalytic with respect to nickel. This oxidative deamination was confirmed by13C NMR, 1H NMR, mass spectrometry, and chemical tests. No evidence of modification of histidine was found. It was demonstrated that the nickel-dependent N-terminal oxidative deamination also occurred in His-2 peptides using potassium peroxymonosulfate (oxone) as an oxidant. When oxone was used, oxygen was not required for the deamination to proceed. The results suggest that both nickel-catalyzed reactions (sulfite and oxygen, and oxone) produce an imine intermediate that spontaneously hydrolyzes to form the free carbonyl. These findings may provide a physiologically relevant model for oxidative carbonyl formation in vivo, as well as a useful method for producing a site-specific carbonyl on peptides and proteins.
Excessive exposures to both nickel and sulfite are known to produce serious toxic and carcinogenic effects in animals and humans (1)(2)(3)(4). The generation of potentially harmful radicals from sulfite oxidation by transition metal catalysis has been well established (5). Nickel salts are physiologically redoxinactive, and therefore nickel toxicity in vivo is inferred to involve activation by coordination with peptides and proteins. A variety of catalytic oxidative properties of Ni(II)/peptide complexes have been reported in the literature. These include decarboxylation of the Ni(II)-Gly-Gly-His complex in the presence of molecular oxygen (6), the ability of Ni(II)-histidyl peptide complexes to function as Fenton reaction catalysts (7,8), and the ability of the Ni(II)-Gly-Gly-L-His complex to catalyze the oxidation and cleavage of DNA (9,10) and to promote the formation of intermolecular protein cross-links (11,12). Muller et al. (13) and Liang et al. (14) have recently reported that autooxidation of sulfite catalyzed by the Ni(II)-complexed Lys-Gly-His-amide tripeptide can oxidatively damage DNA. They postulate in situ formation of monoperoxysulfate, a strong oxidant, as an active intermediate in the damaging effect.
We have recently reported on the identification of a unique Ni(II) binding site on hemoglobin that, in the presence of monoperoxysulfate, produces N-terminal oxidative deamination, as well as intramolecular cross-linking, both specific to the -globins (15). In the present study, we have employed small model peptides in order to verify the structural assignment of oxidative deamination, as well as to identify the minimal sequence requirements for reaction susceptibility. Additionally, we have tested a system that substitutes sulfite (SO 3 2Ϫ ) and oxygen (O 2 ) for potassium peroxymonosulfate (oxone). 1 Our findings show clearly that histidine at position 2 is a fundamental requirement for Ni(II)-catalyzed oxidative Nterminal deamination and that sulfite and ambient oxygen can readily substitute for the potent peroxyl oxidant oxone. Furthermore, by contrasting some of the differences between products produced by the sulfite/oxygen and the oxone reactions with Ni(II) peptides, we can exclude the formation of diffusible monoperoxysulfate (HSO 5 Ϫ ) as a mediator in SO 3 2Ϫ /O 2 -promoted deamination.
Sulfite Reaction Standard Conditions-Unless otherwise noted, reaction mixtures contained 30 mM peptide and 5 mM nickel chloride, to which sodium sulfite was added from a 10ϫ stock to a final concentration of 60 mM, all in 0.1 M sodium phosphate, pH 8.2. 300-l reactions were maintained at room temperature for 24 h under ambient air in 16 ϫ 100-mm glass test tubes protected from light. Reactions were quenched by addition of 1: 10 (v/v) of 0.5 M EDTA, pH 8, and then diluted 1:10 into 2% formic acid in water and placed at 4°C until analysis by reversed phase chromatography.
Oxone Reaction Standard Conditions-Initial reaction mixtures contained 30 mM peptide and 30 mM nickel chloride in 0.1 M sodium phosphate, pH 8.2, to which oxone, also in 0.1 M sodium phosphate, pH 8.2, was added as a 10ϫ stock to a final concentration of 30 mM. After approximately 5 min at room temperature, the oxone reactions were quenched by the addition of (at 1:10 (v/v)) 0.5 M EDTA, pH 8, and then diluted 1:10 into 2% formic acid in water and placed at 4°C until analysis by reversed phase chromatography.
With time, optimized conditions for the oxone reactions were established. Where noted as optimized oxone conditions, the peptides, NiCl 2 , and oxone stocks were made up in 0.2 M sodium bicarbonate, pH 8.2. Additionally, under optimized conditions, the oxone was added in five equal increments 2 min apart, rather than as a single bolus.
Amino Acid Analysis-Peptides were subjected to gas phase hydrolysis at 165°C for 1 h in the presence of HCl containing 1% phenol, using a Savant AP100AminoPrep Hydrolyzer. Amino acids were analyzed using precolumn derivatization with AQC as described previously (16).
Derivatization with 2,4,-Dinitrophenylhydrazine-Derivatizations were performed as described previously (15). * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
LC-MS-Electrospray mass spectrometry was performed using a Finnigan Mat LCQ TM ion trap mass spectrometer interfaced with an HP1090M HPLC. The LCQ™ spectrometer was calibrated as per the manufacturer's recommendations, which provide for a specified accuracy of 0.01% for m/z masses of 100 -2000 Da. Peptides were separated at room temperature using a Zorbax 300SB C18 column with a gradient of 1-40% acetonitrile in 0.1% trifluoroacetic acid/water (v/v) in 20 min at 1 ml/min, following an initial 4-min isocratic (1%) hold.
NMR Analysis-1 H and 13 C NMR spectra were collected on a Varian VXR-300S instrument. Peptides were dissolved in either deuterium oxide or deutero-Me 2 SO. Chemical shifts are reported relative to DSS for deuterium oxide samples or tetramethylsilane for deutero-Me 2 SO samples. 1 H chemical shift versus pH studies were performed using a D 2 O-phosphate system. No corrections of pH measurement for D 2 O content were made.
Reaction of the Ni(II)-complexed Tripeptide Ala-His-Ala with
Peroxymonosulfate Produces N-terminal Oxidative Deamination-Reaction of the tripeptide Ala-His-Ala with Ni(II) and oxone under initial standard conditions resulted in Ͼ60% modification of the peptide. The modified peptide eluted as a broad, asymmetric peak several minutes later than the parent peptide peak when analyzed by reversed phase C18 HPLC (Fig. 1, A and B). The modified peptide displayed significantly increased absorption at 245 nm. Also, the modified peptide displayed a mass of 296.1 Da when analyzed by in-line mass spectrometry. This represents a discrete loss of 1 Da when compared with the parent peptide (296.1 versus 297.1 Da for the parent peptide). MS 2 fragmentation mapped the 1-Da loss to the A 2 , B 2 , and B 3 fragment ions (Fig. 2, A and B). Y-type ions were not detected in either case. The ion of m/z 227, which can be attributed to Y 2 structure, was instead assigned to loss of the C-terminal Ala residue from the AHA tripeptide (17). Ion fragment assignments were confirmed by MS 3 . A significant recovery of the histidine immonium fragment ion was observed (of m/z 110), which further indicated that the histidine residue was unaffected. These findings all suggested that the 1-Da mass loss was located on the the N-terminal alanine, and the histidine residue remained unchanged.
The modified peptide was blocked to Edman protein sequencing, suggesting that a chemical modification had occurred at the N terminus. This finding was corroborated by a lack of reactivity with AQC reagent, which was used to probe for the presence of primary or secondary amines. Amino acid analysis of the modified peptide showed approximately one-half of the yield of alanine relative to histidine that was observed for the parent peptide. Also in contrast to the parent peptide peak, the modified peptide peak reacted quantitatively with DNP, yielding a derivatized peptide with 400 nm absorbance and mass of 476.1 Da (Fig. 3). This represents a mass gain of 180 Da, which is characteristic of a dinitrophenylhydrazone product and therefore indicative of the presence of a free carbonyl on the modified peptide. The presence of a free carbonyl, the apparent loss of free amino group reactivity, and the 1-Da mass decrease all indicated that the modified peptide was oxidatively deaminated at the N terminus, thus resulting in an ␣-ketoamide peptide (Reaction I). 1 H and 13 C NMR analyses of the modified peptide peak were used to further verify this structural assignment. The 1 H spectrum of the (putative) oxidatively deaminated peptide displayed a shift of the N-terminal alanine methyl group downfield from 1.23 to 2.32 ppm along with a resonance splitting change from a doublet to a singlet, consistent with formation of a ketone at the ␣-carbon of Ala-1 ( Table I). The C-1 vinyl proton of the histidine imidazole ring was observed to be significantly deshielded relative to the parent peptide. However, an additional study that examined the effect of pH versus 1 H chemical shift of the histidine C-2 vinyl proton demonstrated no significant change in pK a of the imidazole group following Ni(II)/ oxone oxidation, yielding further evidence that the histidine side chain was not modified in the reaction (Fig. 4). 13 C NMR also indicated the presence of an intact histidine imidazole ring, two amide carbons, one carboxylic acid carbon, and a downfield ketone carbonyl resonance at 196 ppm, all in agreement with oxidative deamination (Table II).
Sodium cyanoborohydride treatment of the modified peptide peak yielded quantitative reduction of the Ni(II)/oxone-oxidized AHA peptide, resulting in a species that eluted earlier in the reversed phase gradient as a doublet of two apparent diastereomers, both exhibiting mass gains of 2 Da (Fig. 1C). These peaks were structurally assigned as diastereomeric al-cohols derived from reduction of the ␣-keto carbonyl. MS 2 fragmentation ( Fig. 2C), as well as 1 H NMR analysis of these peaks, confirmed this assignment.
AQC derivatization of the AHA deamination reaction mixture analyzed by LC-MS yielded evidence of free ammonia released in the reaction. The amount detected was commensurate with the amount of peptide oxidatively modified, showing that deamination was concomitant with the proportional release of free ammonia.
The same oxone reaction on the AHA tripeptide in the absence of added Ni(II) was tested. It was found that when Ni(II) was left out of the reaction, under otherwise identical conditions, about 50% of the peptide was modified. The modified species exhibited an increased absorbance at 250 nm and eluted during the reversed phase HPLC gradient as a sharp, symmetrical peak later than either the parent peptide or the oxidatively deaminated species (Fig. 1D). LC-MS analysis of this peak showed a mass gain of 14 Da over the parent peptide mass (311.1 versus 297.1 Da for the parent). This modified peak was found to be blocked to Edman chemistry sequencing, suggesting that a chemical modification had occurred at the N terminus. In contrast to the oxidatively deaminated AHA peptide peak, this modified peptide peak was unreactive with DNP, suggesting the absence of any free carbonyl group. Fig. 1C; D, modified peptide from oxone reaction without Ni(II) (oxime). Annotation of fragment ions refers to parent peptide. and B 3 fragment ions (Fig. 2D). From these findings, we postulated that this modification was likely the result of chemical oxidation of the terminal amine nitrogen to an oxime (18). 1 H proton NMR analysis confirmed this assignment. The N-terminal methyl resonance was shifted downfield from 1.23 to 1.85 ppm and resonated as a singlet. Also observed was the appearance of a single, sharp resonance far downfield at 11.86 ppm, which is consistent with an oxime proton.
Reaction of the Ni(II)-complexed Tripeptide Ala-His-Ala with Sulfite and Oxygen Produces Identical N-terminal Oxidative
Deamination-Muller et al. (13) have postulated the in situ formation of HSO 5 Ϫ from SO 3 2Ϫ in the presence of ambient oxygen, Ni(II), and the tripeptide Lys-Gly-His-amide. Although Ni(II) complexes of this sequence motif have been reported to exhibit catalytic properties different from that of histidine at position 2 (7), we decided to test for indications of peroxymonosulfate formation from sulfite autooxidation on a peptide with histidine at position 2.
The tripeptide Ala-His-Ala was incubated with sodium sulfite in the presence of nickel under the standard conditions described above. This treatment was observed to produce modification of Ͼ60% of the peptide. The modified species displayed the same elution time, 1-Da mass loss, and MS 2 fragmentation pattern by LC-MS analysis as the oxidatively deaminated ␣-ketoamide species produced using oxone. Additional chemical tests, including Edman sequencing, DNP, and AQC reactivity, as described above for the modified product of AHA using Ni(II) and oxone (excepting proton NMR analysis), confirmed that an identical oxidatively deaminated AHA peptide was produced using Ni(II), sulfite, and ambient oxygen.
The sulfite reaction with AHA in the absence of added nickel was tested. It was found that in contrast to the oxone reaction, no oxime formation was observed. In fact, when nickel was omitted from the reaction of the AHA tripeptide with sulfite, no detectable modification of any kind was observed.
Reaction of the Ni(II)-complexed Tripeptide Ala-Ala-Ala Shows No Evidence of Oxidative Deamination with Either SO 3 2Ϫ /O 2 or Oxone-We have previously postulated that histidine at position 2 is an essential part of an oxidatively reactive Ni(II) binding site on the -chain of human hemoglobin (15). Therefore, as a comparison with the AHA peptide, we examined the reactivity of the tripeptide AAA. Reaction of the AAA tripeptide with Ni(II) and oxone under standard conditions also resulted in Ͼ60% modification of the peptide. This modified peptide eluted primarily as a sharp, symmetrical peak several minutes later than the unmodified parent peptide peak when analyzed by reversed phase C18 HPLC (Fig. 5, A and B). The modified peptide displayed increased absorption at 250 nm and a discrete mass gain of 14 Da when analyzed by LC-MS compared with the parent peptide (245.1 versus 231.1 Da for the parent peptide.) This species was found to be blocked to Edman sequencing. MS 2 fragmentation also indicated that the modification was located on the N-terminal amino acid. Based on these findings, we concluded that this modification was an oxime on the N-terminal nitrogen. When nickel was omitted from the oxone reaction with the Ala-Ala-Ala tripeptide, an identical product was observed (Fig. 5C). Therefore, oxime formation appears to be entirely non-nickel-dependent.
Reaction of the tripeptide Ala-Ala-Ala with Ni(II) and sodium sulfite under standard (SO 3 2Ϫ ) conditions produced no detectable modification of any kind. This finding suggests that in the case of the Ala-Ala-Ala tripeptide, no peroxymonosulfate-like species is generated by that peptide in the presence of Ni(II), SO 3 2Ϫ , and ambient oxygen. Histidine at position 2 therefore appears to be required for both susceptibility to oxidative deamination, as well as the apparent ability to generate a peroxymonosulfate-like species from sulfite and oxygen.
Sequence tions of Ni(II) and a series of small peptides with either SO 3 2Ϫ / O 2 or oxone as the oxidant. We examined the reactions for any evidence of modification of the parent peptide. The results of these experiments are shown in Table III.
Almost all of the peptides with histidine at position 2 demonstrated significant susceptibility to oxidative deamination under both sets of conditions, whereas none of the peptides lacking histidine at position 2 demonstrated any oxidative deamination under either condition. It should be noted that in the cases of both AHA and GHG, a significant amount of the deaminated product from reaction at 30 mM peptide was found to be in the form of apparently diastereomeric pairs of relatively stable dimers (exhibiting a mass of 592 Da in the case of AHA peptide). These dimers were not observed when the SO 3 2Ϫ / O 2 reaction was performed at a peptide concentration of 3 mM, indicating that their formation was (peptide) concentration-dependent. Further characterization of the dimer by 1 H NMR indicated it to be a product of aldol condensation of two molecules of ␣-ketoamide (data not shown). These dimers were not observed when oxone was used as the oxidant.
Unlike Ala-His, the dipeptides carnosine (-alanyl-histidine) and homocarnosine (␥-amino butyryl-histidine) were found to be nonreactive to Ni(II)/SO 3 2Ϫ /O 2 -promoted deamination. These results suggest that only ␣-amines are susceptible to this pathway of deamination. Interestingly, these peptides showed very little oxime formation in the reaction with oxone.
Peptides with proline as the first residue also produced noticeably different results. N-terminal proline, being a secondary amine, is not likely to be as susceptible to the oxidative release of ammonia as other amino acids. No released ammonia in either reaction with PHA was detected, and in both cases, a modified peptide that was formed demonstrated a loss of 2 Da (321 versus 323 Da for the parent peptide). The modified peptide was unreactive with AQC, indicating that the secondary amine was no longer present. This result would be consistent with formation of a cyclic imine species that was stable against spontaneous hydrolysis.
Peptides GGH and KGH-amide with a histidine residue in position 3 were also tested. Under similar conditions, these peptides have been shown by Muller et al. (13) and others (12,14,19) to produce radicals capable of causing DNA damage and protein cross-linking. No deamination of either the GGH or the KGH-amide peptides were detected in reactions with Ni(II)/ SO 3 2Ϫ /O 2 . The KGH-amide peptide was modified in the reaction to an earlier eluting product exhibiting a mass gain of 80 Da, consistent with the displacement of one proton by an SO 3 H group. This product was not further characterized. In the reactions with oxone, decarboxylation of GGH and other peptide degradation of both peptides were observed, none of which appeared to include deamination. By contrast, the tripeptide KHG-amide (bursin) appeared to readily N-terminally deaminate with both Ni(II)/SO 3 2Ϫ /O 2 and Ni(II)/oxone. However, the ketoamide product appeared to spontaneously form an intramolecular Schiff's base with the ⑀-amine of the lysyl side chain (Reaction II). This was confirmed by LC-MS, Edman sequencing, and reaction with DNP. Under these conditions, no evidence of ⑀-amino side chain deamination was found.
Peptides with methionine and tyrosine as the N-terminal residues also appeared to present exceptions to susceptibility to oxidative deamination. The lack of reactivity of MH using sulfite is possibly due to sulfoxide formation acting as a "sink" for oxidative modification, although no substantial difference in the amount of methionine sulfoxide formed was seen relative to ML with either oxidant (20,21). It is also conceivable that other chemical and electronic properties of the sulfur atom of the methionine side chain could affect the reaction pathway. The lack of deamination of the dipeptide YH is less clear. Stable tyrosine radicals have been reported in proteins (22,23). However, no evidence of dityrosine, a known product of oxidative degradation of tyrosine involved in protein cross-linking, was detected (12,22), and no evidence of tyrosine radical was found by EPR or found to be trappable by the method of Fenwick and English (24). However, with an increased excess of sulfite, we noticed significant accumulation of a product of the YH reac-REACTION II REACTION III tion, which exhibited a mass of 354 Da (gain of 36 Da). Under standard conditions, except using 4:1 ratio of sulfite over peptide, the reaction resulted in a 30% yield of this modification. Mass gain, MS 2 fragmentation, and Edman protein sequencing were all consistent with C-terminal decarboxylation followed by the attachment of the SO 3 H group forming a sulfo-peptide (Reaction III). We believe that such a product would most likely be formed through a radical type of reaction, potentially promoted by the tyrosine side chain (25). Analogous products were detectable with other first residue side chains in dipeptides containing His-2, but with yields at least 1 order of magnitude lower. The YH sulfo-peptide product was not observed when Ni(II) was omitted from the reaction. The Ni(II)/oxone reaction with YH produced a small but detectable yield of oxidative deamination. Additionally, a significant amount of other uncharacterized degradation occurred including formation of a yellow precipitate.
In all cases, oxone produced a significant yield of oxime in peptides lacking histidine at position 2. This finding provided us with a means to look for evidence of the generation of a diffusible peroxymonosulfate species from peptide/Ni(II) reac-tions with sulfite. This could be tested by including the AAA tripeptide in reactions with His-2-containing peptides and monitoring for oxime formation. These conditions resulted in substantial deamination of the susceptible peptides, as was seen with oxone addition. No trace of oxime formation was detectable on the AAA tripeptide when it was combined with the LH dipeptide during standard Ni(II)/SO 3 2Ϫ /O 2 conditions. Therefore, it can be inferred that the oxidative deamination reaction did not generate any appreciable quantities of diffusible peroxymonosulfate. However, in the case of all His-2 peptides, an in situ formation of a peroxymonosulfate-like species, which is either constrained from diffusing or consumed in a subsequent reaction too quickly to do so, is plausible.
Nickel Titration Studies of LH Dipeptide Reacted with Ni(II) and Either SO 3 2Ϫ /O 2 or
Oxone-A nickel titration study of the SO 3 2Ϫ /O 2 reaction was conducted using the dipeptide LH under standard conditions. The results showed that in the absence of nickel, no oxidative deamination occurred. At a Ni(II) to peptide ratio of 1:10, about 40% of the parent peptide was deami- nated (i.e. 4 equivalents of deaminated peptide per 1 equivalent of Ni(II)), indicating a catalytic turnover of 4. Using a 1:10 ratio of nickel, and 4 equivalents of sulfite, the catalytic turnover of Ni(II) increased to about 8.5. Increasing the Ni(II)/peptide ratio above 1:5 did not have a significant effect on the yield of deamination (Fig. 6). Therefore, a 1:5 ratio of Ni(II):peptide was selected as an optimal for the standard deamination reaction with sulfite.
A similar Ni(II) titration study using the LH dipeptide was conducted with oxone. In contrast to the SO 3 2Ϫ /O 2 system, under initial conditions, maximal oxidative deamination required nearly 1:1 Ni(II):peptide when oxone was used. However, under optimized conditions for the oxone reaction, the oxone was added to the reaction in five equal increments, rather than as a single bolus. In this case, 30% deamination was achieved using approximately 1:5 Ni:peptide (catalytic turnover of 1.5), suggesting that redox cycling of Ni(II) can be achieved in the oxone reaction as well (Fig. 6). Chromatograms of this "optimized" oxone reaction, along with a standard condition sulfite reaction, both with the LH dipeptide at 1:5 Ni(II):peptide, are shown in Fig. 7. It can be seen from these that the sulfite reaction results in significantly decreased degradative side products compared with oxone. It appeared that when nickel was limiting, oxime formation was a potentially competing side reaction. We therefore purified the LH oxime species and rereacted it with Ni(II) and oxone under standard conditions. No evidence of oxidative deamination was observed, although significant heterogeneous, uncharacterized peptide degradation occurred. This finding suggested that oxime formation may be a competitive and kinetically relevant "dead-end" side reaction when Ni(II) is limited during reaction with oxone.
Reaction Kinetics for Oxidative Deamination of LH Dipeptide Reacted with Ni(II) and Either SO 3 2Ϫ /O 2 or Oxone-Studies were conducted to examine the kinetics of oxidative deamination of the LH dipeptide comparing the use of either SO 3 2Ϫ /O 2 or oxone (with sequential addition) as oxidants (Fig. 8). As can be seen, the oxidative deamination using oxone as the oxidant was completed within (at most) several minutes of initiation. By contrast, the reaction using SO 3 2Ϫ and ambient oxygen displayed a distinctly lower initial rate of deamination, which appeared to be relatively constant during the first 40 min of reaction. No further increase in deamination was observed beyond 6 h (up to 1 week). When this reaction was repeated under an oxygen-enriched head space (Ͼ95% O 2 at approximately 2 atm), no difference in the rate of peptide modification was observed. This finding suggested that oxygen diffusion is not rate-limiting under the standard conditions with sulfite. However, when oxygen was strictly excluded from the sulfite reaction (under argon), no peptide modification was observed. By contrast, exclusion of oxygen from the oxone reaction did not inhibit oxidative deamination.
Quenching and Inhibition Studies of Deamination of LH Dipeptide Reacted with Ni(II) and Either Sulfite or Oxone-
Further studies were conducted to examine the effect of the addition of various compounds on the yields of oxidative modification in the LH/Ni(II) reactions using either sulfite or oxone. These additives included known chelating agents, radical scavengers, and different anions. The results of these experiments tabulated as percentage of inhibition of LH deamination under the respective standard conditions are listed in Table IV. The lack of inhibition observed in the presence of ethanol, methanol, tert-butyl alcohol, or mannitol argues strongly against the involvement of diffusible SO 4 . or OH ⅐ radicals me- diating the oxidative deamination in either reaction (2,26). The inability of catalase to inhibit either reaction suggests that it is unlikely that oxidative deamination in either case proceeds through formation of diffusible H 2 O 2 followed by a Fenton-type reaction, as in a mechanistic scenario for oxidative protein carbonyl formation proposed by Stadtman (27). However, no test was conducted to determine whether this enzyme was inactivated by the reaction conditions, as has been reported for sulfite inactivation of several copper-dependent monooxygenases (28). The complete inhibition of oxidative deamination by the metal chelating agent EDTA would suggest that sequestration of nickel away from the peptide is the mode of action by which it precludes the reactions. The mechanism for inhibition exhibited by thiourea is less clear, but the differential effects of CN Ϫ and HCO 3 Ϫ on reactions using SO 3 2Ϫ /O 2 versus using
HSO 5
Ϫ begins to suggest that these may act by perturbing the coordination structure of the reactive nickel complexes (29). Such differential effects also begin to suggest that the reactive complex that produces an actively oxidizing species from SO 3 2Ϫ may differ from the one involved in the reaction with preformed HSO 5 Ϫ (oxone). Comparative Sulfite and Oxone Titration Studies with Ni(II)complexed LH Dipeptide-Titration studies under standard conditions using oxone as the oxidant showed that approximately 1:1 oxone:peptide was sufficient to approach maximal deamination of the LH dipeptide (Fig. 9). It was also observed that Ն2:1 oxone:peptide resulted in significant, heterogeneous, uncharacterized degradation of the peptide (much worse than the 1:1 shown in Fig. 7). By contrast, titration with SO 3 2Ϫ under standard conditions reproducibly showed that significantly more than 1 equivalent of SO 3 2Ϫ to peptide was required to approach maximal deamination. With 3 equivalents of sulfite, more than 60% of the peptide was oxidatively deaminated, with less than 20% of degradative side-products observed. pH
Effect Studies of LH Dipeptide Reacted with Ni(II) and Either Sulfite/O 2 or Oxone Show High Relative Yields of Oxidative Deamination throughout a Physiological pH Range-
Reactions with the LH dipeptide/Ni(II) system using oxone or sulfite/O 2 were examined for effects of reaction pH, the results of which are shown in Fig. 10. Although the oxone reaction showed inhibition below pH 6.5, both reactions displayed a high relative yield of oxidative deamination over the physiologically relevant pH range of 6.5-8.5. This finding lends support to the possibility that this model system for oxidative peptide modification may be relevant to mechanisms of nickel and sulfite toxicities in vivo (1, 3).
Conclusions-We have shown that the reaction of peptides containing histidine at position 2 with Ni(II) and oxone produces oxidative deamination of the peptides with a minimum of other significant modifications of the peptide, and we found no evidence of modification of the histidine residue (up to 1 equivalent of oxone, above which other degradative modifications occur). When Ni(II) was omitted from the reaction, oxime formation on the N-terminal nitrogen was produced instead. In peptides lacking histidine at position 2, only oxime formation was observed, regardless of whether Ni(II) was present or not.
The identical oxidative deamination of peptides containing His-2 can be produced by using Ni(II), sulfite, and ambient oxygen. A substoichiometric amount of nickel can serve to deaminate the peptides in high yields, indicative of redox cycling of Ni(II) in the reaction. However, when either nickel, sulfite, or oxygen was excluded, no oxidative deamination or any other modification was observed. No oxidative deamination was observed in any peptides lacking histidine at position 2. Significant modifications other than deamination were observed in reactions with both GGH and KGH-amide peptides. These peptides have been reported to promote protein cross- linking and DNA damage under similar conditions. Additionally, the GGH peptide is known to undergo oxidative degradation, including decarboxylation in the presence of ambient oxygen or other oxidizers (6,14,30). These results, along with our finding of oxidative deamination only on ␣-carbon amines in His-2 peptides, indicate a unique structural specificity for a sequence motif that was first discovered in the -chain of human hemoglobin (15).
The finding that both sulfite/O 2 and oxone produce an identical ␣-ketoamide suggests that both pathways may share a common reactive intermediate. The Ni(II)-catalyzed formation of peroxymonosulfate from sulfite and oxygen would appear to be consistent with these results (13). However, co-incubation of peptides lacking histidine at position 2 exposed to the Ni(II), sulfite, and oxygen system showed no evidence of oxime formation, suggesting that no diffusible peroxymonosulfate is produced during the deamination reaction. Additional quenching studies using well characterized radical scavengers appear to rule out diffusible OH ⅐ or SO 4 . as mediating any of the oxidative deamination observed (2,26). We postulate that the reactions of Ni(II) complexed His-2 peptides with either oxone or an in situ formed, nondiffusing, peroxymonosulfate-like species generated catalytically by sulfite autooxidation produce a common imine intermediate. All of our results would be consistent with a pathway involving imine formation at the N terminus, followed by spontaneous hydrolysis (with the exception of proline, which apparently forms a stable cyclic imine). The finding of ammonia release in all cases of oxidative deamination is consistent with such a pathway. This postulated pathway is shown in Fig. 11. Imine formation in oxidative deamination has been postulated by Stadtman (27) to be preceded by radical formation on the carbon adjacent to the departing amine group. If such is the case for Ni(II)-catalyzed N-terminal oxidative deamination, the radical appears inaccessible to quenching by common radical scavengers, i.e. a type of "caged" radical. Results with YH dipeptide suggest that the (tyrosyl) side chain can redirect the outcome of the reaction of such a complexed radical.
These findings are of interest from several perspectives. They provide a defined, potentially physiologically relevant model system with which to study the mechanism of a metalcatalyzed protein carbonyl formation, a modification known to be a frequent consequence of oxidative damage to proteins in vivo (31,32). Such a defined system may provide an opportunity for further elucidating mechanisms underlying the known toxicities of sulfite, a ubiquitous environmental contaminant (2,3). Additionally, our findings point toward a facile method for the intentional introduction of a carbonyl "handle" into natural and engineered proteins and peptides, under conditions that produce relatively few undesirable side reactions, a technology of significant potential utility (33,34). | v3-fos-license |
2018-04-03T02:49:32.786Z | 2011-04-07T00:00:00.000 | 35150774 | {
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} | pes2o/s2orc | Redetermined structure of diphenylphosphonimidotriphenylphosphorane: location of the hydrogen atoms and analysis of the intermolecular interactions
The title compound, C30H25NOP2, is a bulky phosphazene derivative. Its previous crystal structure [Cameron et al. (1979 ▶). Acta Cryst. B35, 1373–1377] is confirmed and its H atoms have been located in the present study. The formal P=N double bond is about 0.05 Å shorter than the P—N single bond and the large P=N—P bond angle reflects the steric strain in the molecule. An intramolecular C—H⋯O interaction occurs. In the crystal, short C—H⋯O contacts connect the molecules into chains propagating in [011], which are cross-linked via C—H⋯π interactions, generating a three-dimensional network. Aromatic π–π stacking also occurs [shortest centroid–centroid separation = 3.6012 (11) Å].
The title compound, C 30 H 25 NOP 2 , is a bulky phosphazene derivative. Its previous crystal structure [Cameron et al. (1979). Acta Cryst. B35, 1373-1377] is confirmed and its H atoms have been located in the present study. The formal P N double bond is about 0.05 Å shorter than the P-N single bond and the large P N-P bond angle reflects the steric strain in the molecule. An intramolecular C-HÁ Á ÁO interaction occurs. In the crystal, short C-HÁ Á ÁO contacts connect the molecules into chains propagating in [011], which are cross-linked via C-HÁ Á Á interactions, generating a threedimensional network. Aromaticstacking also occurs [shortest centroid-centroid separation = 3.6012 (11) Å ].
Redetermined structure of diphenylphosphonimidotriphenylphosphorane: location of the hydrogen atoms and analysis of the intermolecular interactions R. Betz, T. Gerber, E. Hosten and H. Schalekamp Comment For many main group elements as well as transition and rare earth metals, preferred coordination numbers in coordination compounds are apparent. While coordination numbers of 4, 6 and 8 have been found to be dominant in most cases and, as a consequence, vast structural information has been collected for such compounds in solution and in the solid state, information about other coordination numbers is comparatively limited. Especially for smaller coordination numbers the literature is scant or hitherto completely unknown for many elements. One reason for this certainly is that sometimes challenging synthesis procedures have to be followed and, thus, a general but simple synthetic protocol is desireable. Since such compounds may act as versatile and potent catalysts in many industrial processes and might even show interesting pharmacological properties, we were interested in developing an easy-access-route for their synthesis. Applying bulky ligands might open up a pathway in this aspect. In order to be able to compare metrical parameters in envisioned reaction products, we determined the crystal structure of the title compound. The latter one has already been reported earlier (Cameron et al. (1979)), however, no hydrogen atoms were included in the refinement thus ruling out the possibility to assess the role of C-H···X contacts.
The length of the N-P bonds deviate by 0.05 Å with the -formal -P-N-double bond found at around 1.55 Å. The P-N-P angle was measured at more than 146 °. The marked widening of this angle in comparison to the value expected for a sp 2 -hybridized nitrogen atom can be explained by the repulsive interaction of the phenyl-moieties on both P atoms. The phenyl groups on each phosphorus atom are approximately orientated perpendicular to each other. The least-squares planes defined by their carbon atoms intersect at an angle of 82.19 (6) ° in case of the P(O)Ph 2 -moiety and at angles of 79.82 (5)°, 80.91 (6) ° and 83.28 (6) °, respectively, in case of the PPh 3 -moiety. Due to the formation of an intramolecular C-H···O contact (see below), the least-squares plane defined by the P(O)-N-P motif encloses an angle of only 29.40 (9) ° with one of the aromatic carbocycles on the PPh 3 -moiety ( Fig. 1). For the same reason, both phenyl groups of the P(O)Ph 2 -moiety adopt a slightly ecliptic conformation with respect to the P(O) motif, the respective dihedral angles were found at about 19 ° and 26 °.
In the crystal structure, intermolecular C-H···O contacts are present whose range falls by more than 0.3 Å below the sum of van-der-Waals radii of the atoms participating. These can be observed between one of the H atoms in meta-position of a phenyl group on the PPh 3 -moiety and the O atom of the P(O)Ph 2 -moiety and connect the molecules to infinte chains along [0 1 1] (Fig. 2). Furthermore, intramolecular C-H···O contacts invariably involving hydrogen atoms in ortho-position on one of the phenyl groups of the PPh 3 -moiety as well as both phenyl groups of the P(O)Ph 2 -moiety are present. However, the latter two ones are not very pronounced. Additionally, a set of C-H···π contacts are evident involving H atoms and aromatic systems both on the PPh 3 -moiety as well as the P(O)Ph 2 -moiety. Their details are listed in Table 1 (with C g (1) = C41···C46, C g (2) = C31···C36 and C g (3) = C11···C16). In total, the C-H···O contacts as well as the C-H···π contacts connect the molecules to a three dimensional network. In terms of graph-set analysis (Etter et al. (1990); Bernstein et al. (1995)), the intermolecular C-H···O contacts can be assigned a C 1 1 (8) descriptor on the unitary level while the intramolecular C-H···O supplementary materials sup-2 contact involving the phenyl group of the PPh 3 -moiety necessitates a S 1 1 (7) descriptor. For the other two intramolecular C-H···O contacts, a S 1 1 (5) each is feasible. An analysis of C g ···C g interactions shows the closest distance between two centers of gravity to occur between a phenyl group on the PPh 3 -moiety and a phenyl group on the P(O)Ph 2 -moiety. The distance was measured at 3.6012 (11) Å.
The packing of the title compound in the crystal structure is shown in Figure 3.
Experimental
The compound was obtained commercially (Aldrich). A colourles block suitable for the X-ray diffraction study were taken directly from the provided material. | v3-fos-license |
2017-08-29T10:42:43.117Z | 2013-05-29T00:00:00.000 | 37719644 | {
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} | pes2o/s2orc | Neuroblastoma and Copper : A complex relationship Neuroblastoma
Copper is a trace metal essential to the catalysis of a wide range of enzymatic activities, including those involved in the process of energy production (cytochrome c oxidase), the cell response to oxidant injuries (Cu,Zn-superoxide dismutase), the catecholamine (dopamine βmonooxygenase) and melanin (tyrosinase) production, the remodelling of extracellular matrix (lysyl oxidase), blood clotting processes (Factors V and VIII) and iron metabolism (ceruloplas‐ min and hephaestin) [1]. The catalytic properties of copper are linked to its ability to easily assume the oxidized (Cu2+) and reduced (Cu+) states, but just the metal reactive behaviour can trigger severe cell alterations through the generation of hydroxyl radicals in Fenton-like reactions [2,3]. When the cytosolic copper concentration is above the optimal level, the newly formed reactive oxygen species (ROS) rapidly bind to DNA, thus inducing the breaking of the nucleic acid strands and initiating a series of cascade events that can lead to significant damage to cell structures and function [4].
i.
cancer energy metabolism ii.
tumour vascularization Copper transport systems are gaining growing importance in the studies about the various aspects of the metal role in neuroblastoma, so the peculiar expression pattern will be described before discussing the pathological topics.
Copper transport systems in neuroblastoma cells: Regulation and physiopathological implications
Copper critically regulates the degree of neuroblastoma growth and microvascularization, which determines the tumour aggressive phenotype [19,20]. The importance of this metal is emphasized by the strong presence of specific transport proteins in neuroblastoma cells, that testifies to a lively management of tumour copper stores. Highly variegated mechanisms of regulation of copper homeostasis have been specifically reported for neuroblastoma (some of them reviewed here), that make it difficult to establish the nature of copper involvement: is the ion metabolic disruption a cause or an effect?
Copper import. It is widely believed that copper import in neuroblastoma cells is mediated by hCtr1 [21]. However, recent work from our laboratory in an in vitro neuroblastoma cell model has enlightened a role for the cellular prion protein PrP C in mediating the high affinity copper intake, upon normal metal availability [22]. In addition, we demonstrated that copper shortness induces an up-regulation of PrP C expression in a neuroblastoma cell model, a cell adaptive strategy aimed at restoring the standard copper status [23].
In support of its involvement in tumorigenesis, the PrP C expression is up-regulated in nervous tissues affected by hypoxia, a condition typically occurring during the growth of a solid tumour [24]. The reader is referred to paragraphs 4.2 and 6.2 for a detailed account of the PrP C functions in the tumour spread.
Copper efflux. The ATP7A copper ATPase (full length 170 KDa protein) is strongly expressed by neuroblastoma cell lines [21,23] and subjected to an articulated copper-dependent regulation.
In many cell types this efflux pump delivers copper to the secretory compartments and, when copper should accumulate inside the cytosol, it traffics toward the cell periphery to export the ion excess [25]. However, peculiar regulative mechanisms have been documented in neuroblastoma models.
In fact, it has been demonstrated in the M17 neuroblastoma cell line that fluctuating copper levels (excess/starvation) in the cell microenvironment can favour the interaction of ATP7A proteins with clusterins (apolipoprotein J), the last ones targeting the pumps toward degradation through the lysosomal pathway [26]. This copper-regulated clusterin function may have multiple implications, if we consider that a recent study on neuroblastoma cell lines, mouse models, and human specimens evidenced that this molecular chaperon behaves as a tumour and metastasis suppressor, negatively regulated by N-myc in the most aggressive forms [27].
In our opinion, the copper-clusterin link deserves further exploration in the light of the reported elevation in copper neuroblastoma content observed in N-myc amplified tumours.
If N-myc really down-regulates clusterin (still controversial aspect), one would expect an increase in copper export function and so an overall reduction of the ion cancerogenic action.
This evidently contradicts the N-myc -tumour malignancy binomial association (where copper should exert a prominent role) and minimizes the contribution of clusterin to the copperdependent tumour progression. In fact, considering that N-myc elevates the neuroblastoma copper content, one can suppose that the cytosolic copper lowering due to a down-regulated expression of clusterin is overridden by other cell mechanisms causing the increase of cancer copper levels.
Coming back to main focus of this paragraph, multiple ATP7A spliced variants can be retrieved in human cells, not necessarily related to disease states, with a cell type-specific expression pattern. The expression of a 11.2 KDa splicing product (103 amino acids) has been reported in SY5Y neuroblastoma cells, harbouring a sequence able to bind copper ions [28]. It has been proposed that such spliced product can work as a copper chaperon to direct the cytosolic copper toward the nuclear compartment.
Intracellular copper distribution. Among copper chaperons, the contribution of COMMD1 (Copper Metabolism MURR1 Domain containing 1) to the copper status in neuroblastoma cells is an unexplored issue so far. However, some inputs from the recent scientific literature let us hypothesize an involvement.
Endogenous COMMD1 expression has been reported in the SH-SY5Y neuroblastoma cell line, together with the isoform 3. A punctate cytoplasmic distribution, denser in the perinuclear region, has been shown for COMMD1, while COMMD3 appears more diffused [29]. The role of COMMD1 in neuroblastoma progression is potentially articulated on multiple levels of action, even if direct demonstrations are missing and the following dissertation aims at enlightening some aspects of copper-dependent regulation of the protein fate.
A role in preventing tumour growth and metastasis has been proposed for COMMD1, based on its ability to repress the NF-KB pathway and the HIF1α/β dimerization and so inhibit the expression of genes involved in tumour angiogenesis [30]. However, as documented in N2a neuroblastoma cell line, upon copper excess, COMMD1 can form a hetero-complex with CCS and SOD1, leading to decreased levels of SOD1 dimers and subsequently reduced anti-oxidant activity [31]. In other words, in the presence of high copper, the COMMD1 cell fate can potentially assume a negative connotation.
COMMD1 is also an interacting partner of ATP7A proteins and, analogously to clusterin, can drive their degradation through a proteasomal pathway [32], this indicating a further contribution of this chaperon to the neuroblastoma copper content. However, knowledge about these aspects is still limited.
The COMMD1 involvement in determining the neuroblastoma copper condition is strictly linked to the protein XIAP (X-linked inhibitor of apoptosis). XIAP protective action is due to the prevention of the activation of a subset of cell death proteases (caspases 3, 7 and 9) [33,34], and inhibiton of Fas- [35] and Bax-induced apoptosis [33].
During the last decade, a role for XIAP in controlling the cell copper homeostasis has been described [36]. In fact, the overexpression of XIAP protein (not transcript) selectively reported in chemotherapy-resistant neuroblastomas, but no other tissues [37], may indicate the occurrence of a particular copper status. XIAP is a copper-binding protein that, in the olo-form, favours the ubiquitination and degradation of COMMD1, that in turn interacts with ATP7A to support copper excretion [36]. Where overexpressed, it is reasonable to presume a subsequent consistent reduction of COMMD1 cytosolic protein levels, and so an increase of the cellular copper content.
However, the binding of copper to XIAP negatively impacts the protein stability, so a negative feedback exists [38]. In the case of chemotherapy-resistant neuroblastomas, the protein overexpression probably overcomes the effects deriving from copper-driven XIAP inactivation.
Preclinical evidences of the importance of XIAP as a target to treat neuroblastoma have been recently collected, all based on the lowering of the threshold for the induction of apoptosis through the depression of XIAP expression. The use of Thymoquinone, a bioactive compound from nigella sativa, has been shown to selectively down-regulate XIAP in neuroblastoma cells, but not in normal neuronal cells, with an expected higher copper efflux [39]. Smac (Second mitochondria-derived activator of caspase) mimetics (e.g. LBW242) have been reported to sensitize chemotherapy resistant and XIAP-overexpressing neuroblastomas, by favouring the degradation of XIAP and TNF-α expression [37].
Copper-dependence of neuroblastoma metabolic changes
The oxygen partial pressure within a solid tumour ranges from 5-10 mmHg in highly vascularized regions to absence (anoxia) around the necrotic areas [40,41]. Most cancer cells tend to adapt to the intra-tumour hypoxic microenvironment by activating a pro-survival signalling, a pro-angiogenic pattern of gene expression and through the metabolic switching from the oxidative phosphorylation to the glycolytic pathways (Warburg effect) [42].
Currently, there is not a homogeneous view on the causative events, but two major factors are usually indicated as responsible, the Hypoxia-Inducible Factors 1 and 2 (HIF1,2), and p53 transcription factor.
HIF-1α and HIF-2α are major actors in the cell adaptive response to hypoxic conditions and control the expression of distinct, but functionally converging genes [45]. Each cell type exhibits a peculiar profile of HIF-1α and 2α expression and their functions may also differ. In the case of neuroblastoma, at a careful analysis of the expression pattern, tumour stage, and copper status, it can be observed that copper heavily influences the response to hypoxia and that the tumour progression and the evolution of copper metabolism go hand in hand.
HIF-1α
HIF-1α, but not HIF-2α, is preferentially expressed and up-regulated by moderate hypoxia in N-myc amplified neuroblastoma cell lines and primary tumours, correlating with a poor prognosis [41,46]. In the light of the linear increase of copper neuroblastoma levels with the degree of N-myc gene amplification and the proven role of Cu 2+ ions in stabilizing the structure of the HIF-1α subunit [47], it can be deduced that copper plays in key role in inducing the neuroblastoma metabolic changes.
Cu 2+ ions determine the structural stabilization of the HIF-1α subunit (oxygen-sensitive) through the inhibition of prolyl-4-hydroxilases, which allow the subsequent ubiquitination and degradation of such factor [47].
Interestingly, by this way Cu 2+ indirectly promotes the synthesis of ceruloplasmin, a plasma and liquor copper chaperon with a ferroxidase activity, whose expression is typically under HIF-1 control [48]. Being ceruloplasmin a major copper vehicle, such mechanism can be interpreted as cancer "self-nourishing". It must be added that HIF-1 target genes also include VEGF (Vascular Endothelial Growth Factor), a recognized chemotactic and mitogen factor [49], and VEGFR-1 (VEGF Receptor-1) [50], both involved in the positive regulation of the sprouting of blood vessels within the primary tumour.
Further, White et al. (2009) demonstrated that the up-regulation of the hypoxia inducible factor HIF-1α causes the selective distribution of copper ions to the secretory pathway. They observed in tumour-associated macrophages that the hypoxic stress can influence the intracellular distribution of copper ions, determining an increased ion entry through the high affinity channel Ctr1 and then an elevated efflux through the ATP7A pump [51].
All these experimental evidences underline the prominent role of copper in sustaining the HIF-1α-dependent adaptation to hypoxia in N-myc amplified neuroblastomas, as well as the hypoxia-stimulated activation of copper transport activities.
HIF-2α
HIF-2α, but not HIF-1α, has been shown to be highly expressed in neuroblastoma vascularized areas, and this pattern seems to be associated with an unfavourable patient outcome, due to the occurrence of distal metastasis [41]. In addition, a small subset of neuroblastoma cells strongly HIF-2α-positive has been described, which could represent the cancer stem cells [52]. To our knowledge, no precise data are available about the copper-dependent activity/ activation of HIF-2α, however some molecular evidences collected in other cell models strongly point at a potential existence of such a link.
As an example, Menkes copper ATPase (Atp7a) gene expression has been demonstrated to be strongly induced by HIF-2α in mammalian intestine [53]. HIF-2α has been also demonstrated to induce the expression of DMT1 and Ctr1 (by about 25%) copper importers in human intestinal cells, so determining a parallel increase (fivefold) in the processes of cellular copper uptake [54].
These findings confirm that, independently on the involvement of HIF-1α or HIF-2α, tumour hypoxia activates a series of processes functional to distribute copper toward the secretory pathway (enzyme-complexed) or make it available in the extracellular medium. Here, copper may function as a signalling molecule and sustain the angiogenic processes, essential to the neuroblastoma growth.
The scientific literature also suggests that the HIF-2α prolonged response to hypoxia can be alternatively mediated by a high affinity copper-binding protein, namely the cellular prion protein PrP C [55] (Box 1). Accordingly, the PrP C expression degree is elevated in hypoxic nervous tissues [56], and its overexpression has been shown to confer a highly invasive phenotype to tumour cells [55,57].
By virtue of a direct involvement of PrP C in the cell copper import [22,58,59], an elevated protein expression under hypoxia could represent a cancer cell strategy to assure the neuroblastoma growth through the enhanced copper intake [23]. In fact, copper stimulates neuroblastoma cell proliferation [60].
Interestingly, although it has been demonstrated that the up-regulation of PrP C in human colorectal carcinoma cells induces the glucose transporter-1 (Glut-1) expression and a subsequent increase in the glycolytic rate via Fyn-HIF-2α pathway [55], the transfection of a plasmid expressing wild-type HIF-2α in N-myc amplified neuroblastoma cells has been demonstrated to be marginally involved in the regulation of glycolytic genes [46]. Surprisingly, notwithstanding a rise in Glut-1 expression, the glucose influx was not increased [46].
Conclusively, to reinforce the concept of an autonomous cancerogenic role of copper, it can be observed that elevated HIF levels have been observed even under normoxic conditions, meaning that other factors than hypoxia, e.g. copper, can sustain the aerobic glycolysis and induce the expression of HIF-targeted genes.
p53
p53 transcription factor is a key tumour suppressor protein, whose functions contribute to prevent cancer progression. Mutated p53 gene products or defects in the integration of proteins with which p53 is connected, are associated with the malignant progression of the majority of human tumours [61]. Neuroblastoma rarely shows mutated p53 at diagnosis, thus therapies result effective at first. However, gene mutations, p53 cytosolic sequestration, or deregulated p53/MDM2 (ubiquitin protein ligase -E3-for p53) pathways have been reported during neuroblastoma relapses or therapies, thus conferring high-level multidrug resistance [62][63][64][65].
Loss of p53 function seems to impair the efficiency of mitochondrial respiration by hampering the insertion of copper ions as cofactors into the cytochrome c oxidase enzymatic complex [66].
That would cause the switching from cell respiration to aerobic glycolysis (Warburg effect), typical metabolic change observed in cancer cells.
In detail, p53 directly regulates the expression of the SCO2 (Synthesis of Cytochrome c Oxidase) gene, coding for a protein that facilitates the copper delivery to the subunit II of cytochrome c oxidase, determining the assembly of the enzymatic complex [66].
As suggested in [67], given the essential role of copper in determining the Warburg effect in cancer cells, it cannot be excluded that deregulated p53 pathways may affect the expression or function of other proteins involved in cell copper acquisition and utilization.
Copper promotes the neuroblastoma survival and growth by sustaining the anti-oxidant enzyme activities
Cutting copper supply can represent a valuable therapeutic strategy for neuroblastoma, as the induced mitochondrial impairment and oxidative stress can make neuroblastoma cells vulnerable. Accordingly, even under unstressed environment, mitochondria in this cell type exhibit a high rate of protein oxidation, this indicating a consistent susceptibility to the oxidative injury [68,69]. The positive connotation of a drop in the neuroblastoma cell copper content has been demonstrated and emphasized by a rich literature showing that copper chelation (triethylene tetramine tetrahydrochloride) can effectively promote the apoptosis of neuroblastoma cells [70,71].
Here follow some argumentations from the literature around the negative impact of copper starvation on neuroblastoma cell survival, extrapolated from in vitro preclinical studies.
SH-SY5Y neuroblastoma cells have been widely used as a model to dissect the molecular basis of the tumour sensitivity to copper.
In particular, the continuous exposure (up to three passages) of SH-SY5Y neuroblastoma cells to the copper-chelating agent Trien has been demonstrated to induce the expression of antioxidants and a 40% apoptotic cell loss at the end of the third passage [70]. Copper has been shown to be important in keeping a critical level of ATP. In fact, the relevant Cu,Zn SOD and cytochrome c oxidase activities were reduced by, respectively, 80 and 68% [70]. Another report has confirmed these findings, indicating that copper starvation by Trien impairs the antioxidant defences of neuroblastoma cells, with obvious implications with respect to the therapeutic inhibition of the tumour growth [71]. Arciello et al. (2011) further characterized the effects of Trien treatment in SH-SY5Y neuroblastoma cells [72]. SOD1 (cuproenzyme) expression decline was associated with a reduction of the enzyme activity, mainly due to copper shortness rather than to a decreased protein expression. In fact, copper replenishment was able to reactivate the apo-form of the enzyme, in agreement with previous observations [73]. Copper depletion also favoured the entrance of the SOD1 apo-form (not metallated) into the mitochondria [72], where it was retained due to a partial unfolded and obviously inactive configuration. The authors also observed an increased expression of CCS (Box 1), finalized to optimize the copper intracellular distribution [72].
It the light of these findings, it can be observed that the neuroblastoma commitment to the apoptotic death was not due to an irreversible mitochondrial damage, even considering that the loss of the mitochondria-associated SOD1 was much less evident than observed for the cytosolic one [72]. However, it is plausible that the absence of copper prevented SOD1 from counteracting the oxidative-mediated damage to mitochondrial proteins [74]. Accordingly, it has been shown that brain tissues exhibit a SOD1 localization inside the mitochondrial matrix with an antioxidant function [75].
In our laboratory we analysed the anti-oxidant response to copper starvation in a rat neuroblastoma model (B104), investigating in parallel the expression of copper membrane trans-porters [23]. A significant increase of caspase-3 activity was detected in copper-starved cells, indicating the activation of a cell death program through the induction of oxidative stress. In agreement, the total Cu,Zn SOD activity resulted half-reduced with respect to normal conditions, as expected in consideration of the role of copper as a cofactor [23]. Interestingly, the cellular prion protein expression in copper-starved neuroblastoma cells was heavily induced. This finding was reconsidered in the light of a rich literature showing that the 64 Cu loading and the enzymatic activity of Cu,Zn SOD from the brain of P-rnp 0/0 mice result 10-50% reduced with respect to the wild-type genotype [76][77][78].
A special attention has been dedicated to the adaptive response actuated by PrP C , that is physiologically and consistently localized on the outer surface of neurons at synapses and gliocytes [79,80]. Under normal conditions, PrP C binds copper ions with high specificity and affinity (femto-to nanomolar range), by the repeated sequences present on its N-terminal region. By virtue of this property and the ability to undergo endocytosis upon copper binding, PrP C is believed to drive the cellular copper intake [22, 58,59].
The up-regulation of PrP C upon copper limitation has been interpreted as a compensatory mechanism to re-establish the standard cell copper status through a direct transport activity. It has been also demonstrated to be responsible for the ability of copper-starved cells to almost completely recover the SOD enzyme function upon re-exposure to standard growth conditions. The authors conclusively demonstrated that the PrP C neuroprotective action in neuroblastoma cells is due to its ability to translocate copper ions into the cytosol. Here, they can act as cofactors in Cu,Zn SOD activation [23].
Critical role of copper transporters in neuroblastoma vascularization and spread
Most pro-angiogenic factors implicated in neuroblastoma progression need copper to properly work or exert their own functions by activating copper-dependent pathways and enzymes.
The best known pro-angiogenic mediator, namely the Vascular Endothelial Growth Factor (VEGF), has been demonstrated to be overexpressed in high-risk neuroblastomas at the time of diagnosis and to be a bad prognostic marker [81]. The elevated copper levels detected in malignant neuroblastoma are expected to heavily sustain the VEGF tumour angiogenesis, since this metal is a potent inducer of VEGF expression and reinforces the stimulating effect exerted by hydrogen peroxide [82].
The growth of neuroblastoma is anyway sustained by multiple pro-angiogenic factors other than VEGF [10], including Platelet Derived Growth Factor-A (PDGF-A), Fibroblast Growth Factor-2 (FGF-2), and Angiopoietin-2 (Ang-2), as documented in 22 neuroblastoma cell lines and 37 tumour samples [10]. Many among these factors share an intimate relationship with copper, known to variously enhance their angiogenic action through direct (physical interaction) or indirect (expression/release) ways.
As an example, the specific binding of copper to angiogenin, a major angiogenic factor, is able to largely increase its efficiency of interaction with endothelial cells [83,84]. This metal is also fundamental for the release of another pro-angiogenic factor involved in angiogenesis, Fibroblast Growth Factor (FGF) 1, as a part of a multiprotein aggregate (FGF1-p40 Syt1-S100A13) [85].
If on one hand high copper levels can facilitate the tumour development, on the other the stimulation of copper uptake and egress has been associated with the sprouting of new blood vessels within solid tumours, this depicting a high complex picture. A prominent role of copper transport systems emerges.
Potential role of ATP7A and Ctr1 copper transporters
Several experimental evidences point to a crucial role of copper in tumour angiogenesis [86]. Its ability to stimulate the endothelial cell proliferation, migration and sprouting mainly grounds on its role as a powerful inducer/enhancer of the expression of several angiogenic mediators, including VEGF 165 and interleukins [82,87], and a stabilizer of the angiogenin interaction with its receptor [83]. Surprisingly, well-characterized pro-angiogenic factors as VEGF 165 and bFGF, if administered to microvascular endothelial cell cultures, have been shown to rapidly promote the relocalization of the intracellular copper stores (about 80-90%) toward the cell periphery, where the ion efflux occurs, presumably by the ATP7A transport activity [88]. Such process may result contradictory in the light of the discussed role of copper as a powerful pro-angiogenic mediator. Nevertheless, this mechanism may be considered "cancer self-sustaining", making copper available in the tumour microenvironment (paracrine loop).
In addition, it must be observed that the vascular remodelling and the stimulation of cell migration depend on the activity of copper-dependent secreted enzymes (Lysil Oxidase, LOX), so the released metal is probably mostly carried by proteins.
In support of such hypothesis, a report from Ashino et al. (2010) illustrated how the proangiogenic Platelet Derived Growth Factor (PDGF) determines in vascular smooth muscle cells the translocation of the ATP7A copper transporter from the Trans Golgi Network toward special membrane domains (lipid rafts), where the pump is essential for the correct release of copper bound pro-LOX [89]. The authors also demonstrated that the membrane recruitment of Rac-1, a GTPase involved in the extension of lamellipodia, is dependent on copper and on the expression of the high affinity importer Ctr1 (Copper Transporter 1), this further confirming the existence of a solid link between the tumour metastasis and copper homeostasis.
Potential role of the cellular prion protein PrP C
To our knowledge, a few data are reported in the literature around the prion protein role in defining the neuroblastoma aggressiveness. Nevertheless, the substantial expression level observed within the nervous system, which is further elevated by pathological conditions, testifies to a possible involvement of prion protein in the nervous response to cell injuries. In detail, this particular protein may have major implications in modulating the biological cascade leading to metastasis in patients with cancer, mainly by virtue of its presumed ability to sustain cell survival and exert a pro-angiogenic action.
A modest literature discusses a likely role of prion proteins in influencing the angiogenic processes, given a large disagreement about its actual expression in endothelial cells. In fact, although prion protein has been detected in the capillaries of the intestinal mucosa and kidney [90], normal endothelial cells derived from the umbilical cord and other vessels in the adults do not show detectable prion protein amounts in vivo [91]. However, prion protein seems to be up regulated in some pathological circumstances, such as in advanced carotid plaques, in association with the endothelial marker CD105, increasingly expressed in activated endothelia [92], and in brain tissues affected by ischemia [93,94]. By virtue of the latter studies, prion protein could reasonably play a key role in brain tumour progression, being the related gene responsive to the ischemic/hypoxic injury [94]. Accordingly, a neuroprotective action has been described for prion proteins in this context, based on the following evidences: i. prion protein is bound to caveolin-1 and, by recruiting Fyn tyrosine kinase, it can activate the signalling promoting cell survival and angiogenesis events [95]; ii. prion protein co-localizes with the VEGF receptor 2 (KDR), that indicating that prion protein may have a role in VEGF-driven angiogenesis [96].
TNP-470
The administration of angiogenic inhibitors has been introduced as a complement to traditional therapies, in order to hinder the tumour spread.
Several anti-angiogenic therapeutics have been incorporated into clinical trials. Among them, in the '90s, TNP-470, an angiogenesis inhibitor, has emerged as a promising adjuvant in dormancy therapies for high-risk neuroblastoma. In particular, its effectiveness in arresting hepatic metastasis of neuroblastoma has been documented in [97] and [98]. In the light of [99], the antiangiogenic activity of TNP-470 is reasonably linked to its interference with the hepatic copper metabolism. In fact, the continuous administration of TNP-470 in both normal and tumourbearing rats has been shown to increase the serum copper levels, as a consequence of a limited hepatic retention [99]. This feature has been associated with a reduced density of hepatic tumour capillaries [99]. Accordingly, when the administration of TNP-470 was interrupted, angiogenesis was activated and at the same time the serum copper levels fell down [99].
Retinoids target the ATP7A gene expression
Among the most promising possibilities, retinoids (Vitamin A derivatives) may be of help in arresting the cancer growth and delaying the occurrence of recurrences, because of their proven ability to induce cell differentiation and inhibit the VEGF and FGF-2-induced endothelial activation [100]. Interestingly, a recent report from Bohlken et al. (2009) demonstrated that retinoids are able to starve neuroblastoma cells of copper through a significant increase in the ion efflux processes [60]. In fact, the retinoic acid receptor β (RARβ) up-regulates the expression of ATP7A copper efflux pump in BE(2)-C and SH-SY5Y human neuroblastoma cell models, but not in other cell types.
Cell copper transporters modulate the neuroblastoma sensitivity to chemotherapy
Cisplatin-based chemotherapy is commonly employed for neuroblastoma treatment at an advanced stage [101], but the development of resistance to the drug can affect the therapeutic efficacy. Highly diversified mechanisms have been proposed to explain this behaviour, although a definitive understanding has not been achieved. It has been demonstrated that Cisplatin-resistant neuroblastoma cells undergo an increase in the DNA methyltransferase activities that would depress the transcription of specific and widely undefined genes [102]. In fact, it is known that an acute Cisplatin administration can alter the genome methylation status in neuroblastoma cells [103].
Increasing evidences point out a central role of (broad substrate spectrum/specific) drug transporters to explain the onset of Cisplatin resistance. In detail, Haber et al. (1999) observed that malignant neuroblastoma forms, carrying the N-myc oncogene amplification, show an up regulation of the Multidrug Resistance-associated Protein (MRP) gene, associated with a poor sensitivity to low affinity substrates, including Cisplatin [104].
Interestingly, it has been widely demonstrated that Cisplatin shares with copper the pathways of cellular efflux and entry [105,106]. In particular, the cellular uptake of cisplatin (water soluble) is mediated by a member of the SLC (Solute Carrier) group, namely the copper transporter 1 (SLC31A1) [105][106][107], by mechanisms that partially overlap with those copperspecific [105,108]. Candidate Cisplatin-binding sequences have been identified in the extracellular region of hCtr1, this providing further evidence of the Cisplatin transport activity by this channel [109].
Further, the copper efflux transporters, ATP7A and ATP7B, are known to regulate the efflux of cisplatin, and so their expression may be also predictive of drug sensitivity [110].
Neuroblastoma cells are known to express both hCtr1 import and ATP7A export proteins, this suggesting that copper transport systems may participate in determining the development of cisplatin resistance. In support of such hypothesis, a recent study on microRNAs expression pattern in variously N-myc amplified and cisplatin resistant neuroblastoma cells, led to the identification of eight microRNAs, each one targeting at least one of the two cited copper transporters [111]. Furthermore, it has been demonstrated that ATP7A expression may be a target to sensitize cancer cells to Cisplatin [112].
In the light of these findings, it has been argued that an increased cisplatin sensitivity may arise from the upregulation of Ctr1 transporter or by downregulation of the copper/cisplatin efflux transporter ATP7A. In this sense, a therapeutic regimen combining a preconditioning by a copper chelating agent (i.e. Tetrathiomolybdate) and platinum-containing drugs has been proven to enhance the Cisplatin efficacy in a mouse model of cervical cancer, without affecting the integrity of healthy tissues [113].
Another copper-dependent mechanism of resistance to cisplatin involves metallothioneins, a family of low molecular weight copper-binding proteins, whose expression is metal-induced in neuroblastoma cell models [114] and elevated in cisplatin-resistant cell lines [115]. When cisplatin enters a cancer cell, it is vulnerable to metallothionein-inactivation [116]. This mechanism assumes a prioritary connotation if we consider that N-myc amplified neuroblastomas show an increased copper content, that translates in a remarkable induction of metallothioneins and reduced efficacy of Cisplatin-based therapies.
Conclusion
Multifaceted pathophysiological features determine the progression of neuroblastoma malignancies. Mainly on the basis of in vitro and pre-clinical studies, copper, playing a key role within the human nervous system, is candidate to be the actual target of novel therapies. Accordingly, high copper levels seem to underline the development of tumour malignancies, even if we honestly observe that the scientific literature does not offer so many clear cues about the nature of in vivo copper involvement in neuroblastoma. The conclusive impression is that copper interacts with the neuroblastoma microenvironment at various levels, and the effects may be profoundly different, depending on the interested cell type (e.g. endothelial, neuroblast). The overall effects arise from the sum of specific and sometimes discordant copperdriven processes.
If few clinical data are currently available in this regard, the challenge toward the development of a copper-targeting therapy has anyway been launched. On the other hand, recent studies have recognized for neuroblastoma patients the benefits of preconditioning therapies based on the use of copper chelating agents (i.e. tetrathiomolibdate). Such intriguing approach would modulate the expression and/or subcellular localization of copper transport systems, and so both the cancer metal levels and chemoresistance. However, caution is needed in this sense, since the comprehension of copper metabolism in neuroblastoma cancer cells is still preliminary and the routes of copper transport are currently partially known. Significantly, it is only recently that an anion exchanger has been proposed as an additional copper importer in mammalian cells [117]. | v3-fos-license |
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} | pes2o/s2orc | From Synthesis to Characterization of Site-Selective PEGylated Proteins
Covalent attachment of therapeutic proteins to polyethylene glycol (PEG) is widely used for the improvement of its pharmacokinetic and pharmacological properties, as well as the reduction in reactogenicity and related side effects. This technique named PEGylation has been successfully employed in several approved drugs to treat various diseases, even cancer. Some methods have been developed to obtain PEGylated proteins, both in multiple protein sites or in a selected amino acid residue. This review focuses mainly on traditional and novel examples of chemical and enzymatic methods for site-selective PEGylation, emphasizing in N-terminal PEGylation, that make it possible to obtain products with a high degree of homogeneity and preserve bioactivity. In addition, the main assay methods that can be applied for the characterization of PEGylated molecules in complex biological samples are also summarized in this paper.
INTRODUCTION
The binding of proteins, peptides, enzymes, antibody fragments, oligonucleotides, or small synthetic drugs to polymers has become a very useful method for improving therapeutic activity or decreasing the toxicity of these biological agents (Mishra et al., 2016). Among the polymeric materials, polyethylene glycol (PEG) is the most used for these purposes, mainly due to its high biocompatibility, low toxicity, and limited side effects (Gauthier and Klok, 2008). PEGs are water-soluble polymers approved by the Food and Drug Administration for use in oral, topical, and intravenous formulations (D'souza and Shegokar, 2016). It presents a structure of repeated units of polyether diols (either linear or branched) chemically formulated as HO-(CH 2 CH 2 O)n-CH 2 CH 2 -OH ( Figure 1A), where each ethylene oxide residue has a molecular weight (MW) of 44 Da (Roberts et al., 2012). PEGylation refers to the covalent or non-covalent attachment of PEG to different molecules, such as proteins, macromolecular carriers, oligonucleotides, vesicles, and others to improve the pharmacokinetic (Milton Harris et al., 2001;Lee et al., 2013) and pharmacodynamic properties (Abbina and Parambath, 2018). The conjugation to PEG generates an increase in the hydrodynamic volume of the biomolecule of interest, creating a shield around it (Gokarn et al., 2012). This effect enables clearance by the renal system to be reduced, and therefore, the half-life is increased in the bloodstream (Milton Harris and Chess, 2003) concomitant with the increases in PEG molecular weight (Hamidi et al., 2006). Additionally, this approach has been used to improve the stability of some proteins (Yang et al., 2007;Jevševar et al., 2010;Lawrence and Price, 2016;Santos et al., 2019), as well as decrease the immune response against several biomolecules (Soares et al., 2002;Zheng et al., 2012;Sathyamoorthy and Magharla, 2017;Wu et al., 2017).
Since the 1990s several PEGylated biopharmaceuticals (see Table 1) have been approved by the FDA, and some more are currently undergoing clinical trials (information available at https://clinicaltrials.gov/ct2/results?term=Pegylated&Search=App ly&recrs=d&age_v=&gndr=&type=&rslt=). Most of the approved PEGylated proteins were synthesized by non-site-specific chemical conjugation strategies, resulting in heterogeneous mixtures of multi-PEGylated (polydisperse) proteins due to the presence of several reactivity sites on the protein surface (Alconcel et al., 2011), requiring complex separation steps. In addition, protein PEGylation can lead to the loss of protein activity through several mechanisms that include the direct PEGylation of the active site or receptor binding site (Schiavon et al., 2000), the steric entanglement imposed by PEG chains that cause restricted movements (Kubetzko, 2005;Xiaojiao et al., 2016) and conformational changes in proteins (Chiu et al., 2010), among others. Also, recent research has revealed certain shortcomings related to highly PEGylated forms, such as activation of the immune system, nondegradability, and possible accumulations with high molecular weight PEGs. These are strong reasons that support the need to find site-selective PEGylation techniques, yielding homogenous mono-PEGylated products, a field that has garnered considerable in recent years. Although certainly the in vivo potency of therapeutic proteins can be affected by the PEGylation process, this decrease in activity can be largely balanced by their prolonged half-life in the circulation (Oclon et al., 2018). Site-selective PEGylation has been a very useful strategy for introducing PEG at specific amino acid sites in various proteins. Some methods like pH-controlled N-terminal selective acylation (Chan et al., 2006;Chan et al., 2012) or reductive alkylation (Kinstler et al., 1996;Marsac et al., 2006), the use of oxidizing agents (Kung et al., 2013;Obermeyer et al., 2014), the chemo-selective capability of catechol (Song et al., 2016) and transamination reaction (Gilmore et al., 2006) have been used to perform PEGylation at the N-terminus of proteins. Additionally, in recent years there has been a lot of work on using "grafting from" approaches to grow PEG from the surface of proteins via ATRP and RAFT polymerization methods (Quémener et al., 2006;Ameringer et al., 2013;Gody et al., 2015;Tucker et al., 2017). These approaches involve the direct generation of conjugates containing high molecular weight polymers (like PEGs) by directly growing the polymer from the protein surface (Wallat et al., 2014;Obermeyer and Olsen, 2015). An illustrative example of success in chemical site-selective PEGylation is the case of Neulasta ® , which is an N-terminally mono-PEGylated granulocyte colony-stimulating factor bearing a 20-kDa PEG (Molineux, 2004). The improved pharmacokinetic behavior of this biopharmaceutical allows administration only once per chemotherapy cycle compared to the first generation, Neupogen ® , which is administered daily (Cesaro et al., 2013;Zhang et al., 2015).
Despite their robustness, chemical methods usually involve the use of excessive amounts of reagents and careful working conditions. Site-specific PEGylation of peptides and proteins has been approached successfully not only from the chemical point of view but also enzymatically. Several studies report the use of enzymes to conjugate PEG to peptides, proteins, and oligonucleotides (Sato, 2002;Mero et al., 2009;Da Silva Freitas et al., 2013;Sosic et al., 2014).These enzymes usually catalyze the reaction between the biomolecule of interest and a substrate analog containing a functional group (Dozier and Distefano, 2015), which can be the case of PEG. There are a number of more recent approaches aimed at achieving site-selective modification including the use of the Spytag/Spycatcher system (Schoene et al., 2014;Reddington and Howarth, 2015;Cayetano-Cruz et al., 2018;Kim et al., 2018); however, there is as yet insufficient information focused on these methods, and some studies are being developed in this direction.
The structural changes in protein characteristics after the attachment to PEG influence the subsequent characterization of PEGylated proteins. These changes result in an analytical challenge due to the heterogeneity of the PEGylation products and the degree of PEGylation, coupled with the complex protein structure (Hutanu, 2014). Several studies have reported the use of analytical techniques with differing degrees of difficultyfrom colorimetric methods to more complex techniques such as computational approaches-for the characterization of PEGylated peptides and proteins.
In the present review, we have focused on summarizing both classic and novel chemical and enzymatic tools used for the covalent attachment of PEG in site-specific regions of peptides and proteins, as well on the main analytical methods for PEGylated molecule characterization.
Chemical Approaches for Site-Selective Pegylation
For the selective modification of specific amino acids in peptides and proteins, the knowledge of some characteristics about their The square represents PEG functional group that covalently binds to the terminal amine of the protein. PEG chain is end-capped with a terminal methoxy group to prevent reactivity and enzymatic attack upon administration in mammals.
primary structure is needed. An important physicochemical feature in proteins is the difference in pKa between the amino group of an N-terminal amino acid residue (~7.6) and the amino groups in the side chains of lysine (~10.5) and arginine (~12) (Roberts et al., 2012). This difference allows the selective N-terminal modification of proteins based on pH control and the use of reductive agents like sodium cyanoborohydride. A useful strategy for the specific conjugation of peptides and proteins is based on the amino acid ratio in a protein being variable. Moelbert et al. reported the accessibility index on the surface of the 20 essential amino acids, which makes it possible to know the expression of these amino acids in different areas of the proteins in relation to their natural abundance (Moelbert, 2004). It has also been reported that short peptides/proteins (less than 50 residues) tend to over-represent glutamine and cysteine in the N-terminal region (Villar and Koehler, 2000). It is well known that single-chain proteins possess only one N-terminal residue, having a uniquely reactive site for chemical modification (Rosen and Francis, 2017). Therefore, as virtually all proteins present these functional groups, a number of valuable reactions have been developed for their selective modification (Boutureira and Bernardes, 2015).
The use of potassium ferricyanide as an oxidizing agent in o-aminophenol-performing N-terminal PEGylation has also been shown (Obermeyer et al., 2014). In 2016 Song et al. described an alternative strategy for PEGylation at the N-terminus of several proteins as well as two peptides based on the chemoselectivity of catechol (Song et al., 2016). More recently, Rosen and Francis described classical methods for the selective modification of N-terminal amino group under pH control. These methods include the selective acylation and alkylation of N-terminal amines at low-to-neutral pH and also transamination using pyridoxal-5′-phosphate aldehyde, which undergoes condensation with ε-amines from lysine side chains and N-terminal α amines to form imines (Gilmore et al., 2006;Rosen and Francis, 2017). Chen et al. demonstrated the ability of benzaldehyde to selectively modify native peptides and proteins on their N-termini. Preservation of the positive charge on the N-terminus of the human insulin A-chain through reductive alkylation instead of acylation leads to a 5-fold increase in bioactivity. They showed that under mild conditions, aldehyde derivatives and carbohydrates can site-specifically react with peptide and protein N-termini, providing a universal strategy for site-selective N-terminal functionalization in native peptides and proteins . PEG-isocyanate is in the group of PEG reagents used for the site-specific modification of different proteins (Berberich et al., 2005;Sharma et al., 2017). The reaction takes place via the amine group to produce a stable thiourea linkage (Ganesan et al., 2015). For example, in 2009 Cabrales et al. generated PEGylated human serum albumin (PEG-HSA) by conjugating PEG-phenylisothiocyanate 3 and 5 kDa at primary amine groups of the HSA, enhancing the hydrodynamic volume of the protein and restoring intravascular volume after hemorrhagic shock resuscitation (Cabrales et al., 2008). Furthermore, Chen and He reported in 2015 the achievement of nanophosphors coated with PEG-isocyanate and polylactic acid (PLA) for paclitaxel delivery, resulting in a significant improvement and serving as a platform in the field of drug development (Chen and He, 2015). Lee et al. synthesized a dual functional cyclic peptide gatekeeper attached on the surface of nanocontainers by using PEG-isocyanate as a linker to enhance dispersion stability and biocompatibility. This allowed the active targeting of cancer cells with high CD44 expression together with the ability of triggered drug release . It is important to note that specific PEG-reagents like isocyanates have a short half-life in aqueous solutions (Erfani-Jabarian et al., 2012); thus, a stoichiometric excess of these reagents is necessary, causing difficulties in the removal of the remaining PEG.
A relevant report for one-step N-terminus-specific protein modification showed the stable and selective imidazolidinone product at the N-terminus, with 2-pyridinecarboxaldehyde (2PCA) derivatives (Macdonald et al., 2015). The main basis of this reaction is the nucleophilic attack of the neighboring amide nitrogen on the electrophilic carbon of the initially formed N-terminal imine (Koniev and Wagner, 2015). As an example, a 2PCA-functionalized polyacrylamide-based hydrogel has been developed for the immobilization of extracellular matrix proteins through the N-terminus to study their biochemical and mechanical influence on cells .
In the next section, we provide an overview based on reactions which can be used to selectively modify specific amino acids. Keeping that in mind, in some cases the described modification does not refer to the PEGylation itself, but the concept could be applied if the introduction of PEG reagents is desired. A mechanism corresponding to N-terminal PEGylation has been illustrated in Figure 1B, while general mechanisms of the siteselective chemical reactions are shown in Figure 2.
Targeting Cysteine
Cysteine residues are interesting targets for residue-specific modification of peptides/proteins due to their low apparition frequency (Harvey et al., 2000). These are often found partially or fully covered within the protein structure, limiting their accessibility to chemical reagents (Thordarson et al., 2006). Proteins with N-terminal cysteine have been successfully modified through native chemical ligation (NCL) when, on the first and reversible step, a thioester intermediate is formed, which then undergoes a spontaneous S-to-N acyl shift and yields an amide bond (Johnson and Kent, 2006;Rosen and Francis, 2017). This methodology has been useful in the preparation of high complexity protein-polymer conjugates. For example, Zhao et al. described a PEGylated human serum albumin (HSA) in a site-specific method by taking advantage of the unusual chemical reactivity of the only one free Cys34 of the HSA molecule and the high specificity of PEG-maleimide for the protein sulfhydryl (-SH) groups. Targeting the distinctive free Cys34 through this site-specific PEGylation could generate a chemically welldefined and molecularly homogeneous product and may be also convenient in preventing dimerization (Zhao et al., 2012). Another technique which plays a major role in modern chemical biology and has been used for many applications is known as expressed protein ligation (EPL) (Mitchell and Lorsch, 2014;David et al., 2015;Liu et al., 2017). EPL constitutes an improvement for NCL, and in this case selectivity over lysine acylation was achieved through pH control, by using benzaldehyde derivatives bearing selenoesters to acylate N-terminal positions through acyl transfer (Raj et al., 2015). As N-terminal cysteines are rare in nature, they frequently need to be introduced by genetic engineering (Nguyen et al., 2014a;Uprety et al., 2014;Gunnoo and Madder, 2016). Methionine aminopeptidase can take out the first methionine to liberate an N-terminal cysteine (Gentle et al., 2004), and some proteolytic enzymes that specifically cleave in the presence of cysteine residues in a protease recognition sequence (Busch et al., 2008;Wissner et al., 2013) have been used as strategies for the exposure of N-terminal cysteine and its subsequent bioconjugation.
Targeting Serine and Threonine
The presence of an N-terminal serine or threonine offers unique opportunities due to the high susceptibility of 1, 2-aminoalcohols to periodate oxidation, resulting in the formation of a glyoxylyl group, which can be used to form several linkages (Xiao et al., 2005). It has been shown that the extra periodate used to oxidize the N-terminal residues of proteins carries the risk of oxidizing other residues, such as cysteines and methionines, as well as causing unwanted oxidative cleavage of protein glycosyl groups . This is mainly the approach applied in classical research, based on targeting serines or threonines at the N-terminal position, which uses periodate oxidation to generate a glyoxylyl group. Gaertner et al. performed site-selective PEGylation of an N-terminal serine residue, which was oxidized using sodium periodate followed by subsequent oxime ligation with an aminooxy and hydrazyde PEG derivative (Gaertner and Offord, 1996). The modified proteins, interleukin (IL)-8, granulocyte colony-stimulating factor (G-CSF) and IL-1rα, fully retained their activity after PEGylation (Krall et al., 2016).
Targeting Tyrosine
Francis et al. Have Reported a Number of Efficient strategies where tyrosine residues were modified via a three-component Mannich-type reaction, alkylation of the residue and coupling with diazonium reagents (Tilley and Francis, 2006). However, Jones et al. were the first to describe direct polymer conjugation, including PEGylation, to tyrosine residues. These authors developed a general route to polymer-peptide biohybrid materials by preferentially targeting peptide tyrosine residues using diazonium salt-terminated polymers. Also, aniline derivatives are attractive molecules for tyrosine-targeted protein modifications with 4-aminobenzoyl-N-PEG 2000 -OMe through either diazonium coupling or three-component Mannich-type reactions (Jones et al., 2012). Recently, the first study to apply Mannich reaction modification and reactive coloration in fibrous proteins was developed, providing promising future applications for the reactive dyeing process of silk (Chen et al., 2019).
Targeting Tryptophan
Peptides containing N-terminal tryptophan residues may be modified using the Pictet-Spengler reaction with aldehydes in glacial acetic acid. The Pictet-Spengler reaction is based on the oxidation of the N-terminal amino group to an imine, where an aldehyde undergoes cyclic condensation with the α-amine and the indole side chain of a tryptophan residue, forming a new stable C-C bond (Agrawal et al., 2013;Mittal et al., 2014). Li et al. applied the Pictet-Spengler reaction to peptide ligation using peptide segments containing an aldehyde at the C-terminal and a Trp at the N-terminal. The main advantage of this reaction is the formation of a product with a stable C-C bond in a single step (Li et al., 2000). Also, Sasaki et al. applied the Pictet-Spengler reaction to the N-terminal labeling of horse heart myoglobin with an N-terminal glycine, employing tryptophan methyl ester and tryptamine as the coupling partners (Sasaki et al., 2008).
As an alternative to chemoselective modification, recombinant methods have also been used to incorporate unnatural amino acids (UAA) into proteins as chemical handles for a bio-orthogonal conjugation reaction (Liu and Schultz, 2010). The transfer of nonnatural amino acids with azide and ketone functional groups at the N-terminus of proteins bearing N-terminal arginine residues using leucyl/phenylalanyl (L/F)-tRNA-protein transferase has proven efficient, both in the presence of other peptides and in crude protein mixture (Taki and Sisido, 2007). Although considerable progress has been made, an improvement in the existing N-terminal strategies is needed as none of the methods reported to date offer universal sequence compatibility.
ENZYMATIC TOOLS FOR SELECTIvE PEGYLATION OF PROTEINS
Enzyme-mediated bioconjugation has gained a lot of attention in recent years because of the ability of biocatalysts to modify specific molecular tags under mild conditions. In this section, we briefly explore some enzymatic tools used for selective PEGylation purposes. Among these, sortase A (SrtA) from Staphylococcus aureus has been the most widely applied enzyme for protein bioconjugation in academic research (Tsukiji and Nagamune, 2009;Popp and Ploegh, 2011;Schmidt et al., 2017;Wang et al., 2017). It catalyzes a transpeptidase reaction between an N-terminal amino group derived from glycine and a specific internal amino acid sequence on a protein, usually LPXTG (where X can be any amino acid) (Rosen and Francis, 2017) (Figure 3A). Although the sortase A is applied for labeling the peptides and proteins among them, the approach of sortase-mediated PEGylation has been used to label large macroscopic particles with PEG-stabilized proteins to the surface of cells (Tomita et al., 2013). More recently, Li et al. took advantage of the mutated sortase A enzyme, which can enzymatically ligate the universal α-amino acids to a C-terminal tagged protein, allowing specific modification of the C-terminus of human growth hormone (hGH) with PEG. This site-specific bound PEG-hGH has similar efficacy as wild-type hGH (Shi et al., 2018). Despite there being as yet no approved PEGylated drugs derived from sortagging, it could be a promising advancement for improving the performance of traditional PEGylated drugs.
Microbial Transglutaminases
Microbial transglutaminases (mTGases) are another class of enzymes that has frequently been used for protein conjugation (Figure 3B). Several excellent reviews covering applications of microbial transglutaminase have been published previously (Mariniello and Porta, 2005;Rachel and Pelletier, 2013;Adrio and Demain, 2014;Strop, 2014). In general terms, TGases catalyze the acyl transfer reaction between the c-carboxyamide group of a protein-bound Gln residue and a variety of linear primary amines, such as the amino group of Lys (Griffin et al., 2002). In terms of site selective PEGylation this approach could be ineffective due to promiscuity in the amine substrates for these enzymes (Rachel and Pelletier, 2013). Nevertheless, Pasut et al. examined how the properties of PEGylated human growth hormone (hGH) changed depending on whether it was generated by chemical modification at the N-terminus or enzymatically using transglutaminase. Enzymatic labeling of hGH was carried out using TGase and a PEG reagent incorporating a primary amine. The study shows that although hGH carries 13 glutamine residues, 63.3% of the reaction product was a monoPEGylated form at position 141, showing a certain degree of site selectivity (Da Silva Freitas et al., 2013). Spolaore et al. studied the reactivity of IFN α-2b to microbial mTGase to obtain a site-specific conjugation of this biopharmaceutical. Characterization by mass spectrometry of the conjugates indicated that among the 10 Lys and 12 Gln residues of the protein only Gln101 and Lys164 were selectively conjugated with a PEG-NH 2 for Gln101 and a PEG modified with carbobenzoxy--glutaminyl-glycine for Lys164 derivatization, with activity retention and improvements at pharmacokinetic levels (Spolaore et al., 2016). A mono-PEGylated derivative of filgrastim (granulocyte colonystimulating factor) was also prepared using mTGase. The conjugation yielded an active protein with a single conjugation site (Gln135) that exhibited good in vivo stability (Scaramuzza et al., 2012). Although in the previous examples the PEGylation sites do not correspond to the N-terminal amino acid, they do illustrate a partial selectivity of mTGase despite its tendency toward substrate promiscuity. Also, these results indicate the potential of mTGase in the future of specific PEGylation and the development of innovative biopharmaceuticals. More recently, Braun et al. obtained an insulin-like growth factor 1-PEG (IGF1-PEG) conjugate for release in diseased tissue by using a combination of enzymatic and chemical bioorthogonal coupling strategies. In this interesting example, mTGase was used for the ligation at the level of the N-terminal lysine of IGF1 to a PEG30 kDa modified protease-sensitive linker (Braun et al., 2018).
Subtiligase
Subtiligase is a redesigned peptide ligase based on the modification of the active site of subtilisin. It was engineered by converting catalytic Ser221 to Cys, thereby increasing the ligase activity compared to amidase activity, and Residue Pro225 was converted to Ala to reduce steric assembling (Haridas et al., 2014). Subtiligase facilitates the ligation between a peptide C-terminal ester and a peptide N-terminal α-amine, without requiring a recognition motif at the termini of any reaction partners (Lin and He, 2018) (Figure 3C). The selective modification of the α-amine using subtiligase is a powerful approach in proteomics to enrich new N-termini arising from protease recognition and cleavage (Wiita et al., 2014), because 80% and 90% of wild-type eukaryotic proteins are acetylated at the N-terminal position (Polevoda and Sherman, 2003). This advantage could be exploited for the selective attachment of PEG-modified peptides as an innovative application to improve either the conjugation efficiency or the originality in the development of therapeutics.
Butelase 1
Butelase 1 is an enzyme isolated from the medicinal and ornamental plant Clitoria ternatea, which is a high-yielding asparagine/ aspartate-specific cysteine ligase (Nguyen et al., 2014b) ( Figure 3D). In spite of being C-terminal-specific for Asx, this enzyme accepts most N-terminal amino acids to mediate intermolecular peptide and protein ligation (Nguyen et al., 2016). Although it was recently discovered, butelase 1 has been used for several purposes, such as protein modification and engineering, peptide/ protein ligation and labeling, peptide/protein macrocyclization, and living-cell surface labeling (Lin and He, 2018). No work has yet reported butelase 1 as being used for PEGylation reactions. However, some recent experiences with the enzyme, such as the method developed by Nguyen et al. for butelase-mediated ligation using thiodepsipeptides, have been applied in N-terminal labeling of ubiquitin and green fluorescent protein (GFP). The ligation yield of > 95% could be achieved for the model peptide and ubiquitin with a small substrate excess. This result anticipates a wide-ranging application and the perspectives of using butelase 1 for N-terminal modification of peptides and proteins (Nguyen et al., 2015).
Lipoid Acid Ligase
Lipoid acid ligase (LplA) is an alternative enzyme that has also been exploited for protein bioconjugation. This enzyme is able to recognize a specific LplA acceptor peptide (LAP) and catalyze the attachment of a lipoate moiety to a lysine residue in LAP (GFEIDKVWYDLDA) through an ATP-dependent reaction (Puthenveetil et al., 2009;Zhang et al., 2018) (Figure 3E). Regarding PEGylation, Plaks et al. used LplA for multisite clickable modification based on the incorporation of azide moieties in GFP at the N-terminal and two internal sites. Modification of the ligated azide groups with PEG, α--mannopyranoside and palmitic acid resulted in highly homogeneous populations of conjugates, being a potential approach, for instance, for site-specific multipoint protein PEGylation, among other modifications (Plaks et al., 2015). Additionally, other studies have been conducted using LplA-mediated enzymatic protein labeling followed by subsequent bio-orthogonal reactions (Hauke et al., 2014;Drake et al., 2016;Gray et al., 2016), allowing site-specific labeling of N-or C-terminus, even at the internal regions of a target protein.
There are other enzymes that have also been exploited for protein bioconjugation, including tubulin tyrosine ligase, which catalyzes the ATP-dependent addition of a tyrosine residue to the C-terminus of a-tubulin yielding a peptide bond (Schumacher et al., 2015;Zhang et al., 2018); N-myristoyltransferase, leading the transference of myristate from myristoyl-CoA to the N-terminal glycine of protein substrates, resulting in an amide linkage (Wright et al., 2010;Zhang et al., 2018); and biotin ligase, another ATP-dependent enzyme, catalyzes the conjugation of biotin derivatives onto proteins (Howarth and Ting, 2008;Fairhead and Howarth, 2015).
ANALYTICAL METHODS FOR CHARACTERIZATION OF PEGYLATED PROTEINS
The evidence indicates that the use of PEG to improve the properties of biopharmaceuticals or diagnostic agents will increase. This is supported by the growing number of proposals in clinical evaluation each year. In order to achieve high-quality products, it is necessary to take into account the implementation of accurate methods for the analysis of some parameters that provide a higher level of characterization of the molecule under study. It is important to note that none of the techniques on their own allows for the most complete characterization of the PEGylated proteins, but in many cases the combination of these is necessary to obtain more accurate results. This section provides an overview of the most frequently used analytical methods for the characterization of PEGylated peptides and proteins.
High-Performance Liquid Chromatography-Mass Spectrometry
High-performance liquid chromatography (HPLC) has been used for the separation and quantitation of free PEG and its PEGylated-protein form (PEG-conjugate). Some features of the PEGylated protein such as conjugate molecular weight, polymer mass distribution, or the degree and sites of PEGylation can be measured by HPLC methods. Lee et al., using SEC (sizeexclusion chromatography) and RP-HPLC (reversed phasehigh-performance liquid chromatography) mapping, assessed N-terminal PEGylated EGF, demonstrating the formation of a PEGylated macromolecule and that PEGylation occurred at the N-terminal position, respectively (Lee et al., 2003). Also, Brand et al. performed the separation of N-terminal PEGylated retargeted tissue factor tTF-NGR by using HPLC-based gel filtration, revealing pure elution fractions with the mono-PEGylated protein, which were represented by one clear band in SDS-PAGE and Western blotting (Brand et al., 2015). Although generally useful, the HPLC conditions and detection method must be improved for each compound based on the specific properties of the conjugated proteins.
To improve HPLC performance in the characterization of PEGylated proteins and to provide a more detailed characterization, the solution of coupling liquid chromatography to mass spectrometry was adopted. For decades, mass spectrometry (MS) has been the technique of choice for PEGylated protein characterization in terms of accurate average molecular weight and degree of PEGylation (Hutanu, 2014). A comparison of PEGylated and un-PEGylated counterparts by MS and peptide mapping is used to identify and quantify PEGylation sites and characterize impurities that occasionally go undetected by simpler techniques (Caserman et al., 2009). Collins et al. performed N-terminal amine PEGylation to stabilize oxytocin formulations for prolonged storage. Conjugation was confirmed by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) MS, where a clear shift in molecular weight was observed in the MALDI-TOF spectrum from the NHS ester polymer to the polymer-peptide conjugate (Collins et al., 2016). In another study conducted by Qin et al. following MALDI-TOF MS, PEG modification sites were determined through comparative analysis of peptide mapping between rhGH (recombinant human growth hormone) and PEG-rhGH. The use of MS makes it possible to discriminate positional isomers, with PEGylation sites potentially located at the N-terminus and nine lysine residues of rhGH (Qin et al., 2017). However, the exact determination of the PEG attachment site(s) continues to be highly challenging, especially in a mixture composed of products with differing degrees of PEGylation (Gerislioglu et al., 2018). On the other hand, ESI-TOF has overcome some disadvantages related to polydispersion and the overlapping protein charge pattern of PEGylated proteins (Forstenlehner et al., 2014). Furthermore, ESI-MS is preferred to MALDI due to automated workflow and reduced sample preparation time (Hutanu, 2014). Several studies have reported applying the approach of the line-up of liquid chromatography to MS (LC-MS) for the sensitive quantitation of free PEG in biological fluid samples (Pelham et al., 2008;Yin et al., 2017) or tissues (Gong et al., 2014), as well as clear detection and identification of the positional isomers formed upon PEGylation (Gerislioglu et al., 2018;Shekhawat et al., 2019), obtaining significant structural information in a heterogeneous sample of PEGylated proteins Muneeruddin et al., 2017), among others.
Dynamic Light Scattering
Dynamic light scattering (DLS) is an additional technique also convenient for the molecular weight evaluation of PEGylated proteins, as it can measure the molecular radii of the samples (Kusterle et al., 2008;Gokarn et al., 2012), and discriminate between linear and branched PEGs (Wan et al., 2017). This method, among others, was used by Vernet et al. in 2016 to assess the first large-scale study with the site-specific mono-PEGylation of 15 different proteins and characterization of 61 entities in total (Vernet et al., 2016). In addition, Khameneh et al. conducted a study in which site-specific PEGylated hGH was prepared by using microbial transglutaminase. Physicochemical properties, size and zeta potentials of native and PEGylated hGH, were evaluated by DLS, indicating that the size and zeta potentials of the protein were increased and decreased respectively by PEGylation, enhancing the stability of the protein (Khameneh et al., 2016). Recently, Meneguetti et al. applied DLS for the characterization of a novel N-terminal PEGylated asparaginase, showing that the PEGylation of ASNase caused an increase in the hydrodynamic diameter of the protein related to the increase in the amount of PEG attached to the protein (Meneguetti et al., 2019). The DLS approach has been used in the characterization not only of PEGylated proteins, but also of PEGylated organic nanotubes, revealing that PEGylation dramatically improves the dispersibility of the nanotubes in saline buffer (Ding et al., 2014). Despite its wide use in the characterization of the hydrodynamic radius of PEGylated proteins, this methodology presents certain disadvantages in its application, such as the presence of large particles that can also be detected during the analysis; low resolution when the populations are close in size or a highly polydispersed sample; light absorption by the dispersant can interfere with detection because of their viscosity as well as the density of the particles. These are important parameters to take into account when carrying out this type of analysis.
Nuclear Magnetic Resonance
1 H nuclear magnetic resonance (NMR) spectroscopy is useful to quantify PEGylated species in complex biological fluids with advantages of time and simplicity in the sample preparation (Alvares et al., 2016). The application of this technique for the structural characterization of conjugates with PEG (Kiss et al., 2018) has been being useful in the quantitative determination of the degree of PEGylation (Zaghmi et al., 2019), the assessment of the higher-order structure of PEGylated therapeutic proteins (Cattani et al., 2015;Hodgson and Aubin, 2017) or even the behavior of free PEG in serum samples (Khandelwal et al., 2019). More recently, solid state NMR has been used for the structural characterization of large PEGylated proteins such as asparaginase (Giuntini et al., 2017;Cerofolini et al., 2019). The combination of NMR with other techniques such as LC-MS/MS has enabled the accurate quantification of isobaric glycan structures, even in the picomolar order (Wiegandt and Meyer, 2014), an approach that could be used for a better characterization of high complexity PEGylated molecules.
Immunoenzymatic Assays
Enzyme-linked immunosorbent assay (ELISA) is a powerful tool for measuring the concentration of PEGylated proteins in serum samples . This technique permits the study of the effects of PEGylation in protein immunogenicity as well as the anti-PEG immune response (Wan et al., 2017). While direct ELISA has the advantage of lacking only one specific antibody for compound detection, it cannot distinguish between PEGylated and unPEGylated proteins (Cao et al., 2009). On the other hand, sandwich or indirect ELISA employs two antibodies: one to capture the analyte on a solid surface and a second to determine the concentration of the captured analyte (Gan and Patel, 2013). Bruno et al. used a quantitative sandwich ELISA to analyze the pharmacokinetics of Pegasys and PEG-Intron using two mouse monoclonal antihuman IFN-α antibodies that recognize different epitopes of IFN-α (Bruno et al., 2004), and a similar ELISA was used for the measurement of Neulasta ® (Roskos et al., 2006) and Mircera ® (Macdougall et al., 2006). Su et al. produced secondgeneration monoclonal antibodies attached to PEG (AGP4/ IgM and 3.3/IgG) that also bind to the repeating subunits of the PEG backbone, but with greater affinity than those of first-generation AGP3 and E11 (Su et al., 2010). Since then, they have produced a range of specific anti-PEG IgG and IgM monoclonal antibodies for use in ELISA, FACs, IHC, and flow cytometry, which can be found under anti-PEG in the Institute of Biomedical Sciences at Academia Sinica, Taiwan.
Bioinformatics Methods
With the advent of the era of bioinformatics, computational methods have been effectively employed for an easier designing, engineering, and characterization of proteins, which supports experimental methodologies and, in many cases, saves time and materials. At present, computational analysis is highly recommended to select the proper position on the protein for site-selective PEGylation (Rouhani et al., 2018). In 2013 Mu et al. conducted a bioinformatics study in which four forms of PEGylated staphylokinase obtained by site-specific conjugation of PEG to the N-and C-termini of SK, respectively, were structurally evaluated to provide greater molecular insight into the interaction between the PEGylated protein and its receptor (Mu et al., 2013). The results suggested that the PEG polymer wraps around the protein providing steric shield, and this effect depends on the PEG chain length and PEGylation site of the protein (Rouhani et al., 2018b). Also, Mirzaei et al. (2016) applied computational and non-glycosylated systems to define an artless methodology for site-selective (cysteine) PEGylation of erythropoietin analogs. The results showed that using an in silico approach together with the experimental methodologies can be a strategy to optimize the parameters of PEGylation (Mirzaei et al., 2016). Recently, Xu et al. (2018) used interferon (IFN) as a representative model system to characterize the molecular-level changes in IFN introduced by several degrees of PEGylation through molecular dynamics simulations. The simulations generated molecular evidence directly linked to improved protein stability, bioavailability, retention time, as well as the decrease in protein bioactivity with PEG conjugates, providing an important computational approach in the improvement of PEGylated protein drug conjugates and their clinical performance (Xu et al., 2018a). However, and in spite of the advances obtained in this field, there are still some drawbacks that must be solved, such as the computational cost in terms of infrastructure, and many times, it could be hard to explain what the biological or clinical meaning of features identified using bioinformatics analysis.
RECENT APPROACHES IN THE SITE-SELECTIvE CONJUGATION OF PROTEINS
The chemistry of natural amino acids has been a highly exploited approach in the bioconjugation of proteins. However, there is often poor control over the site and various modifications and incompatibilities with complex mixtures or living systems (Reddington and Howarth, 2015). Since the manipulation of proteins is at the core of biochemical research, the search for new strategies in efficient and specific bioconjugation has been an objective developed by the scientific community through protein engineering. These strategies include for example the SpyTag/ SpyCatcher system.
The Spytag/Spycatcher System
The SpyTag/SpyCatcher system allows the specific and covalent conjugation of proteins through two short polypeptide tags (Zakeri et al., 2012). The larger partner, the SpyCatcher, adopts an immunoglobulin-like conformation that specifically binds the SpyTag (γ-carbon of Asp-117), leading the formation of an extremely resistant intermolecular bond between two amino acid side chains (Gilbert et al., 2017). In this extremely fast method, no exogenous enzymes need to be added or removed (Fisher et al., 2017) and despite its recent description, this system has already been used in the production of synthetic vaccines (Brune et al., 2016), thermo-stable enzymes (Schoene et al., 2016;Wang et al., 2016), and other applications Dovala et al., 2016;Lakshmanan et al., 2016). Take advantage of this system, Gilbert et al. described how the XynA enzyme was genetically encoded to covalently conjugate in culture media, providing a novel and flexible strategy for protein conjugation exploiting the substantial advantages of extracellular self-assembly (Gilbert et al., 2017). Recently, Cayetano-Cruz et al. published a study in which the α-glucosidase Ima1p enzyme of Saccharomyces cerevisiae was attached to the surface of virus-like particles (VLPs) of parvovirus B19 using the SpyTag/ SpyCatcher system. This approach made it possible to obtain a more thermostable enzyme and the modified VLPs were also able to act on glycogen. Hence, these particles may be developed in the future as part of the therapy for the treatment of diseases caused by defects in the human acid α-glucosidase (Cayetano-Cruz et al., 2018). SpyCatcher is large and may be difficult to attach to polymers; therefore, the final product contains a large SpyCatcher protein sequence (Zakeri et al., 2012). It could be a reason why no study to date has been reported using this system to modify proteins with PEGs. However, this is a promising mechanism to create PEGylated proteins, taking advantage of the fact that SpyTag can be placed at the N-terminus, at the C-terminus and at the internal positions of a protein (Zakeri et al., 2012), and previously bound, for instance, to the polymers (PEG) being conjugated.
Ring Opening Polymerization
Ring opening polymerization (ROP) is a reaction, in which the terminal end of a polymer chain acts as a reactive center where additional cyclic monomers can react by opening its ring system, forming a longer polymer chain (Jenkins et al., 1996) with the occurrence of two main reactions: initiation and growth (Penczek and Pretula, 2016). In 2013, Spears et al. used the approach of ROP for first time for the in situ controlled branching of polyglycidol and formation of BSAglycidol bioconjugates with "PEG-like" arms (Spears et al., 2013;Qi and Chilkoti, 2015). Since then, ROP has been used as a methodology to modify various molecules as well as to obtain different varieties of polymers. Ma et al. prepared a cross-linked fluorescent polymer through ROP and performed a subsequent ring opening PEGylation with 4-arm PEG-amine, yielding polymeric nanoparticles in aqueous solution with hydrophilic PEG groups covered at the surface (Ma et al., 2015). Also, Tian et al. (2018) developed smart polymeric materials based on biomimetic PEGylated polypeptoids by combining ring-opening polymerization and a post-modification strategy (Tian et al., 2018). Furthermore, the usefulness of this approach has also been established in the preparation of PEGylated and fluorescent nanoprobes for biomedical applications (Wan et al., 2015;Xu et al., 2018b) and the development of polymeric gene vectors with high transfection efficiency and improved biocompatibility (Xiao et al., 2018). All this demonstrates the potential that ROP could have in the design of PEGylated proteins of biopharmaceutical interest or other molecules used in the diagnosis of different diseases.
Click Chemistry
Click chemistry is another method widely used for PEG attachment to proteins for different purposes (Jølck et al., 2010;Leung et al., 2012;Li et al., 2012;Xu et al., 2015;Huang et al., 2018;Lou et al., 2018). Here, azide and alkyne groups react selectively with each other in the presence of Cu 1+ as the catalyst (Rostovtsev et al., 2002) through the initial reaction of reduced thiols with a maleimide compound containing a click-reactive alkyne moiety. Then, a large PEG molecule containing a complementary clickreactive azide moiety is selectively conjugated to the click-tagged thiols (van Leeuwen et al., 2017). This method is versatile, fast and simple to use, easy to purify, site-specific, and gives high product yields (Hein et al., 2008); however, its drawback is related to the toxicity of copper, even in small amounts. This could limit the development of pharmaceuticals using this methodology; as a result, PEGylation via copper-free click reaction has gained more attention nowadays (Debets et al., 2010;Koo et al., 2012;Lou et al., 2018). The reaction conditions are extremely mild and do not cause protein denaturation, nor are any metals, reducing agents or ligands required.
Non-Covalent PEGylation
Non-covalent PEGylation is an innovative approach in which a chemical reaction between protein and PEG is avoided (Reichert and Borchard, 2016). It is based on the mechanisms of hydrophobic interactions (Mueller et al., 2011a;Mueller et al., 2011b;Mueller et al., 2012), ionic interactions (Khondee et al., 2011), protein polyelectrolyte complex (Kurinomaru and Shiraki, 2015;Kurinomaru et al., 2017), or chelation (Mero et al., 2011). The main advantage of this technique is that it eliminates a potential loss of product due to additional purification processes (Reichert and Borchard, 2016). However, the release of the protein during storage is an important shortcoming for this approach (Santos et al., 2018).
CONCLUDING REMARKS
The covalent attachment of peptides and proteins to polyethylene glycol remains a preferred method for modifying the pharmacokinetic and immunological properties of therapeutic molecules, supported not only by the introduction of PEGylated drugs on the market but also by the increasing number of currently ongoing clinical studies. The chemical versatility of polyethylene glycol derivatives enables the synthesis of various PEGylated protein structures, with a trend to target-specific amino acid residues located at the terminal ends (N or C-terminus) of the peptides or protein of interest, which contributes to obtaining homogeneous and well-defined conjugates. These site-selective modifications must preserve the biological activity of the PEGylated molecule. As part of the development of the science of PEGylation, new methods continue to be implemented based on new approaches, as well as faster and more efficient techniques, such as enzymatic ligation or the development of bio-orthogonal chemistry. As the number and location of PEG chains attached to a protein can affect its activity, it is critical to uncover these important structural details. Thus, strong analytical methods must be developed, allowing for a qualitative and quantitative characterization with a greater degree of robustness and accuracy. In this sense, the computational tools (predictive models based on molecular dynamics) are a great help in clarifying interactions, binding sites or stability of PEGylated proteins in the unending search for and design of new, more effective biopharmaceuticals.
AUTHOR CONTRIBUTIONS
LB and CR-Y: writing of the topic related to the chemical reactions of site-specific pegylation. JBL and ML-E: writing of the topic related to enzymatic pegylation. BE and RC: writing of the topic related to the characterization of pegylated proteins. AP and JF: critical revisions and corrections of the manuscript. | v3-fos-license |
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} | pes2o/s2orc | Evaluation of the Correlation between Focal Adhesion Kinase Phosphorylation and Cell Adhesion Force Using “DEP” Technology
Dielectrophoresis (DEP) is the phenomenon in which a particle, such as a living cell, is polarized and moved by electrical gravity in a non-uniform electric field. In the present study, the DEP force is utilized to act on the cells to induce spatial movement for investigating the correlation between the cell adhesion force and activation level of focal adhesion kinase (FAK). The DEP force produced by the non-uniform electric field was used to measure the cell adhesion force of ECV304 cells, on type 1 collagen (COL1)- and fibronectin (FN)-coated polydimethylsiloxane (PDMS) membranes. For COL1-coating, ECV304 cells revealed weak and variable adhesion force (0.343–0.760 nN) in the first eight hours of incubation. Interestingly, the cell adhesion force of ECV304 at two and five hours of cultivation was significantly high and matched their FAK activation level. In comparison, ECV304 on FN-coated membrane had higher and more stable cell adhesion force (0.577–2.053 nN). FN coating intensified the cell adhesion force of ECV304 with culture time and similar outcome was present on the activation level of FAK. Therefore, this study demonstrated a relationship between cell adhesion force and FAK activation level that was dependant on the choice of the extracellular matrix (ECM) component. Subsequently, two tyrosine kinase inhibitors (AG18 and genistein) and one PI3K inhibitor (LY294002) were applied to study the influence of protein phosphorylation on the cell adhesion force. FAK plays an important role on cell attachment and DEP force measurement is a useful technique for studying cell adhesion.
to microfluidic systems for large-scale separation of thousands of cells [17][18][19]. Like gel electrophoresis, which moves particles in a uniform, constant field has been widely applied for the separation and analysis of a variety of biological particles such as cells, DNA, and viruses, DEP may provide a new technique in cell adhesion measurement. In our present study, we demonstrated that DEP can be used to investigate the interaction between cells and ECM components and FAK regulates cell adhesion force under the stimulus of COL1 and FN.
Materials
Human bladder epithelial cells, ECV304 was obtained from the American Type Culture Collection (ATCC). SYLGARD ® 184 silicone elastomer kit was purchased from Dow Corning (Taipei, Taiwan). All culture materials were purchased from Gibco (Grand Island, NY, USA) and all chemicals of reagent grade were obtained from Sigma (St Louis, MO, USA). Polydimethylsiloxane (PDMS) membranes were prepared with SYLGARD ® 184 silicone elastomer base and SYLGARD® 184 silicone elastomer curing agent in the ratio of 10 to 1. After the polymer mixture was poured into the mould, the mould was placed in a vacuum chamber for 30 min to remove air bubbles and heated to 100 °C within an hour for PDMS solidification. After 1 min of plasma treatment, 50 μL of type 1 collagen (100 mg/mL, 1% w/v) or fibronectin (100 mg/mL, 1% w/v) were spreaded on PDMS membrane for COL1 or FN coating. Finally, we measured the contact angle of PDMS membranes to ensure that the COL1 or FN coating was formed. This was shown by a reduction in the contact angle from 107.6° to 0°.
Theoretical Background on DEP Force
DEP force is a phenomenon in which a force is exerted on a dielectric particle when it is subjected to a non-uniform electric field. The movement of the particles (cells) depends on the cellular properties, working solution, and the strength of the electrical field. The dielectrophoresis force DEP acting on a homogeneous dielectric ellipsoidal particle is [20,21]: where v is the particle (cell) volume, m is the permittivity of the suspending medium, |E rms | 2 is gradient of the root mean square value of the electric field squared, and K n (ω) is a frequency dependent factor. Re[K n ()] is the real part of K n (ω). The frequency dependent factor is given by: (3) and where * and * are the complex permittivities of the suspending medium and particle, respectively. In this case, the cell is akin to a particle and glucose solution is the suspending medium.
A general complex permittivity is defined as * = -j(/) with permittivity ε and conductivity σ.
Where j = √ 1 and ω is the frequency, A n is depolarising factor for the axis n, and s is an arbitrary distance for integration, and r 1 , r 2 , r 3 are the half lengths of the major axes n (n = 1, 2, 3) of the particle. For a sphere, r 1 = r 2 = r 3 and A 1 = A 2 = A 3 = 1/3, the frequency dependent factor in Equation (2) is: where K(ω) is referred to as the Clausius-Mossotti factor. Definitions of the other terms in Equation (4 The direction of the force (positive or negative DEP) is governed by the value of Re[K()], which can be positive or negative, respectively. If Re[K()] is positive, particles move to regions of highest field strength (positive dielectrophoresis). In contrast, negative DEP (Re[K()] < 0) forces cause particles to be repelled from the maximum electric field gradient. In this study the liquid medium is glucose (2% w/v) and Re[K()] was calculated to be +0.999.
Cell Culture
The human bladder epithelial cells, ECV304, were cultured in Medium 199 supplemented with 10% w/v fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin and 100 g/mL streptomycin. For cell adhesion measurement and analysis of FAK phosphorylation, ECV304 cells were cultured on 1% w/v COL1-and 1% w/v FN-coated polydimethylsiloxane (PDMS) membranes and the seeding density was 5,000 cells/cm 2 . Due to the poor hydrophilicity of PDMS, the PDMS membranes required oxygen plasma pretreatment to improve surface tension for the coating of COL1 and FN.
Measurement of Cell Adhesion Force
Cell adhesion force was measured by dielectrophoresis. Figure 1 shows the experimental setup consisting of an optical microscope (Model CK30, Olympus, Japan), a charge coupled device (CCD) camera (Model E-330, Olympus, Japan), a function generator (Model 33220A, Agilent, USA), a power amplifier (Model HSA4012, NF Corporation, Japan), and aluminum (Al) electrodes. The experiment employed micro-processing technology to etch a glass plate with two electrodes of different widths (80 m and 100 m) to produce a non-uniform electric field. To produce the aluminum electrodes, a photo resistor rotary coater was used to create a positive photo resistor S1813 coating (step 1: 500 rpm/5s, step 2: 6,000 rpm/30s) on a glass cover slip, which was subsequently baked in an oven at 115 °C for 25 min. After baking, the cover slip was exposed for 90 s and developed for 40 s, followed by rinsing with deionized (DI) water and aluminum etching with a solution containing 70% phosphoric acid, 3% nitric acid, 14% acetic acid, and 13% water. The photo resistor was finally removed with acetone ( Figure 2(a)). The Al electrodes were about 300 nm thick, with an 80 μm gap between the electrodes. The cover slip of the fabricated electrode was immersed in 95% alcohol for 1 h and then in DI water for 2 h before starting the experiment. For DEP force generation, an electrical potential was applied to the electrodes by a function generator coupled to a power amplifier to produce a non-uniform electric field.
For measurement of cell adhesion force, a single cell was cultured on a 1% COL1-or FN-coated PDMS membrane (Figure 2(c)). At certain time intervals after cell culture, the PDMS membranes were covered with the electrode plate. The cell adhesion force was determined by the cell's movement away from the seeding position when an external electrical field was applied to cause dielectrophoresis. The external AC electrical field (1 MHz) was set at 0 V at the beginning of the experiment and was increased steadily until the cell moved. During the measurement of cell adhesion, the electrical potential was increased at 1/3 V/s from 0 to 10 V and then changed to 1/10 V/s until the focal cell was detached from the PDMS surface ( Figure 2(d)). Cellular detachment was observed on the optical microscope and the images of the cell were recorded through a CCD camera connected to a computer. The cell radii and spreading area were measured by image analysis using the ImageJ software (National Institutes of Health, USA). The electrical potential at which cell detachment occurred was recorded and subsequently converted to the cell adhesion force by a previously published procedure using Equations (1)-(4) [22]. Thirty cells were individually tested at each time point in triplicate.
Immunoprecipitation
For immunoprecipitation from the membrane fraction, the cells were washed twice with ice-cold PBS containing 0.1 M sodium orthovanadate and resuspended in lysis buffer (10 mM Tris-HCl/ 150 mM NaCl/1 mM EDTA). The samples were centrifuged for 15 min at 80,000 g. The pellets were resuspended in lysis buffer with 1% Nonidet P-40 and sonicated for 30 seconds. After centrifugation for 90 min, the supernatants (1 mg of protein per mL) were incubated with anti-FAK antibody for 2 h at 4 °C with gentle shaking. The immune complex was then incubated with protein A/G agarose for 1 h and then collected by centrifugation. The agarose-bound immunoprecipitates were washed and incubated in boiling sample buffer containing 62 mM Tris-HCl (pH 6.7), 1.25% w/v sodium dodecyl sulfate (SDS), 10% v/v glycerol, 3.75% v/v mercaptoethanol, and 0.05% w/v bromophenol blue. The samples were then subjected to Western immunoblotting by a previously published procedure [23].
Statistical Analysis
The results are expressed as mean ± standard error of the mean (SEM). Student's t-test was performed to compare two groups of data, whereas analysis of variance (ANOVA) followed by Scheffe's test were conducted for multiple comparisons. * p values < 0.05 were deemed to be significant.
The Effect of ECM Components (COL1 and FN) on Cell Adhesion Force
Human bladder epithelial cells, ECV304, were used to investigate the influence of FAK on cell adhesion force. The cell detachment voltage was recorded when the cell was completely detached from the membrane surface. When the electric field was supplied on an adhesive cell, the cell morphology was deformed from a spindle figure (cell adhesion stage, Figure 2(c)) to a round shape (after cell detached from membrane surface, Figure 2(d)). In this study, the 3D finite element field modeling software COMSOL 3.4 Multiphysics was used to calculate the electrical field gradient (E) and then converted it to cell adhesion force (Table 1). The cell adhesion force was significantly high at 2, 5 and 7 h of cultivation on the COL1-coating but was variable over the first eight hours (Figure 3(a)). On the contrary, cell adhesion force on FN-coated membrane gradually increased from 0.577 nN to 2.989 nN with time (Figure 3(b)). Thus, FN showed a more direct effect on ECV304 cell adhesion strength.
The Effect of ECM Components (COL1 and FN) on FAK Phosphorylation
ECV304 cells cultured on COL1-coated membrane had high expression of phosphorylated FAK (pFAK) at 2, 5, and 7 h of cultivation (Figure 4(a)). In comparison, the effect of FN coating on FAK phosphorylation of ECV304 was strongly expressed from the fourth hour of cell cultivation and the expression of pFAK was remained high thereafter (Figure 4(b)). The chronological trends of FAK phosphorylation followed closely with those of cell adhesion force for both types of coated membranes. Thus cell adhesion was closely associated with FAK activation.
The Effect of Tyrosine Kinase Inhibitors on FAK Phosphorylation and Cell Adhesion Force of ECV304 Cells
Two general tyrosine kinase inhibitors, AG18 and genistein were used to investigate the correlation between FAK phosphorylation and cell adhesion force [25,26]. Since the effect of COL1-coating on FAK activation was significant at 2 and 5 h of cultivation, FAK expression inhibition and cell adhesion force reduction were investigated at those times on COL1-coated membranes. Similarly, FAK phosphorylation at 5 and 8 h was investigated in FN-coated membranes. In both cases, FAK expression at the first hour was the control.
As shown in Figure 5(a,b), AG18 (100 M) inhibited the phosphorylation of FAK at 2 and 5 h of cultivation on COL1-coated membranes and at 5 and 8 h of cultivation on FN-coated membranes. Similarly, genistein (60 M) showed the complete inhibition of FAK phosphorylation in both COL1-or FN-coated membranes. Thus both AG18 and genistein effectively inhibited FAK phosphorylation. Furthermore, Heinz et al. reported that the FAK-induced cell movement must be completed by phosphatidylinositol-3-kinase (PI3K; 110 kDa) [27]. Thus, PI3K has an important role in cell migration. Though this study primarily focused on the activation of FAK, PI3K also showed signs of activation at different time points during cultivation. However, the addition of LY294002 (PI3K Inhibitor, 50 M) only inhibited FAK phosphorylation at the fifth hour of activation in COL1-coated membranes (Figure 6(a)). Moreover, LY294002 had no inhibitory effect on FAK phosphorylation in FN-coated membrane (Figure 6(b)). AG18 showed a significant decrease in cell adhesion force at the second hour of cell cultivation in COL1-coated membranes ( Figure 5(c)). On the other hand, cell adhesion force was reduced at the eighth hour of cell cultivation in FN coated membranes ( Figure 5(d)). The addition of genistein also decreased cell adhesion force in both COL1-and FN-coated membranes at the times investigated. The reduction of cell adhesion force through tyrosine kinase inhibitors confirmed that cell adhesion was highly correlated with FAK phosphorylation. However, LY294002 (PI3K inhibitor) did not lead to an obvious decrease in cell adhesion force ( Figure 6(c,d)). Thus cell adhesion may not be mediated by PI3K.
Discussion
Cell adhesion is intimately related to cellular characteristics and processes such as morphology, migration, growth, and differentiation. There is increasing evidence that FAK is a key factor in mediating cell adhesion force [6][7][8]. Michael et al. used sheer stress to study the relationship between cell adhesion force and FAK expression. FAK expression in FAK-null cells promoted cell adhesion force by integrin activation [8]. In the present study, DEP force measurement and quantitative biochemical methods were employed in the present study to examine the relationship between FAK activation and cell adhesion force in different ECM coatings. The adhesion force of ECV304 cells in COL1-coated membranes was variable (Figure 3(a) and Table 1). Cell adhesion force was high at 2, 5, and 7 h of cell cultivation, with maximal force achieved at 2 h. On the contrary, cell adhesion force in FN-coated membranes increased with time (Figure 3(b) and Table 1). Cell adhesion force was high at 2, 4, and 7 h of cultivation, reaching maximum at 7 h. In addition, cells cultured in both coating systems showed consistent trends between cell adhesion force and FAK phosphorylation at various time points. Tyrosine kinase inhibitors, AG 18 and genisteine, not only inhibited FAK phosphorylation, but also reduced cell adhesion. Cell detachment force of indicated time points was detected by DEP force measurement system normalized to cell adhesion strengthening at first hour cultivation (which was assigned a value of 1 arbitrary unit). * p < 0.05 versus control (first hour).
Generally, ECM components such as COL1 and FN bind different types of integrins on the cell surface to influence many aspects of cellular behavior, including differentiation, motility, growth, and survival. Therefore, linkage between integrins and ECM components mediates the adhesion between cells and their environments (e.g., surface materials). Previous studies showed that an increase in cell spreading area was accompanied by higher adhesion force [28]. Thus, the cell radius and spreading area of ECV304 on COL1 and FN coated membranes was recorded each hour by optical microscopy (Table 1). Interestingly, the cell morphologies on COL1-and FN-coated membranes were different but there was no major variation on cell radius or spreading area. In addition, the radii of ECV 304 cells increased from 12-15 μm in the first hour to 12-19 μm in the second hour, with no significant change thereafter. Although cell radii are factors in the calculation of cell adhesion force (Equation (1)), the variation in the radii in the present study may not be significant enough to influence cell adhesion force or FAK phosphylation level. The present study showed that ECV304 cells on FN-coated membranes had stronger cell adhesion (0.577-2.053 nN) than the COL1 counterparts (0.343-0.760 nN) over the first eight hours of cell cultivation. There were higher fluctuations in cell adhesion force for COL1-coated membranes whereas that in FN-coated ones increased with time. Gallant et al. and Elineni et al. mentioned that cell adhesion force was contributed by focal adhesion size, integrin binding and focal adhesion assembly [29,30]. These findings indicate that different ECM components may vary cell adhesion strength through focal adhesion size, distinct types of integrin binding and focal adhesion assembly for cell spreading, migration and growth.
Cell adhesion force has previously been measured with different methods. Michael et al. (2009) measured it by detaching human dermal fibroblasts from PDMS surfaces using a spinning disk and obtained an adhesion force of 10.5 nN (converted from 232 dyn/cm 2 for a cell radius of 12 μm). Weder also measured it by detaching human osteosarcoma cells, Saos-2 from cell culture dishes using AFM, reporting results of 0.88-1.2 nN [31]. Similar adhesive forces (1-3 nN) between trophoblasts and uterine epithelium were measured by AFM [32]. The cell adhesion forces of ECV304 cells measured in the current study are within the same orders of magnitude to those obtained in the aforementioned studies (Table 1). On the other hand, the DEP force measurement system can record the cell morphology in real-time and measure many samples in a short time more efficiently than the spinning disk and AFM methods. Therefore, DEP force measurement is a viable alternative method to quantify cell-ECM adhesion.
The importance of FAK in regulating cell adhesion force provides new insights on cellular surface-binding models. FAK was identified as a signal transducer mediating the physical properties of the substrate in fibroblasts [33]. In addition, FAK is required for the adhesion of colon cancer cells to liver sinusoids and lung capillaries [34,35]. In the present study, FAK activation was demonstrated to be highly correlated to cell adhesion force. These findings are similar to those reported by Michael et al. (2009). When AG18 was added, FAK activation at the second hour of cell cultivation in COL1-coated membranes and the eight hour of cell cultivation in FN-coated ones were inhibited. These observations were mirrored by reductions in cell adhesion force at the corresponding time points. Similarly, genistein inhibited the activation of FAK and cell adhesion in both coating systems. Conversely, LY294002 did not lead to a significant decrease in cell adhesion force. In fact, cell adhesion force increased at certain time points. These results indicated that LY294002 did not affect cell adhesion.
Conclusions
This study demonstrated the feasibility of using DEP force measurement to investigate cell adhesion force. The data indicated that cell adhesion force of ECV304 cells was highly associated with FAK phosphorylation levels through the addition of inhibitors, AG18 and genistein. Furthermore, the effect of the extracellular matrix (ECM) component on cell adhesion strength and stability can be quantified efficiently by DEP force measurement. | v3-fos-license |
2019-08-06T13:03:15.904Z | 2019-08-05T00:00:00.000 | 199436336 | {
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} | pes2o/s2orc | Poly (ADP‐ribose) polymerase inhibition protects against myocardial ischaemia/reperfusion injury via suppressing mitophagy
Abstract Myocardial ischaemia/reperfusion (I/R) injury attenuates the beneficial effects of reperfusion therapy. Poly(ADP‐ribose) polymerase (PARP) is overactivated during myocardial I/R injury. Mitophagy plays a critical role in the development of myocardial I/R injury. However, the effect of PARP activation on mitophagy in cardiomyocytes is unknown. In this study, we found that I/R induced PARP activation and mitophagy in mouse hearts. Poly(ADP‐ribose) polymerase inhibition reduced the infarct size and suppressed mitophagy after myocardial I/R injury. In vitro, hypoxia/reoxygenation (H/R) activated PARP, promoted mitophagy and induced cell apoptosis in cardiomyocytes. Poly(ADP‐ribose) polymerase inhibition suppressed H/R‐induced mitophagy and cell apoptosis. Parkin knockdown with lentivirus vectors inhibited mitophagy and prevented cell apoptosis in H/R‐treated cells. Poly(ADP‐ribose) polymerase inhibition prevented the loss of the mitochondrial membrane potential (ΔΨm). Cyclosporin A maintained ΔΨm and suppressed mitophagy but FCCP reduced the effect of PARP inhibition on ΔΨm and promoted mitophagy, indicating the critical role of ΔΨm in H/R‐induced mitophagy. Furthermore, reactive oxygen species (ROS) and poly(ADP‐ribosylation) of CypD and TSPO might contribute to the regulation of ΔΨm by PARP. Our findings thus suggest that PARP inhibition protects against I/R‐induced cell apoptosis by suppressing excessive mitophagy via the ΔΨm/Parkin pathway.
| INTRODUC TI ON
Reperfusion therapy is the most effective treatment for acute myocardial infarction, reduces ischaemic injury and limits the infarct size.
However, reperfusion can independently induce myocardial injury including cardiomyocyte death, leading to expansion on the scope of myocardial infarction. 1 Myocardial ischaemia/reperfusion (I//R) injury may account for up to 50% of the infarct size. 1 Despite great effort to optimize reperfusion conditions, the means of reducing I/R injury-associated cell death are very limited. 2 Several critical factors mediate the detrimental effects of I/R-induced injury, including oxidative stress, intracellular Ca 2+ overload, inflammation and mitochondrial permeability transition pore (MPTP) opening. 1,2 The opening of the MPTP is related to poly(ADP-ribose) polymerase (PARP) activation, and PARP inhibition prevents myocardial I/R injury. 3,4 PARP is a highly conserved DNA-binding nuclear enzyme family that can be activated by DNA damage. Poly(ADP-ribose) polymerase plays an important role in the regulation of multiple physiological cellular functions including DNA repair, transcription, cell cycle, cell death and genomic integrity. 5 The activation of PARP initiates an energy-consuming cycle by transferring ADP-ribose units from nicotinamide adenine dinucleotide (NAD) to form long branches of ADP-ribose polymers (PAR) on glutamic acid residues of a number of target proteins. 6 Mitochondria generate most of the energy for the heart via oxidative phosphorylation. To maintain a healthy and functional mitochondrial network, dysfunctional or damaged mitochondria are eliminated via a process known as mitochondrial autophagy or mitophagy, which is triggered by starvation, hypoxia and reactive oxygen species (ROS). 7,8 Mitophagy has been classified into canonical and non-canonical pathways in the heart. 8 The Parkin-dependent pathway is the main form of canonical mitophagy. 9 Poly(ADP-ribose) polymerase activation has been shown to prevent mitophagy in xeroderma pigmentosum group A-deficient cells, and PARP inhibition by the inhibitor olaparib induces mitophagy in BRCA1 and BRCA2 mutant breast cancer cells. 10,11 However, whether PARP activation regulates mitophagy in I/R-injured cardiomyocytes remains unclear. This study showed that PARP inhibition attenuated I/R-or hypoxia/oxygenation (H/R)-induced mitophagy and cell apoptosis in vivo and in vitro.
| Reagents and antibodies
The Parkin-siRNA lentivirus and control lentivirus were con-
| Mice experiments
Acute myocardial I/R model was performed on adult male C57BL/6 mice (10-12 weeks) by ligating the left anterior descending artery (LAD). Mice were anaesthetized with isoflurane and ventilated using a Rodent Anesthesia Machine. After taped to a heating pad in the supine position, mice chest was opened at the third intercostal space and the heart was exposed by squeezing. A 6-0 silk suture was passed under the LAD 1-2 mm from the tip of the left atrium. Left anterior descending artery was ligated with a slipknot. The occlusion was maintained for 30 minutes and then the knot was released to reperfuse the heart for 120 minutes. 12 To determine the myocardial infarct size, hearts were collected and sectioned into 2-3 mm slices.
| Electron microscopy
Sections of myocardium or cell on coverslips were fixed in 2.5% glutaraldehyde overnight. After rinsing in 0.1 mmol/L cacodylate buffer with 1% tannic acid, the samples were immersed in 1% osmium tetroxide in 0.1 mmol/L cacodylate buffer for 1 hour. After being rinsed again, the samples were dehydrated with alcohol and embedded in Epon 812. Then, the samples were examined using a transmission electron microscope.
| Mitochondria isolation
Mitochondria from cultured H9C2 cells were isolated with the Mitochondria Isolation Kit (Thermo Scientific) referred to the manufacturer's instruction.
| Measurement of mitochondrial membrane potential (ΔΨm)
ΔΨm was determined using a commercial assay kit by incubation with JC-1(Beyotime) in serum-free medium for 20 minutes at 37°C. Then cells were washed with JC-1 staining buffer and imaged under a fluorescence microscope (Olympus). Normal mitochondria produce red fluorescence, and depolarized or inactive mitochondria produce green fluorescence. ΔΨm was calculated by the red/green fluorescence ratio.
| ROS production
Reactive oxygen species were determined with the Reactive Oxygen Species Kit (Beyotime). According to the instruction, cells were incubated with DCFH-DA for 20 minutes at 37°C. The ROS level was examined in a Thermo Fisher Varioskan Flash spectral scanning multimode reader.
| Immunoprecipitation
Poly(ADP-ribosylation) of CypD and TSPO was detected using immunoprecipitation method. 13 Lysates of H9C2 cells were prepared with NP-40 Lysis Buffer (Bosterbio). For the immunoprecipitation studies, 800 μg proteins were incubated with 10 μg PAR antibody followed by incubation with 60 μl protein A/G agarose (Santa Cruz Biotechnology) and the pellets were washed four times. Then, beads were added 40 μl 2 × SDS-PAGE loading buffer and boiled for 5 minutes to elute the immunocomplexes. Supernatants were subjected to SDS-PAGE and analysed for CypD and TSPO. Similar procedures were performed to determine the protein-protein interaction between PARP-1 and CypD or TSPO.
| NAD and ATP measurements
Nicotinamide adenine dinucleotide was measured with a NAD/ NADH Assay Kit (Beyotime), and ATP was detected with an ATP Assay Kit (Beyotime). The level of NAD and ATP was normalized to total protein content which was determined by the bicinchoninic acid method.
| Statistical analysis
All data were presented as the mean ± SEM and analysed by either one-way ANOVA or a two-tailed Student's t test. The null hypothesis was rejected at P < .05.
| PARP inhibition reduces infarct size and suppresses mitophagy in I/R-injured hearts
The role of PARP inhibition in myocardial I/R injury was investigated using the LAD ligation model. I/R-induced PARP activation and the inhibitor, DPQ, effectively inhibited PARP activity, as indicated by PAR expression ( Figure 1A). As expected, PARP inhibition limited the increase of the infarct size compared with the I/R group ( Figure 1B).
Moreover, PARP inhibition improved the cardiac function after IR injury, as indicated by LVEF ( Figure 1C)
| PARP inhibition prevents cell apoptosis and mitophagy in H/R-treated cardiomyocytes
To test the effects of PARP inhibition on cell apoptosis and mitophagy after I/R injury in vitro, we subjected H9C2 cells to hypoxia/reoxygenation (H/R) treatment. Figure 2A
| Knockdown of Parkin prevents mitophagy and cell apoptosis in H/R-treated cardiomyocytes
To determine the relationship between mitophagy and cell apoptosis in H/R-treated H9C2 cells, Parkin was knocked down with lentivirus-RNAi in H9C2 cells. Figure 3A
| PARP inhibition prevents H/R-induced mitophagy by regulating the mitochondrial membrane potential (ΔΨm)
Opening of the MPTP causes the loss of ΔΨm, which triggers PINK1/ Parkin-mediated mitophagy. 15,16 ΔΨm was obviously reduced by H/R injury, and mitochondrial membrane depolarization was restored by PARP inhibition (Figure 4A,B). To ensure the critical role of ΔΨm in mitophagy in H/R-injured H9C2 cells, we pre-incubated H9C2 cells with cyclosporin A, which is a potent inhibitor of the MPTP. Figure 4C shows that cyclosporin A prevented the disruptive effect of H/R on ΔΨm. Similar to PARP inhibition, cyclosporin A prevented mitophagy in H/R-injured H9C2 cells ( Figure 4D,E). FCCP is an oxidative phosphorylation uncoupler that depolarizes the mitochondrial membrane. 17 It was found that the effect of PARP inhibition on ΔΨm
| ROS contributes to the PARP-mediated decline in ΔΨm
Excessive ROS trigger the opening of the MPTP and activate PINK1/ Parkin-mediated mitophagy. 18 To investigate whether ROS play a role in PARP-mediated changes in ΔΨm, we measured ROS production in H/R-treated H9C2 cells. The production of ROS was significantly increased in H/R-treated H9C2 cells compared with that in control cells, and PARP inhibition partially prevented the increase in ROS production after H/R ( Figure 5A). The reductant NAC reduced the production of ROS ( Figure 5B). More importantly, we found that NAC prevented the H/R-induced decline in ΔΨm ( Figure 5C,D). Thus, ROS have an effect, at least partially on the changes in ΔΨm mediated by PARP after H/R.
| Poly-ADP-ribosylation of CypD and TSPO may also contribute to the PARP-mediated decline in ΔΨm
Poly(ADP-ribose) polymerase activation induces poly-ADP-ribosylation of mitochondrial proteins after I/R. 19 Immunoblotting indicated that more PAR was found in proteins of isolated mitochondria after H/R injury ( Figure 6A) Figure 6B,C). However, we did not find a direct interaction between PARP-1 and CypD or TSPO ( Figure S4). These data demonstrate that PARP might regulate the opening of the MPTP by directly modifying CypD and TSPO by poly-ADP-ribosylation.
| D ISCUSS I ON
In the present study, we found that PARP inhibition prevents I/R injury-induced mitophagy and cell apoptosis in cardiomyocytes. Consistent with previous studies, 23,24 we also found that PARP inhibition prevented I/R-induced cell apoptosis in vivo and in vitro.
Mitophagy inhibition due to knockdown of Parkin decreases
Mitophagy facilitates the normal turnover of mitochondria and becomes more important during exposure to stress including I/R injury. Although the role of mitophagy in myocardial I/R injury has been discussed in several studies, it needs to be further explored because of conflicting conclusions. 25 ΔΨm. 18 = 3). B, Immunoprecipitation using a PAR antibody showed that CypD was modified by poly(ADP-ribosylation). (n = 5). C, Immunoprecipitation using a PAR antibody showed that TSPO was modified by poly(ADP-ribosylation). (n = 5) However, it is unclear whether MPTP opening is related to the poly-ADP-ribosylation of relative proteins. The protein components of the MPTP still need to be explored. CypD and TSPO are potent components that act as regulators of MPTP opening. 21,34 Down-regulation of CypD prevents MPTP opening and protects against the loss of ΔΨm. 35 Conversely, the loss function of TSPO causes MPTP opening and leads to a reduced ΔΨm. 21,36 Our results showed that both CypD and TSPO could be poly-ADP-ribosylated, but the effects of this modification on the functions of CypD and TSPO were not studied. As reported, poly-ADP-ribosylation can serve as a marker for ubiquitinproteasomal system (UPS)-dependent protein degradation. 37 Thus, we speculate that the function of CypD and TSPO might be changed by poly-ADP-ribosylation, which should be investigated in the future.
In conclusion, our study is the first to report that PARP inhi-
CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest. Pan and Feng Xu analysed the data. All authors read and approved the version of the manuscript to be published.
DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request. | v3-fos-license |
2019-04-04T13:04:41.968Z | 2008-01-01T00:00:00.000 | 93301696 | {
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} | pes2o/s2orc | Effects of expander conditioning on the nutritional value of diets Effects of expander conditioning on the nutritional value of diets with dried distillers grains with solubles in nursery and finishing with dried distillers grains with solubles in nursery and finishing pigs pigs
production rate was greater ( P < 0.005) for the corn-soybean meal diets than for diets with DDGS. However, pellet durability was improved ( P < 0.001) by addition of DDGS to the diets. Pigs fed corn-soybean meal diets had better ( P < 0.03) overall ADG and F/G than pigs fed diets with DDGS. Ex-pander conditioning did not affect ADG ( P > 0.83) but improved overall F/G and dressing percentage ( P < 0.007). In Exp. 3, 192 finishing pigs (average weight 222 lb) were assigned to 16 pens to determine nutrient di-gestibility. Treatments were the same as in Exp. 2. Feed and water was consumed ad libitum during a 6-d adjustment period; then, feces were collected for 2 d. Corn-soybean meal diets had greater ( P < 0.001) digestibility of DM, N, and GE than diets with DDGS, and expander conditioning improved ( P < 0.02) digestibility of DM, N, and GE compared with standard conditioning. However, the improved digestibility of DM with expander conditioning was apparent primarily for the DDGS diets (diet × conditioning interaction, P < 0.01). In conclusion, expanding diets improved ADG, F/G, and nutrient digestibility in nursery pigs and F/G, dressing percentage, and nutrient di-gestibility in finishing pigs fed diets without and with DDGS.
Summary
Three experiments were conducted to determine the effects of expander conditioning on nutritional value of diets without and with corn-and sorghum-based dried distillers grains with solubles (DDGS).In Exp. 1, 180 nursery pigs (average weight 29 lb) were assigned to 30 pens.Treatments were arranged as a 3 × 2 factorial with main effects of diet formulation (corn-soybean meal vs. 30% cornor sorghum-based DDGS) and conditioning (standard steam vs. expander) prior to pelleting.Pigs fed corn-soybean meal diets had better (P < 0.005) ADG, F/G, and digestibility of DM, N, and GE than pigs fed diets with DDGS.Diets with corn-based DDGS supported better (P < 0.03) ADG, F/G, and digestibility of DM and N than diets with sorghum-based DDGS.Expander processing improved (P < 0.009) ADG, F/G, and digestibility of DM, N, and GE compared with standard conditioning.Pigs fed diets with sorghumbased DDGS showed the greatest response in F/G to expander conditioning leading to a DDGS source × conditioning interaction (P < 0.008).In Exp. 2, 176 finishing pigs (average weight 164 lb) were assigned to 16 pens.Treatments were arranged as a 2 × 2 factorial with main effects of diet formulation (cornsoybean meal vs. 40% sorghum-based DDGS) and conditioning (standard steam vs. expander) prior to pelleting.Net electrical en-ergy required for feed processing was lower (P < 0.001) and production rate was greater (P < 0.005) for the corn-soybean meal diets than for diets with DDGS.However, pellet durability was improved (P < 0.001) by addition of DDGS to the diets.Pigs fed corn-soybean meal diets had better (P < 0.03) overall ADG and F/G than pigs fed diets with DDGS.Expander conditioning did not affect ADG (P > 0.83) but improved overall F/G and dressing percentage (P < 0.007).In Exp. 3, 192 finishing pigs (average weight 222 lb) were assigned to 16 pens to determine nutrient digestibility.Treatments were the same as in Exp. 2. Feed and water was consumed ad libitum during a 6-d adjustment period; then, feces were collected for 2 d.Corn-soybean meal diets had greater (P < 0.001) digestibility of DM, N, and GE than diets with DDGS, and expander conditioning improved (P < 0.02) digestibility of DM, N, and GE compared with standard conditioning.However, the improved digestibility of DM with expander conditioning was apparent primarily for the DDGS diets (diet × conditioning interaction, P < 0.01).In conclusion, expanding diets improved ADG, F/G, and nutrient digestibility in nursery pigs and F/G, dressing percentage, and nutrient digestibility in finishing pigs fed diets without and with DDGS.Key words: digestibility, dried distillers grains with solubles, expander conditioning
Introduction
The U.S. Renewable Fuel Standard mandates that 15 billion gal of ethanol from grain starch will be needed by 2015.Thus, it seems certain that coproducts from the ethanol industry, such as dried distillers grains with solubles (DDGS), will continue to increase in supply and use in diets for pigs.Dried distillers grains with solubles have about three times as much fiber as the cereals from which they are produced.In the 2007 Swine Day Report of Progress, (Feoli et al.,page 126 and Feoli et al.,page 131), we suggested that addition of high levels of DDGS in diets for nursery and finishing pigs had negative effects on nutrient digestibility and growth rate.Previous experiments from our laboratory have shown that conditioning wheat midds-based diets high in fiber in an expander prior to pelleting improved nutrient digestibility in nursery and finishing pigs.Therefore, the objective of the present experiments was to determine the effects of expander conditioning on the nutritional value of diets with and without DDGS.
Procedures
For Exp. 1, 180 nursery pigs (42 d old and initially 29 lb) were used in a 14-d growth assay.The pigs were sorted by sex and ancestry, blocked by weight, and assigned to pens.There were 3 gilts and 3 barrows in each pen and 5 pens per treatment.The pigs were housed in an environmentally controlled nursery having 4-ft × 4-ft pens with woven-wire flooring.Each pen had a self-feeder and nipple waterer to allow ad libitum consumption of feed and water.
3 Used as an indigestible marker.
Diets were either steam conditioned to 180°F or expanded conditioned (302°F, 200 PSI) before passing into a pelleting press (30 HD Master Model, California Pellet Mill, San Francisco, CA) equipped with a 7/8-in.-thickdie having 5/32-in.openings.Samples of the processed diets were collected, and pellet durability index (PDI) was determined by using the tumbling-box technique (ASAE S269.4 DEC1991).Additionally, the PDI procedure was modified to induce more stress on the pellets by adding 5 hexagonal nuts into the tumbling box.
Pigs and feeders were weighed on d 0 and 14 to allow calculation of ADG, ADFI, and F/G.Feces were collected on d 4 and 5 from no less than 3 pigs per pen to allow determination of apparent digestibility for DM, N, and GE.
Data were analyzed as a randomized complete block design (initial weight as a covariate) by using the MIXED procedure of SAS.Orthogonal contrasts were used to separate treatment means with comparisons of (1) control vs. DDGS diets, (2) corn-vs.sorghumbased DDGS, 3) standard vs. expander conditioning, (4) corn-soy vs. DDGS × standard vs. expander conditioning, and (5) corn-vs.sorghum-based DDGS × standard vs. expander conditioning.
For Exp. 2, 176 finishing pigs (initially 164 lb) were used in a 54-d growth assay.The pigs were sorted by sex and ancestry, blocked by weight, and assigned to pens.There were 11 pigs per pen and 4 pens per treatment.The pigs were housed in an environmentally controlled finishing facility having 6-ft × 16-ft pens with half solid and half slatted concrete flooring.Each pen had a self-feeder and nipple waterer to allow ad libitum consumption of feed and water.
Treatments were arranged as a 2 × 2 factorial with main effects of diet formulation (corn-soybean meal vs. 40% sorghum-based DDGS) and conditioning (standard steam vs. expander) prior to pelleting.All diets (Table 2) were formulated to 0.90% lysine, 0.60% Ca, and 0.50% P. Feed was processed as in Exp. 1, but 6 batches of feed were made, and more extensive processing data were collected.Voltage and cone pressure of the expander were kept constant at 250 volts and 200 PSI, respectively.Then, motor load and production rate for the pellet mill, net electrical consumption for the pellet mill and the expander, and PDI were measured and analyzed as a randomized complete block design by using the MIXED procedure of SAS with batch as the blocking criterion.Orthogonal contrasts for a 2 × 2 factorial were used to separate means for the main effects of diet formulation and conditioning.
Pigs and feeders were weighed on d 0, 26, and 54 to allow calculation of ADG, ADFI, and F/G.The pigs were slaughtered (average weight 287 lb) at a commercial slaughter facility, and carcass data were collected.Growth performance and carcass data were analyzed as a randomized complete block design by using the MIXED procedure of SAS with initial weight as the blocking criterion and pen as the experimental unit.Hot carcass weight was used as a covariate in analysis of data for dressing percentage, carcass lean percentage, backfat thickness, and loin depth.Orthogonal contrasts for a 2 × 2 factorial were used to separate treatment means with main effects of diet formulation and conditioning.
For Exp. 3, 176 finishing pigs (initially 222 lb) were used in an 8-d digestibility study.The pigs were sorted by sex and ancestry, blocked by weight, and assigned to pens.There were 11 pigs per pen and 4 pens per treatment.The pigs were housed in an environmentally controlled finishing facility having 6-ft × 16-ft pens with half solid and half slatted concrete flooring.Each pen had a selffeeder and nipple waterer to allow ad libitum consumption of feed and water with pigs and feeders weighed on d 0 and 8. Feces were collected on d 7 and 8 from no less than 6 pigs per pen.Concentrations of DM, N, GE, and Cr in the diets and feces were determined to allow for calculation of apparent digestibilities.Treatments and diets were the same as in the growth assay (Exp.2).
Data were analyzed as a randomized complete block design by using the MIXED procedure of SAS with initial weight as the blocking criterion and pen as the experimental unit.Orthogonal contrasts for a 2 × 2 factorial were used to separate means for the main effects of diet formulation and conditioning.
Results and Discussion
In Exp. 1 (Table 3), the corn-soybean meal diets supported better (P < 0.005) ADG, AD-FI, F/G, and digestibility of DM, N, and GE, than diets with DDGS.Also, pigs fed diets with corn-based DDGS had better (P < 0.03) ADG, F/G, and digestibility of DM and N than pigs fed diets with sorghum-based DDGS.Expander conditioning improved (P < 0.009) ADG, F/G, and digestibility of DM, N, and GE compared with standard conditioning.However, expander conditioning tended to improve digestibility of DM most in diets with DDGS as indicated by the corn-soybean meal vs. DDGS × standard vs. expander conditioning interaction (P < 0.08).Finally, pigs fed diets with sorghum-based DDGS had greater improvement in F/G with expander conditioning than pigs fed diets with corn-based DDGS (corn-vs.sorghum-based DDGS × standard vs. expander; P < 0.008).
Milling data (Table 4) for the finishing diets used in Exp. 2 indicated that addition of 40% DDGS decreased pellet mill throughput (i.e., production rate) and increased energy used in the pelleting process (P < 0.005).However, contrary to some reports, high inclusion of DDGS improved PDI (P < 0.001).As for pig growth (Table 5), adding 40% DDGS to diets for finishing pigs reduced (P < 0.02) overall ADG and ADFI and increased (P < 0.03) overall F/G.Expander conditioning improved (P < 0.002) F/G, but this response was consistent for diets with and without DDGS (i.e., no diet formulation × expander conditioning interaction; P > 0.41).
Pigs had lower (P < 0.001) HCW when fed diets with 40% DDGS.Even when corrected to a constant HCW (via covariate analysis), dressing percentage (P < 0.03) and loin depth (P < 0.06) were greater for pigs fed the corn-soybean meal diets than for pigs fed the DDGS treatments.However, half the loss in HCW and all the loss in dressing percentage were recovered when diets with DDGS were expander processed prior to pelleting.
Nutrient digestibility in finishing pigs (Table 6) was greater (P < 0.001) for pigs fed diets without DDGS.Expander conditioning improved (P < 0.02) digestibility of N and GE compared with standard conditioning, but digestibility of DM was improved with expander conditioning only in the DDGS diets (diet × conditioning interaction, P < 0.01).
In conclusion, adding 30 and 40% DDGS to nursery and finishing diets decreased growth performance and nutrient digestibility compared with a corn-soybean meal control.However, expanding diets improved ADG, F/G, and nutrient digestibility in nursery pigs and F/G, dressing percentage, and nutrient digestibility in finishing pigs fed diets without and with DDGS.
Table 1 . Composition of nursery diets
1 Dried distillers grains with solubles.
Table 4 . Effects of expander conditioning of finishing diets with corn-and sorghum-based dried dis- tillers grains with solubles (DDGS) on production efficiency (Exp. 2) 1
Each diet was replicated by manufacturing a new batch of feed 6 times. 2 Standard conditioning prior to pelleting.3Expanderconditioningpriortopelleting.4Measuredattheexit of the standard conditioner and at the expander cone.5Dashesindicate P > 0.15. 6Pellet durability index (ASAE S269.4 DEC1991). 7Modified by adding 5 hexagonal nuts (1/2-in.diameter) to the tumbling box. | v3-fos-license |
2020-01-30T09:13:55.385Z | 2020-01-26T00:00:00.000 | 210945741 | {
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} | pes2o/s2orc | Deficiency in Dipeptidyl Peptidase-4 Promotes Chemoresistance Through the CXCL12/CXCR4/mTOR/TGFβ Signaling Pathway in Breast Cancer Cells
Dipeptidyl peptidase (DPP)-4, a molecular target of DPP-4 inhibitors, which are type 2 diabetes drugs, is expressed in a variety of cell types, tissues and organs. DPP-4 has been shown to be involved in cancer biology, and we have recently shown that a DPP-4 inhibitor promoted the epithelial mesenchymal transition (EMT) in breast cancer cells. The EMT is known to associate with chemotherapy resistance via the induction of ATP-binding cassette (ABC) transporters in cancer cells. Here, we demonstrated that deficiency in DPP-4 promoted chemotherapy resistance via the CXCL12/CXCR4/mTOR axis, activating the TGFβ signaling pathway via the expression of ABC transporters. DPP-4 inhibition enhanced ABC transporters in vivo and in vitro. Doxorubicin (DOX) further induced ABC transporters in DPP-4-deficient 4T1 cells, and the induction of ABC transporters was suppressed by either the CXCR4 inhibitor AMD3100, the mTOR inhibitor rapamycin or a neutralizing TGFβ (1, 2 and 3) antibody(N-TGFβ). Knockdown of snail, an EMT-inducible transcription factor, suppressed ABC transporter levels in DOX-treated DPP-4-deficient 4T1 cells. In an allograft mouse model, however, the effects of DOX in either primary tumor or metastasis were not statistically different between control and DPP-4-kd 4T1. Taken together, our findings suggest that DPP-4 inhibitors potentiate chemotherapy resistance via the induction of ABC transporters by the CXCL12/CXCR4/mTOR/TGFβ signaling pathway in breast cancer cells.
Introduction
Diabetes is associated with an increased risk of diverse cancers, and diabetic medicine may influence cancer biology [1][2][3][4]. In particular, diabetic patients need to take diabetic medicine for a long period of time and establishing a safety profile of diabetic drugs is essential for diabetic research. The dipeptidyl peptidase (DPP)-4 inhibitor increases glucagon-like peptide (GIP-1) and promotes insulin secretion in type 2 diabetic patients [5]. However, DPP-4 cleaves many growth factors, chemokines and neuropeptides that may promote human malignancies, and DPP-4 inhibitors potentially increase these substances [6][7][8][9].
There are controversial discussions regarding the biology of DPP-4 in malignancies such as human glioma [7], prostate cancer [10], melanomas [11] and nonsmall cell lung cancer (NSCLC) [12]. C-X-C motif chemokine 12 (CXCL12), the substrate of DPP-4, and its receptor CXCR4 have been associated with the biology of cancer, such as tumor proliferation, survival, invasion and angiogenesis [13]. In this context, DPP-4 could cleave/inactivate CXCL12 and downregulate the CXCL12/CXCR4 axis, subsequently inhibiting cancer cell growth, invasiveness and metastasis [14,15]. Furthermore, a study suggested that DPP-4 might be involved in increased sensitivity of epithelial ovarian carcinoma cells to chemotherapy [16]. We have recently shown that a DPP-4 inhibitor accelerated the epithelial mesenchymal transition (EMT) and lung metastasis via the CXCL12/CXCR4/mTOR axis in breast cancer cells [17]. Signaling through mTOR is vital for proliferation and survival in cancers and plays a central role in adaptive resistance to inhibitors of oncogenic signaling pathways [18,19]. Transforming growth factor-β (TGFβ) signaling, as a tumor suppressor, inhibits cell proliferation but blocks chemotherapy sensitivity. Recent studies have presented evidence that TGFβ signaling acts as a novel antidrug therapeutic target in breast cancer [20] and colorectal cancer [21].
Worldwide, breast cancer is the most common cancer and the leading cause of cancer death in women [22]. Chemotherapy is widely used to treat breast cancer patients [23], and most cancer-related deaths are linked to resistance to chemotherapy. Multidrug resistance (MDR) has been considered as a major determinant in cancer chemotherapy [24][25][26]. ATP-binding cassette (ABC) transporters are the largest family of transmembrane proteins and play a vital role in the development of multidrug resistance and chemoresistance [27] by removing a wide array of commonly employed chemotherapeutic drugs from cancer cells [28]. The overexpression of ABC transporter genes can induce chemoresistance in several cancers [29][30][31]. Recently, researchers focused on the subset of ABC transporters such as P-glycoprotein (also known as ABCB1 or P-gp), MDR-associated protein 1 (MRP1; also known as ABCC1) and ABCG2 (also known as breast cancer resistance protein, BCRP) [32]. The EMT is related to cancer drug resistance and contributes to metastasis after chemotherapy treatment [33]. Regarding breast cancer, cells undergoing the EMT overexpress ABC transporters [34] and are associated with cellular resistance to drug-induced apoptosis [35]. Here, we hypothesize that DPP-4 inhibition increases ABC transporters in the presence of chemotherapy to promote drug resistance in breast cancer.
Chemotherapy in DPP-4-Deficient Breast Cancer Cells Facilitated the Expression of ABC Transporters
To explore the effects of DPP-4 suppression on chemoresistance in breast cancer cells, we utilized the DPP-4 inhibitor KR62436 or DPP-4 knockdown by specific shRNA (DPP-4-kd) in 4T1 cells. Western blot analysis revealed that the expression of P-gp and ABCG2 had no obvious change in KR-treated 4T1 cells compared with that of the control group. The levels of P-gp and ABCG2 were significantly increased in 4T1 cells treated with doxorubicin (DOX) and were further increased in cells treated with KR and DOX in combination ( Figure 1A). DPP-4-kd 4T1 cells exhibited similar DOX-induced P-gp and ABCG2 protein expression ( Figure 1B). In MCF-7 cells, KR promoted the expression of ABCG2 and MRP1 either with or without DOX ( Figure 1C). A similar trend was observed in the highly metastatic human breast cancer cell line MDA-MB-231; the combination of KR with DOX induced the highest expression levels of ABC transporters ( Figure 1D).
The Role of EMT in DPP-4 Deficiency-Induced ABC Transporters
EMT-related transcription factors (TFs) have been shown to play vital roles in regulating ABC transporter expression [36]. As shown previously [17], DPP-4 deficiency induced the EMT, which was characterized by the suppression of E-cadherin and the induction of α-SMA in 4T1 cells, and the trends were much more prominent in KR+DOX treated cells ( Figure 3A) and DOX-treated DPP-4-kd 4T1 cells ( Figure 3B). Immunofluorescence analysis revealed that primary tumors of DPP-4-kd 4T1 cells exhibited EMT markers ( Figure 3C). Snail is a key transcription factor that induces the EMT. Snail knockdown by specific siRNA (siRNA snail) diminished snail levels, resulted in the suppression of the EMT (restoration of E-cadherin and suppression of α-SMA) and was associated with the suppression of ABC transporters P-gp and ABCG2 in 4T1 cells ( Figure 3D). However, E-cadherin knockdown-induced EMT did not reverse AMD-3100 suppression of ABC transporters and smad3 phosphorylation in KR+DOX-treated cells ( Figure 3E), suggesting that the EMT-inducible transcription factor snail was required for ABC transporter expression induced by DPP-4 deficiency and DOX-treatment; the EMT, as indicated by the suppression of the E-cadherin and the induction of α-SMA, was not sufficient to induce ABC transporters under the same conditions. All densitometric quantification relative to β-actin levels and P-smad3 protein expression relative to smad3 levels (n = 3 per group) were performed by using ImageJ.
The Effects of DPP-4 Deficiency on Chemotherapy-Induced Apoptosis in Breast Cancer Cells
To validate that DPP-4 deficiency-induced ABC transporters were relevant to chemotherapy resistance, we performed an apoptotic assay. DOX and docetaxel (DOC) induced early apoptosis in 4T1 cells, as revealed by an annexin V assay; the proportion of early apoptotic cells was significantly reduced in cells treated with KR combined with either DOX or DOC ( Figure 4A,B). As expected, N-TGFβ diminished the KR-induced chemoresistance, suggesting that N-TGFβ sensitized the cells to chemotherapy ( Figure 4C), as described previously [20]. KR significantly diminished DOX-induced cleavage of caspase-3 ( Figure 4D). Such suppressive effects of KR on the induction of caspase-3 cleavage in DOX-treated cells were diminished by N-TGFβ, AMD3100 and rapamycin ( Figure 4E-G).
DPP-4 Deficiency Induced the Expression of ABC Transporters and Was Associated With Chemoresistance in the Allograft Breast Cancer Model
Finally, we tested whether DPP-4 deficiency in tumors was associated with chemoresistance in vivo. DPP-4-kd 4T1 cells displayed accelerated tumor growth when compared to that of shRNA-control 4T1 (control) tumors. DOX significantly suppressed tumor growth in both control and DPP-4-kd 4T1 tumors, but DOX-mediated suppression was less trend in DPP-4-kd 4T1 tumors ( Figure 5A; weight suppression rate (%) by DOX: control 42.8% vs. DPP-4-kd 29.7%). DPP-4-kd 4T1 tumors exhibited increased expression of P-gp, ABCG2 and MRP1 in primary tumors compared with that of control tumor-bearing mice, and this trend was enhanced in the presence of DOX ( Figure 5B and Figure S2).
Bouin staining of the lung revealed that DPP-4-kd 4T1 tumor-bearing mice exhibited more lung metastasis when compared to control mice with or without DOX; DOX treatment in both control and DPP-4-kd 4T1 tumor-bearing mice displayed some trend of induction in lung metastasis due to an extremely higher incidence of lung nodules in some mice ( Figure 5C). However, the difference was not reached significance and was not obvious as well. 3 , mice were intraperitoneally injected with DOX (5 mg/kg, once a week). Twenty-one days after treatment, the mice were sacrificed, and the primary tumors and lungs were analyzed. (A) The tumor volume in each group was measured ever day during treatment (* p < 0.05). (B) Immunofluorescence analysis of DPP-4 expression in control and DPP-4-kd primary tumors. Western blot analysis of P-gp and ABCG2 expressions in the primary tumor tissue. Densitometric analysis of protein expression relative to β-actin levels (n = 6 per group) was performed by using ImageJ. (C) The lung surface metastases (left panel) were imaged, and the quantification of lung metastases (right panel) was performed by Bouin staining. Black arrows indicated lung surface metastases. The data in the graphs was shown as mean ± SEM; n = 9.
Discussion
Patients with diabetes and cancer have a poor prognosis after treatment with chemotherapy or surgery with a high mortality compared with those without diabetes [2,3]. Chemotherapy is the primary treatment choice for metastatic patients, and chemoresistance is associated with metastasis in breast cancer [37]. Chemoresistance is associated with the EMT. Based on our previous report showing the potential of EMT and metastasis-promoting effects of DPP-4 inhibitors, which are widely prescribed drugs to treat diabetes, we tested whether DPP-4 inhibitors could induce chemoresistance in breast cancer cells.
Cancer cells undergo EMT, resulting in an enhanced capacity for invasion, metastasis and chemotherapy resistance [38] that is associated with the induction of ATP-binding cassette (ABC) transporters, which are responsible for diverse drug resistances [39]. Ricardo et al. demonstrated that in embryonic development phase, ABC transporters were required for managing the export of a germ cell attractant during directional cell migration in Drosophila [40]. Chemoresistance in cancer cells was associated with the EMT and upregulation of ABC transporters could be an evolutionarily conserved system. The overexpression of MDR1 could be conducive to both initiation and acceleration of the chemotherapy resistance in breast cancer cells [41]. Saxena M. et al suggested that chemotherapeutic drug-induced EMT increased the expression of ABC transporters and induced both the drug resistance and the invasion in breast cancer cells [34]. In recent studies, researchers have revealed that overexpression of EMT-related transcription factors (TFs), such as snail, enhanced the chemoresistance through the induction of P-gp and ABCG2 in breast cancer cells [42,43]. In our previous study, we demonstrated that deficiency in DPP-4 induced EMT in 4T1 cells, MCF-7 cells and MDA-MB-231 cells and accelerated lung metastasis of breast cancer in vivo via the CXCL12/CXCR4/mTOR axis [17]. This signaling pathway could be also relevant in the chemoresistance associated with ABC transporters in DPP-4-deficient cells, and EMT-related TFs played a major role in regulating ABC transporters expression, as described in a recent study. Snail has shown to be a major contributor of the maintenance of malignancy potentials and facilitates cancer metastasis and increases chemoresistance [44]. DPP-4 deficiency breast cancer cells treated with chemotherapy promoted the expression of snail. Our in vitro mouse and human cell lines analysis further supports the rationale of DPP-4 deficiency-associated chemoresistance and certain suggestions for clinical relevance.
When compared to the clearer in vitro data, in vivo data is somewhat puzzling. In our studies, DOX suppressed tumor growth in both control and DPP-4-kd 4T1 tumors, but DOX-mediated suppression was less trend in DPP-4-kd 4T1 tumors (tumor weight (g) suppression rate (%): control 42.8% vs. DPP-4-kd 29.7%), but not yet significant. DOX treatment in both control and DPP-4-kd 4T1 tumor-bearing mice displayed an insignificant increase in lung metastasis due to a variation in each sample by an extremely higher incidence of lung nodules in some mice by DOX treatment (either control or DPP-kd). The reason why such difference in each mouse observed was not identified yet. It is reasonable that there is the threshold for ABC transporter to gain chemoresistance; ABC transporter levels in DPP-4-kd tumor in some mice could not be sufficient enough to reach the threshold to induce chemoresistance. Other chemoresistance mechanisms also would be required in this set of experiment in vivo. DPP-4 displays diverse enzymatic and nonenzymatic action. Therefore, DPP-4-kd absolutely induced ABC transporters; parallelly also DPP-4-kd could be associated with the induction of unknown anti-chemoresistance molecular mechanisms in vivo. To explain the discrepancy between in vitro and in vivo data required further investigations.
Allograft Breast Cancer Mouse Model
Eight-week-old female BALB/c mice were obtained from Inc. Japan (CLEA Japan). The DPP-4 knockdown by specific shRNA (DPP-4-kd) or shRNA-control (control) 4T1 cells (5 × 10 5 cells in 20 µL of PBS) were orthotopically implanted into the mammary fat pads of each mouse using a Hamilton syringe fitted with a 25G needle. Concomitantly, the mice were randomly allocated to one of the following four groups: (1) control; (2) DPP-4-kd-4T1; (3) control +DOX and (4) DPP-4-kd-4T1+DOX. When the tumor volumes reached 80-100 mm 3 , the mice were intraperitoneally injected with DOX (5 mg/kg, once a week). Tumor sizes were measured on every alternate day with a digital caliper, and volumes were calculated using the following formula: tumor volume (mm 3 ) = (width 2 ) × (length/2). To quantify the tumor growth rate, we determined the relative tumor volume compared to the average volume of the starting tumor (0 day) for each group. Twenty-one days after treatment with DOX, the mice were sacrificed, and the primary tumors and lungs were removed and analyzed. The experiments described herein were executed in line with the animal protocols of Kanazawa Medical University (lentiviral shRNA in vivo experiment protocol number 2018-17).
Ethics Statement
The Animal Use Committee of Kanazawa University approved all animal study protocols (2019-15; 2019-06-17), and all experiments were conducted in accordance with the guidelines for the care and use of laboratory animals.
Western Blot Analysis
Western blot analysis was performed according to standard protocols. Total proteins were extracted by RIPA lysis buffer with PMSF, sodium orthovanadate and a protease inhibitor cocktail (Santa Cruz Biotechnology). Then, the protein lysates were boiled in SDS sample buffer at 94 • C for 5 min, separated on SDS-polyacrylamide gels, and transferred to PVDF membranes (Pall Corporation, Pensacola, FL, USA) using the semidry method. After being blocked with TBS-T (Tris-buffered saline containing 0.05% TWEEN 20) containing 5% non-fat dry milk, the membranes were incubated with each primary antibody at 4 • C overnight, followed by incubation with the appropriate secondary antibody for 1 h at room temperature. The signal was developed with an enhanced chemiluminescent substrate detection solution, and the membranes were imaged using an ImageQuant LAS 4000 mini (GE Healthcare Life Sciences, Uppsala, Sweden).
Flow Cytometry
The cells were seeded in a 6-well plate (1 × 10 6 cells/well) and preincubated with KR (K4264) (50 µmol/L). When the cells reached 70-80% confluence, they were treated with DOX (0.425 µmol/L) or DOC (0.9 µmol/L). The incubator was maintained at 5.0% CO 2 and 37 • C. After 24 h of treatment, the cells were washed with cold PBS three times, trypsinized using trypsin-EDTA (Invitrogen) and centrifuged at 2000 rpm for 5 min. The cells were resuspended and washed 2 times with cold PBS, centrifuged at 2000 rpm for 5 min and resuspended in banding buffer, and then annexin V-FITC was added. Subsequently, the cells were incubated at room temperature for 5 min in the dark. Cell fluorescence was measured by flow cytometry (Gallios, Beckman Coulter, Tokyo, Japan).
Immunofluorescence in Mouse Tissue
The frozen tumor sections were fixed with 4% paraformaldehyde phosphate buffer solution for 30 min at 4 • C. Then, the sections were washed twice with PBS and blocked with 2% BSA/PBS for 30 min at room temperature. Then, the tissues were incubated with primary antibody overnight at 4 • C, washed with PBS and incubated with the corresponding secondary antibody for 30 min. The tissues were then gently washed and shaken three times with PBS and mounted with mounting medium containing DAPI. The images were analyzed by fluorescence microscopy (BZ-X710 Viewer, KEYENCE, Osaka, Japan).
Bouin Buffer Staining
Bouin buffer solution was prepared as follows: 10% formaldehyde: 0.9% picric acid:5% acetic acid at ratios of 15:5:1. First, the lung specimens were perfused with 10% formaldehyde and then fixed in Bouin solution for at least 24 h after dissection. Subsequently, surface lung metastasis was quantitated by counting the number of metastatic nodules according to the Bouin staining.
Statistical Analysis
The data were analyzed utilizing one-way analysis of variance, followed by Tukey's multiple comparison test to confirm statistical significance, which was considered p < 0.05, unless otherwise noted. GraphPad Prism software (ver. 7.0f; La Jolla, CA) was utilized for the statistical analysis. The figures display the means and standard deviations (mean ± s.e.m).
Conclusions
In conclusion, we described that (1) DPP-4 deficiency induced ABC transporters in mouse and human breast cancer cell lines; (2) the transcription factor snail was required for DPP-4-deficiencyinduced ABC transporters and E-cadherin suppression with α-SMA induction was not essential; (3) the inhibition of the CXCR4/mTOR axis or TGF-β signaling sensitized 4T1 cells with DPP-4 deficiency to chemotherapy-induced apoptosis and (4) in an allograft model, the effects of DOX in either primary tumor or metastasis were not statistically different between control or DPP-4-kd 4T1 even though some mice displayed extremely high prevalence of metastasis in DOX-treated DPP-4-kd tumor bearing mice. These data further demonstrated the significance of DPP-4 inhibition in the biology of cancer. Diabetes is known to have a higher incidence of cancer, and clinicians are required to treat diabetes during chemotherapy. Clinical evidence related to this topic requires further evaluation; one may be aware of choosing the proper diabetic medicine for cancer-bearing patients, especially patients with chemoresistance and CXCR4-positive cancers. | v3-fos-license |
2019-06-20T13:35:28.690Z | 2019-06-19T00:00:00.000 | 195096794 | {
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} | pes2o/s2orc | Functionalized MoS2-erlotinib produces hyperthermia under NIR
Background Molybdenum disulfide (MoS2) has been widely explored for biomedical applications due to its brilliant photothermal conversion ability. In this paper, we report a novel multifunctional MoS2-based drug delivery system (MoS2-SS-HA). By decorating MoS2 nanosheets with hyaluronic acid (HA), these functionalized MoS2 nanosheets have been developed as a tumor-targeting chemotherapeutic nanocarrier for near-infrared (NIR) photothermal-triggered drug delivery, facilitating the combination of chemotherapy and photothermal therapy into one system for cancer therapy. Results The nanocomposites (MoS2-SS-HA) generated a uniform diameter (ca. 125 nm), exhibited great biocompatibility as well as high stability in physiological solutions, and could be loaded with the insoluble anti-cancer drug erlotinib (Er). The release of Er was greatly accelerated under near infrared laser (NIR) irradiation, showing that the composites can be used as responsive systems, with Er release controllable through NIR irradiation. MTT assays and confocal imaging results showed that the MoS2-based nanoplatform could selectively target and kill CD44-positive lung cancer cells, especially drug resistant cells (A549 and H1975). In vivo tumor ablation studies prove a better synergistic therapeutic effect of the joint treatment, compared with either chemotherapy or photothermal therapy alone. Conclusion The functionalized MoS2 nanoplatform developed in this work could be a potent system for targeted drug delivery and synergistic chemo-photothermal cancer therapy.
Background
Cancer is one of the biggest challenges that threaten human health, among which lung and bronchus are the most common cause of cancer-related death [1][2][3]. Chemotherapy is still one of the frequently used therapeutic modalities for cancer treatment over the past decades, however chemotherapy suffers from several therapeutic bottlenecks, such as severe side effects, low solubility, and the tendency to induce drug resistance [4,5]. Erlotinib (Er) is a clinical anticancer drugs working by selectively and reversibly inhibiting the epidermal growth factor receptor (EGFR) tyrosine kinase [6], thereby inhibiting downstream signaling pathways such as cell proliferation, metastasis, and angiogenesis [7]. Although Er has shown strong clinical therapeutic effect for lung cancer, the tumor therapeutic activity is still limited by above bottlenecks of chemotherapy, especially low solubility, instability and drug resistance. Therefore, it is necessary to develop an Er delivery system to overcome these defects and enhance its bioavailability.
interesting reports that demonstrate encouraging potential of 2D nanomaterial theranostics in the pre-clinical area and targeted delivery of cancer therapeutics [12][13][14][15][16][17]. Since graphene oxide was applied, single layer 2D nanomaterials has drawn much attention in a wide range of areas because of their unique physical and chemical properties. Owing to these excellent properties, more effort has been paid to search for other similar 2D materials [17,18]. MoS 2 nanosheets, as a kind of transition metal dichalcogenides (TMDCs), displayed huge potential applications for nanoelectronic [19][20][21], transistors [22][23][24], energy storage devices [25,26] and catalysis [27][28][29]. Past several years, a few groups have explored the promising application of single-layer MoS 2 sheets in the biomedical field [30,31]. Photothermal therapy (PTT) as a non-invasive therapeutic approach triggered by light, can transfer optical energy into heat, resulting in the thermal ablation of cancer cells [32,33]. As a new type of 2D TMDCs, MoS 2 has exhibited its intrinsic high NIR absorbance as well as outstanding photothermal conversion efficiency, which indicated that MoS 2 could be used as a photothermal agent (PTA) for PTT [31,[34][35][36]. Besides, as a NIR photothermal delivery system, MoS 2 has been reported that could stimulate the drug release triggered by NIR irradiation [37,38]. Hence, the MoS 2 -nanosheets could be used to form a NIR-triggered drug delivery system because of the larger surface area and amazing photothermic. However, MoS 2 nanosheets rapidly aggregate in physiological solution, which hampers the application of MoS 2 nanosheets in the medical field. Therefore, the surface modification of single-layer MoS 2 nanosheet remains a tremendous challenge for the application of the MoS 2 nanosheets in biomedicine. Chou et al. showed that the thiolated molecules could be attached to the MoS 2 nanosheets at the defect sites resulted from chemical exfoliation process and reported that the modified single-layer MoS 2 sheets show outstanding biocompatibility and high absorbance when under the irradiation of NIR laser [34,39]. The novel approach for the surface modification of single-layer MoS 2 nanosheet is urgently needed.
Hyaluronic acid (HA), as a water-soluble biomacromolecule, has great biocompatibility and biodegradability [40,41], Besides, HA is also a natural ligand for CD44 that is often overexpressed by various cancer cells, especially in drug-resistance cancer cells, and has been widely used in active targeting treatment of lung cancer [42][43][44]. MoS 2 nanosheet modified with HA not only enhances its stability in physiological solution, but also could specifically combine with the drug-resistant tumor cells which overexpress CD44 [45]. Hence, this study was designed to demonstrate that Er-loaded MoS 2 modified with HA could be engineered as a photothermal-triggered drug delivery system to specially target the CD44-overexpressing cancer cells, deliver non-water-soluble drug Er into cells, produce NIR-mediated hyperthermia, stimulate drug release triggered by photothermic, resulting in a synergistic cancer therapeutic effect in vitro and in vivo.
Synthesis of MoS 2 nanosheets
MoS 2 nanosheets were synthesized by using the Morrison method [46]. In brief, 0.5 g MoS 2 flakes were stirred with a solution of n-butyllithium in hexane (0.5 mL; 1.6 M) under N 2 atmosphere for 48 h, performed in a nitrogen glove box. After intercalation by lithium, the sample was centrifuged and washed repeatedly with hexane to remove leftover lithium and additional organic residues. Intercalated MoS 2 solution was then dislodged from the glove box and instantly ultra-sonicated in water for 1 h to obtain exfoliated MoS 2 , besides, the unexfoliated MoS 2 and excess LiOH were removed by centrifugation at 3000 rpm. The supernatant containing exfoliated MoS 2 was dialyzed against water for 48 h to remove excess impurities and the finally obtained MoS 2 nanosheets aqueous solution was stored at 4 °C for future use.
Preparation of targeted nanocomposite
200 mg HA was first dissolved in 20 mL deionized water, then 38 mg ethylene dichloride (EDC) and 46 mg N-hydroxysuccinimide (NHS) were added under stirring. Afterwards cystamine dihydrochloride (1.2 g) was added to the mixture and stirred overnight. The product, denoted HA-SS, was obtained after dialyzing against water with a cellulose membrane (MWCO: 1 kDa) for 24 h. 5 mg HA-SS was added to a dispersion of MoS 2 (0.25 mg/mL, 4 mL water) and then ultrasonicated for 30 min. After stirring overnight, the resultant product (MoS 2 -SS-HA) was dialyzed with water against a cellulose membrane (MWCO: 100 kDa). The obtained MoS 2 -SS-HA nanosheets were stored at 4 °C until use.
Erlotinib loading and releasing Erlotinib loading
Erlotinib (Er) loading onto MoS 2 -SS-HA was applied according to the following protocol. In brief, MoS 2 -SS-HA nanosheets watery solution (0.5 mg/mL) was mixed with different concentrations of Er which were dissolved in DMSO solution and stirring the mixture for 24 h (pH = 7.0). Excess Er was removed by centrifugation at 4000 rpm for 20 min. The supernatant was filtered (0.45 μm) to remove the remaining undissolved Er. The obtained solution was centrifuged for 5 times by ultrafiltration (10 kDa MWCO) to remove the dissolved excess Er. Loading amount of Er was detected using UVvis spectra absorbance peak at 343 nm.
Erlotinib releasing
The methods were similar to what we described previous [47][48][49]. Briefly, 2.5 mg MoS 2 -SS-HA-Er nanosheets were suspended in PBS buffer (5 mL, pH = 7.4) or acetate buffer (5 mL, pH = 5.6) and placed into dialysis bags (10 kDa MWCO). After 1 h, the samples were irradiated by 808 nm NIR laser with a power density of 1 W/cm 2 for 20 min. The release assay was performed on a shaking bed at 37 °C at a speed of 100 rpm. Each of 0.5 mL dialysate was collected at designed time points and replaced with the same volume of fresh buffer solution. The released amount of Er from MoS 2 -SS-HA-Er was quantified by UV-Vis spectroscopy. The accumulative amount of Er released from the composites was calculated as follows:
Materials characterization
The morphology of MoS 2 was measured using transmission electron microscopy (TEM; JEM-2100, JEOL). The thickness and size of the MoS 2 particles were determined with a 5500 atomic force microscope (AFM; Agilent). The zeta potential was quantified with a ZS90 Zetasizer instrument (Malvern Instruments). Dynamic light scattering (DLS) was performed with static light scattering instrument (BI-200SM, Brookhaven Instruments). UVvis spectra were obtained on a UV3600 instrument (Shimadzu Corporation). Fourier transform infrared (FT-IR) spectroscopy was recorded on a Vetex70 (Bruker Corp., Germany). The photothermal properties of the composite were examined using a laser device (Shanghai Xilong Optoelectronics Technology Co. Ltd.) at a wavelength of 808 nm.
Biocompatibility of MoS 2 -SS-HA in vitro and in vivo Cytotoxicity assays in vitro
The methods were described in our previous study [48].
Hemolysis assays of MoS 2 -SS-HA in vivo
Hemolysis assay were performed as following: 1 mL blood obtained from rat (Wistar, female, 4-6 weeks old, purchased from Fuzhou Wushi Animal Center) was treated with ethylene diamine tetracetic acid. Then the blood was centrifuged at 1000 rpm for 10 min and removed the upper serum carefully. The lower red blood cells were diluted 30 times with PBS. Next, 0.3 mL of diluted red blood cells was mixed with (i) 0.9 mL of PBS as a negative control, (ii) 0.9 mL of water as a positive control, (iii) 0.9 mL of MoS 2 -SS-HA dispersions at different concentrations (50, 100, 200, 400, 800 μg/mL). Eventually, all the mixtures were kept shaking at 100 rpm for 2 h and then centrifuged at 12,000 rpm for 10 min. The absorbance of supernatants was detected at 541 nm by UVvis spectrophotometry. Hemolysis percentage (%) = (A sample-A negative)/(A positive-A negative) × 100%.
Biocompatibility of assays in vivo
Biocompatibility of MoS 2 -SS-HA was carried out in vivo in accordance with the protocol approved by Institutional Animal Care and Use Committee. BALB/c nude female mice (5-7 weeks old) were purchased from Fuzhou Wushi Animal Center and maintained in cages in a SPF-grade animal room with access to food and water ad libitum. After 1 week of adaptation feeding, nude mice were randomly divided into two groups (n = 5), then nude mice were treated with 100 μL of (i) saline, (ii) MoS 2 -SS-HA in saline (2.0 mg/kg), via tail vein injection. The injection was performed every 2 days. After 3 weeks, all mice were sacrificed and the major organs (heart, liver, spleen, lung and kidney) were obtained and were stained with hematoxylin-eosin (H&E) to observe histopathological changes.
In vitro cellular uptake
Intracellular uptake of the FITC-loading MoS 2 -SS-HA nanosheets was judged by confocal laser scanning microscopy (CLSM, Leica TCS SP8, IL, USA) and flow cytometry (BD FACSAriaIII, BD Bioscience). As a fluorescent probe, the FITC was loaded on the MoS 2 -SS-HA nanosheets in the same protocol as Er (shown in 2.3.1 Er loading procedure). Cells were seeded in 6-well plates (1 × 10 5 cells/well) and cultured for 24 h. The media was aspirated and 2 mL of fresh DMEM containing 0 or 5 mg/mL HA was added. 2 h later, cells were washed twice with PBS and incubated with MoS 2 -SS-HA-FITC (FITC = 5 μg/mL). The cells in all the groups were cultured for another 2 h and then washed and fixed with glutaraldehyde for 30 min. The cell nuclei were stained with DAPI and detected by CLSM and flow cytometry.
Cytotoxicity of MoS 2 -SS-HA-Er in vitro
The cytotoxicity of MoS 2 -SS-HA-Er against lung cancer cells (A549, H1975, PC-9) was assessed by MTT assay. Briefly, cells were seeded in 96-well plates and cultured overnight. Then media was aspirated and a solution of Er (200 μL, [Er] = 1.25, 2.5, 5, 10, 20 μg/mL) were added to each well, and the plates incubated at 37 °C in a 5% CO 2 atmosphere for 24 h. The MTT reagent (10 μL, 5 mg/mL) was then added, followed by incubation at 37 °C in a 5% CO 2 atmosphere for 4 h. The supernatant was then carefully removed and the MTT-formazan produced by live cells solubilized in 150 mL of DMSO for 20 min. Finally, the absorbance at 490 nm was measured using a microplate reader (MULTSIKAN MK3, Thermo Fisher). Cell viability (%) was determined from the absorbance at 490 nm and normalized to a negative control wells containing untreated cells. Experiments were performed in triplicate. A second set of experiments was performed to assess the potential for photochemo-therapies. Cells were cultured in a 96-well plate at 1 × 10 4 cells per well (200 μL of cell suspension per well) for 24 h, and then co-cultured with Er or MoS 2 -SS-HA-Er. Cells were divided into 4 treatment groups at Er concentrations ranging from 1.25 to 20 μg/mL as follows: (i) Saline + NIR; (ii) Free Er + NIR; (iii) MoS 2 -SS-HA-Er + NIR (synergistic therapy); (iv). MoS 2 -SS-HA + NIR. After incubation for 12 h, the cells were washed with 100 μL PBS and 100 μL culture medium was then added to the wells. The cells were irradiated with an 808 nm laser at different power densities (0.5 W, 0.8 W, 1.2 W) for 20 min, before the cells were cultured for a further 24 h and an MTT assay used to measure cell viability.
Detection of cell apoptosis and cell cycle
To evaluate the therapeutic efficacy of the nanosheets, cell cycle and apoptosis were measured by using flow cytometry according to following protocol. Briefly, cells were seeded at a density of 3 × 10 5 cells/well in the sixwell plates. After 24 h, the cells were separated into five groups, as following: control, Er, MoS 2 -SS-HA + NIR, MoS 2 -SS-HA-Er and MoS 2 -SS-HA-Er + NIR ([Er] = 10 μg/ mL). Besides, groups of MoS 2 -SS-HA + NIR and MoS 2 -SS-HA-Er + NIR were exposed to 808 nm-NIR laser with a power density of 1.2 W/cm 2 for 20 min. Finally, the cells were collected and detected by flow cytometry according to the instruction of Apoptosis Assay Kits (Keygen BioTech, Nanjing, China). For cell cycle assay, the cells were collected and washed thrice with ice-cold PBS. Then the cells were fixed with cold 70% ethanol for 24 h at 4 °C. Subsequently, the cells were centrifuged and washed twice with PBS. Ultimately, the staining solution consisting of 1% (v/v) Triton X-100, 0.01% RNase, and 0.05% PI was added to the cells and stained for 30 min in darkness before detection by flow cytometry.
Antitumor effects of MoS 2 -SS-HA-Er in vivo
All animal experiments were carried out in accordance with the protocol approved by Institutional Animal Care and Use Committee. BALB/c nude female mice (5-7 weeks old) were purchased from Fuzhou Wushi Animal Center and maintained in cages in a SPF-grade animal room with access to food and water ad libitum. A549 cells (1 × 10 6 cells/well) were suspended in 100 μL PBS were subcutaneously injected into the left fore limb of each nude mouse to form the tumor model. The tumor volume was measured using vernier caliper and calculated as V = (length × width 2 )/2. The nude mice were randomly divided into six groups (n = 5) when the tumor volume reached up to 90 mm 3 . Afterward, each group of the tumor-bearing nude mice was treated with 100 μL of (i) saline, (ii) Er (2.0 mg/ kg), (iii, iv) MoS 2 -SS-HA in saline (2.0 mg/kg), and (v, vi) MoS 2 -SS-HA-Er (2.0 mg/kg) in saline, respectively, via tail vein injection. After 8 h, the mice of group (iv) and (vi) were treated with 808 nm-NIR laser light (0.5 W/cm 2 ) for 10 min. The infrared ray (IR) images of tumors were observed with an infrared thermal camera (InfReC R500EX, Tokyo, Japan), showing the real-time temperature changes with irradiation. During the treatment, the tumors volume and the weight of mice were recorded every 3 days.
Statistical analysis
Statistical analysis was performed with GraphPad Prism 5.0 (GraphPad software, San Diego, CA). In general, for two experimental comparisons, a two-tailed unpaired Student's t test was used unless otherwise indicated. For multiple comparisons, one-way ANO-VAs were applied. When cells were used for experiments, three replicates per treatment were chosen as an initial sample size. All n values defined in the legends refer to biological replicates. Data were assessed as mean ± SD. Statistical significance was set at *p < 0.05 and high significance was set at **p < 0.01.
Result and discussion
Preparation and characterization of nanocomposites The typical morphologies, structures, and dispersity of MoS 2 , MoS 2 -SS-HA and MoS 2 -SS-HA-Er were analyzed by transmission electron microscopy (TEM). In Fig. 2a, it is shown that MoS 2 nanosheets have a welldefined laminar morphology with a size of around 70 nm, while MoS 2 -SS-HA and MoS 2 -SS-HA-Er have a wrinkled sheets and larger-size (125 nm), which are usually observed for modification of nanosheets. The morphology of prepared MoS 2 -SS-HA was characterized by AFM images, which shows the material to consist of exfoliated sheets (Fig. 2b), the results are consist with the literature of MoS 2 [51]. The AFM data in Fig. 2c, show that the thickness of MoS 2 nanosheets is approximately 0.7 nm, indicating that the acquired nanosheet was single layer [52]. The height of nanosheets increased to about 2 nm after HA coating, revealing the successful coating of HA on the surface of MoS 2 nanosheets. The hydrodynamic . 2f ). The characteristic peak of MoS 2 is between 260 to 300 nm. The characteristic peak of HA is around 192 nm, similar to literatures [54,55]. The characteristic peak of Er is around 335 nm in accordance with references [56,57]. MoS 2 -SS-HA-Er showed both characteristic peaks of HA and Er.
As expected, physiological stability of MoS 2 nanosheets was improved after modified with HA. In addition, the MoS 2 nanosheets generated obvious aggregation among in water, PBS and cell medium within 1 h, while no obvious aggregation was observed in water and other physiological solutions for 1 month after decorated with HA, which demonstrated the good dispersibility of (Fig. 2g). To confirm the combination of Er and MoS 2 -SS-HA sheets, Fourier transform infrared (FT-IR) spectroscopy was conducted. As shown in Fig. 2h, the amide group (-CO-NH-) of HA appears as a characteristic band at about 1600 cm −1 , and the band at 3431.03 cm −1 correspond to the characteristic vibration of the -C≡H bond due to the coupling vibration of Er.
Photothermal activity
It is well known that MoS 2 nanocomposites have good photothermal conversion efficiency [58,59], and hence the photothermal activity of MoS 2 -SS-HA was explored in a series of tests. In Fig. 3a1, the photothermal heating effect of a 50 μg/mL suspension of the MoS 2 -SS-HA composite under irradiation by different laser power is depicted. A laser power intensity dependent photothermal effect was observed, as would be expected. When the laser power was 1.2 W/cm 2 the temperature reached 50 °C within 500 s. The photothermal effect is found to be concentration-dependent under irradiation at 0.5 W/ cm 2 (Fig. 3a2). These results indicate the suitability of MoS 2 -SS-HA for photothermal therapy and potential for the thermal ablation of tumors. After three on-off cycles of irradiation (Fig. 3b), the temperature response of the MoS 2 -SS-HA the laser power was 1.2 W/cm 2 the temperature reached 50 °C within 500 s. The photothermal effect is found to be concentration-dependent under irradiation at 0.5 W/cm 2 (Fig. 3c). These results indicate the suitability of MoS 2 -SS-HA for photothermal therapy and potent suspension remains largely constant. This suggests that MoS 2 -SS-HA has excellent photothermal stability.
In vitro drug release
MoS 2 -SS-HA nanosheet has ultrahigh surface area and a water-soluble biomacromolecule, therefore could be used as a drug carrier [30,60]. We loaded an insoluble chemotherapeutic drug, erlotinib (Er), on the MoS 2 -SS-HA nanosheets to obtain the MoS 2 -SS-HA-Er. It was shown in Fig. 2f that Er had been loaded on the surface of MoS 2 -SS-HA nanosheets by UV-vis spectra.
Drug release from the nanosheets was affected by many experimental factors [61,62], the most commonly studied are pH and NIR irradiation. The in vitro release behavior of MoS 2 -SS-HA-Er was investigated in different pH media (5.6 and 7.4) with and without laser irradiation (Fig. 3c). The release of Er was almost no difference between pH 5.6 and pH 7.4. At a given pH, there is greater Er release with laser irradiation than without. This indicates that NIR light-triggered photothermal heating could promote the release of Er and accelerate the death of cancerous cells. For example, the cumulative release of Er after 12 h at pH 7.4 with laser irradiation (33.3%) was much greater than that at pH 7.4 without laser irradiation (8.9%).
Biocompatibility of MoS 2 -SS-HA in vitro and in vivo
Biocompatibility is an essential concern when it comes to the development of nanomaterials for biomedical application. An ideal drug delivery platform must be biocompatible, non-toxic and not be associated with incidental adverse effects [62,63]. Herein, before conducting further experiments in vitro and in vivo, the cytotoxicity of MoS 2 -SS-HA nanosheets against cells was measured by MTT assay. As shown in Fig. 4a, the viability of all cells remained over 80% at high concentration of MoS 2 -SS-HA, indicating the ultralow cytotoxicity of MoS 2 -SS-HA against lung cancer cells (PC-9, A549 and H1975) and HELF cells after 24 h incubation. In addition, hemolysis assay results indicated that negligible hemolysis was observed in MoS 2 -SS-HA groups, even the concentration reached to 800 μg/mL, demonstrating that the MoS 2 -SS-HA nanosheets has excellent blood compatibility (Fig. 4b). Further, as shown in Fig. 4c, H&E staining results revealed little histopathological changes between saline group and MoS 2 -SS-HA group. Hence, good compatibility of the MoS 2 -SS-HA nanosheets both in vitro and in vivo indicate the great application potential in the cancer treatment.
Cellular uptake in vitro
HA-coated nanoparticles could target the tumor site [64]. The targeting ability of the nanocomposites was investigated by CLSM. A549, H1975 and PC-9 which are HA-receptor positive, while HELF is HA-receptor negative, were cultured with MoS 2 -SS-HA-FITC and MoS 2 -SS-HA-FITC + HA, as shown by the coincident presence of DAPI (blue) and FITC (green) fluorescence (Fig. 5a). Relatively little MoS 2 -SS-HA-FITC is taken up by HA-receptor negative cell line HELF, and a little amount is taken up by erlotinib sensitive cell line PC-9. In contrast, the uptake of MoS 2 -SS-HA-FITC by erlotinib resistant cell line A549 and H1975 was similar, both exhibiting much greater FITC fluorescence than HELF. This arises because of the lack of an HA receptor on HELF. In order to clarify the cellular uptake mediated by HA receptor, additional HA was added to block CD44 (HA receptor). After HA added, negligible FITC green fluorescence was detected in all cell lines. The quantitative flow cytometry data (Fig. 5b) were consistent with the confocal imaging results, which further indicated MoS 2 -SS-HA is an excellent CD44-mediated cancer cell targeting drug delivery platform [43,44,[65][66][67][68].
Cytotoxicity of MoS 2 -SS-HA-Er in vitro
The therapeutic efficacy of free Er and MoS 2 -SS-HA-Er against cancer cells was investigated by MTT assays after incubation with PC-9, A549 and H1975 (Fig. 6a). The cell viability of erlotinib-sensitive cell line PC-9 was below 40% with treatment of free Er (20 μg/mL), while the viability of erlotinib-resistant cell line A549 and H1975 Fig. 5 The intracellular uptake of MoS 2 -SS-HA using FITC as a fluorescent probe. Confocal fluorescence images (a) and quantitative flow cytometric analyses (b) of A549, H1975, PC-9, and HELF cells which were incubated with MoS 2 -SS-HA-FITC for 2 h ("+HA" means pretreat with 5 mg/mL HA for 2 h). Intensity of FITC fluorescence was obviously decreased with pretreatment of HA, demonstrating the HA-mediated intracellular uptake. A549 (EGFR wide-type, erlotinib-initially resistant), H1975 (EGFR-mutated subtype, L858R/T790M double mutations, erlotinib-acquired resistant) and PC-9 (EGFR-mutated subtype, exon 19 deletion, erlotinib-sensitive). The bar represents 25 μm is above 60% and 80%, respectively, with treatment at high concentrations of 20 μg/mL free Er, indicating free Er revealed poor therapeutic efficiency against erlotinibresistant cell lines. The excitement is the viability of erlotinib-resistant cell line (A549 and H1975) decreased below 40% with treatment of MoS 2 -SS-HA-Er at a concentration of 20 μg/mL. Considering the cell viability at equivalent doses of Er, in the case of A549 and H1975 cells the MoS 2 -SS-HA-Er group has markedly lower viability than cells treated with the free drug, the reason may be that the nanosystem is embedded in lysomal vesicles by CD44 receptor-mediated endocytosis [69]. To further explore the cell-killing effect of MoS 2 -SS-HA-Er combined with hyperthermia, cells were incubated with saline (control), Er, MoS 2 -SS-HA and MoS 2 -SS-HA-Er at the same Er concentration ([Er] = 10 μg/mL) exposed to the various density of NIR laser irradiation (Fig. 6b). NIR irradiation alone was found to have no effect on cell viability: A549 and H1975 cells showed essentially identical cell viability (> 85%) when they were treated with laser irradiation at a density from 0.5 to 1.2 W/cm 2 . For all cell types, the viability of cells treated with MoS 2 -SS-HA-Er ([Er] = 10 μg/mL) and laser irradiation are lower than those receiving MoS 2 -SS-HA-Er alone, indicating the PTT effect of the MoS 2 nanocomposites. The erlotinibresistant A549 and H1975 cell viabilities in the combined therapy group are much lower than the monotherapy groups (chemotherapy or PTT), showing the synergistic benefits of simultaneous PTT and chemotherapy. This leads to enhanced chemotherapy. Given that the cytotoxic effect of MoS 2 -SS-HA-Er with or without laser irradiation was higher with A549 and H1975 cells than PC-9 cells, there is also the potential for selective killing of drug-resistance cancer cells. All these results confirm that the multifunctional drug delivery system constructed in this work is effective in killing tumor cells and promising in chemo-photothermal combined cancer therapy.
Cell cycle and cell apoptosis
The result of cells apoptosis assay was illustrated in Fig. 6c. The apoptosis percentages of the cells incubated with MoS 2 -SS-HA nanosheets were higher than the control group (saline treatment) after irradiation, exhibiting that the MoS 2 -SS-HA could lead to cell apoptosis depending on the hyperthermia generated by the irradiation.
Meanwhile, cells incubated with MoS 2 -SS-HA-Er were significantly less than the free Er group, verifying the stronger apoptosis effect on CD44-positive cancer cells caused by the MoS 2 -SS-HA nanosheets. Considering the hypotoxicity of MoS 2 -SS-HA nanosheets, we believed that the targeting ability played an important role in antitumor effect. Furthermore, after treated with MoS 2 -SS-HA-Er + NIR, the apoptosis rates of all cells (A549, PC-9, and H1975) were the highest among all groups, demonstrating that the combined therapy remarkably promoted the cell apoptosis. In the cell apoptosis result, the ratio of apoptosis cells in A549, PC-9 and H1975 increased to 24.6%, 36.3% and 23.6%, respectively. The apoptosis cells percentage of MoS 2 -SS-HA-Er + NIR group was significantly higher than MoS 2 -SS-HA-Er group and MoS 2 -SS-HA + NIR group, due to the synergy of chemotherapy and photothermal.
To further explore the mechanism of cell death, the result of cell cycle tested by flow cytometry was analyzed. As indicated in Fig. 6d, compared with the control group, the proportion of cells treated with MoS 2 -SS-HA + NIR in G0/G1 had no obvious difference, suggesting that the photothermal therapy alone had slight influence on the cell cycle distribution. Cells incubated with MoS 2 -SS-HA-Er showed the higher arrest percent of G0/G1 compared to the group of free Er, owing to the enhanced intracellular drug uptake mediated by the targeting of HA. After exposed under the 808 nm laser, the cells treated with MoS 2 -SS-HA-Er + NIR showed the highest ratio of G0/G1-phase because of the synergistic therapeutic effect. In summary, MoS 2 -SS-HA-Er could enhance the arrest of G0/G1 phase and prevent DNA replication, especially with 808 nm NIR irradiation, while the MoS 2 -SS-HA + NIR had no such effect. The results of the apoptosis ratio and cell cycle arrest were consistent with the conclusion of MTT assay, suggesting that the MoS 2 -SS-HA-Er + NIR induced the death of cancer cells through the G0/G1-phase arrest and cell apoptosis induction.
Anti-tumor efficacy in vivo
Encouraged by the synergistic therapeutic effect of MoS 2 -SS-HA-Er + NIR in vitro, comparative studies of inhibiting tumor effectiveness in vivo was further investigated. In the thermal images (Fig. 7a) = 10 μg/mL, power = 1.2 W/cm 2 ). Significant differences between groups are labeled with *p < 0.05 and **p < 0.01 injected mice quickly increased and could readily reach a level (ΔT = 21 °C) which could induce hyperthermia and heat-induced drug release to kill the tumor. However, the tumor temperature of control (treated with saline) and free Er shows insignificant change (ΔT = 4 °C). Moreover, due to high toxicity always leading to a significant weight loss, the body weight of these mice was measured during the treatments, and no obvious weight loss was observed (Fig. 7b), indicating the low toxicity of the treatments in vivo. The tumor volumes of each group were measured and were then plotted as a function of time (Fig. 7c). Compared with the control group, efficient inhibition of tumor growth is observed for the group treated with MoS 2 -SS-HA-Er + NIR. Especially, the mean tumor volume in the MoS 2 -SS-HA-Er + NIR group is the smallest among all treated groups, which demonstrates that MoS 2 -SS-HA-Er can effectively inhibit tumor growth under the NIR laser irradiation. The reason could be attributed to (i) HA functionalized MoS 2 -SS-HA-Er targeting tumor site, and (ii) enhanced on-demand release of Er from MoS 2 -SS-HA-Er after laser irradiation, ultimately inhibiting tumor growth, further (iii) the combination of photothermal with chemotherapy therapy, both of which were activated simultaneously by 808 nm laser. Fig. 7 Comparative investigation of inhibiting tumor effectiveness in vivo. a NIR thermal images of A549 tumor-bearing mice injected with saline, Er, MoS 2 -SS-HA, and MoS 2 -SS-HA-Er + NIR. Mice body weight curves (b) and tumor growth curves of tumors (c) after various treatments for five groups. Significant differences between groups are labeled with *p < 0.05 and **p < 0.01
Conclusions
In summary, a multifunctional MoS 2 -based drug delivery system (MoS 2 -SS-HA-Er) was successfully synthesized in this work and shown to allow tumor-targeting synergistic chemo-photothermal therapy. MoS 2 nanosheets were modified with the targeting and water-soluble biomacromolecule HA to enhance biocompatibility. The nanocomposite had a uniform diameter (125 nm), and could be loaded with the insoluble anti-cancer drug Er. The release of Er is accelerated under near infrared light irradiation, which is promising for controllable drug delivery system. The nanocomposites can be specifically delivered into cancerous cells via a receptor-mediated endocytosis pathway using hyaluronic acid targeting. The nanocomposites were found to be able to induce the death of cancerous cells while leaving healthy cells unaffected. Furthermore, the MoS 2 -SS-HA-based drug delivery system can be used for synergistic cancer therapy associated with NIR-mediated hyperthermia and heat-induced local drug release in vitro and in vivo. An effective treatment of lung cancer in vivo under NIR irradiation is obtained, indicating that synergistic efficacy of hyperthermia and chemotherapy is better than hyperthermia or chemotherapy alone. | v3-fos-license |
2021-07-27T00:06:05.627Z | 2021-05-20T00:00:00.000 | 236330237 | {
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} | pes2o/s2orc | Determination of the Elemental Composition of Aromatic Plants Cultivated Industrially in the Republic of Moldova Using Neutron Activation Analysis
: The mineral contents of roots, leaves, stalks, and inflorescences of the aromatic plant species Anethum graveolens L., Coriandrum sativum L., Lavandula angustifolia Mill., Levisticum officinale W.D.J. Koch, and Salvia sclarea L. were studied by means of neutron activation analysis. The contents of 36 major and trace elements were determined and biological transfer coefficients were calculated. Among major and minor elements, K with a content in the range of 9230–59,600 mg/kg and Fe in the range of 69–3420 mg/kg were the most abundant elements in the studied plants. The content of the toxicant As ranged between 0.14–0.79 mg/kg; however, in the leaves (1.3 mg/kg) and inflorescences (1.0 mg/kg) of L. angustifolia there was found to be about 1 mg/kg, equal to the guideline maximal level recommended for food by the WHO. By comparing the data to Markert’s Reference Plant, “chemical fingerprints” were identified for each species. High contents of the elements Al, Hf, Se, Sc, Na, Ta, Th were determined in all studied plants. Collocated soil samples from the cultivation field were analyzed to calculate the biological accumulation coefficients for 35 of the elements determined in the plants. Considering the levels of chemical elements, the medicinal herb samples investigated are considered as relatively safe for human consumption.
Introduction
Medicinal aromatic plants have been used widely throughout human history, mainly due to their ease of access, affordability, and perceived therapeutic efficacy combined with absence of adverse side effects [1]. According to Fabricant and Farnsworth [2], of the 120 active compounds isolated from higher plants currently used in modern medicine, a positive correlation between modern therapeutic applications and traditional uses is found in 80%. Medicinal plants are often consumed as herbal preparations (infusions, essential oils, etc.) or spices, and are considered valuable sources of dietary supplementation.
A large body of research has been dedicated to studying the essential oils compositions of the species under discussion [8][9][10][11]. They have been popularly used in pharmaceuticals, medicine, cosmetic products, the tobacco industry, agriculture, and perfumery; for aromatherapy; food preparation, preservation, and flavoring; and as melliferous and ornamental plants [12][13][14][15][16][17][18][19][20]. In general, medicinal plants are important in pharmacological research and drug development, especially in cases where they are used as starting materials [21].
Furthermore, there has been an ever-increasing interest in determination of the elemental composition of medicinal plants [22,23], which stems from the fact that trace elements are essential for higher plants and for the human organism only in trace amounts [24,25]. Minerals at supra-optimal levels could be toxic, and information about the inorganic content of raw plant materials has been requested for the purposes of quality control of herbal supplements and preparations. Consumer protection organizations and regulatory bodies have been involved following reports of poisoning with arsenic, cadmium, lead, and mercury from traditional Indian and Chinese herbal medicines [26][27][28].
The processes of mineral transportation and accumulation are influenced by the chemical properties of the elements and compounds (e.g., solubility, bioavailability), by factors of the environment (soil characteristics, climatic conditions, distance to pollution sources, agricultural practices such as fertilization, etc.), and by the plant survival mechanisms [29][30][31]. Since the selected medicinal plants are vascular, the transfer of minerals from soil can be considered one of the main pathways of their accumulation in different compartments of the plant [32]. This merits further studies of the associations between soil content and the elemental distribution among the plant organs.
The aim of this study was (i) to investigate the elemental compositions of five popularly used medicinal plants cultivated industrially on unfertilized chernozem soil in the Republic of Moldova, using neutron activation analysis; and (ii) to evaluate element uptake from soil and accumulation in different morphological parts of plants (roots, leaves, stalks, inflorescences).
Study Area
The plant samples were collected during the flowering stage directly from the fields located near the towns of Causeni and Glodeni. The following plant samples were collected near Causeni: coriander (on 03 June 2019 in Cainari, geographically located at 46 • The plants were grown in natural conditions on chernozem soil and were not fertilized. The climate in the region is continental, characterized by cold winters and warm and dry summers, with temperatures in the range from −15 • C in January to 35 • C in July. According to the State Hydrometeorological Service, in 2019, the annual average temperature was +10.6-+12.6 • C and the average precipitation was about 404 mm.
Sampling and Sample Preparation
Aromatic plants of the species C. sativum, S. sclarea, A. graveolens, L. officinale, and L. angustifolia were collected in the summer of 2019 at the full flowering stage. All analyzed plants are essential oil crops and were collected during the full flowering stage, when the volatile oil content and potential therapeutic properties are expected to be maximum. For each of the studied species, approximately 100 g of dry raw material was obtained from plants collected over the entire area of the fields. After drying, each individual plant was separated into parts: roots, leaves, stalks, and inflorescences. The sorted plant parts were used to prepare compound samples.
Two associated soil samples were collected in close proximity to the plants, at depths from 10 to 20 cm as to avoid topsoil pollution arising from the surrounding environment. In the studied area, chernozem soil of brownish grayish color predominates, with pH levels around 6.0.
The samples were dried and stored in paper bags prior to analysis. In the laboratory, the plant components (organs) were homogenized separately, in a homogenizer, to obtain average samples. The soil samples were air-dried for 24 h and sifted through a 2 mm stainless-steel sieve.
For neutron activation analysis, all samples were dried at 40 • C to constant weight, and subsamples of about 0.3 g for vegetation and 0.1 g for soil were packed in polyethylene foil bags for short-term irradiation and in aluminum cups for long-term irradiation.
Neutron Activation Analysis (NAA)
The elemental contents of the analyzed herbs were determined by means of neutron activation analysis performed in the radioanalytical laboratory REGATA, at the IBR-2 reactor of the Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Dubna, Russia. Descriptions of the irradiation channels and the pneumatic transport system of the REGATA installation can be found elsewhere [33]. The concentrations of elements based on short-lived radionuclides: Al, Mg, Cl, Ca, Ti, V, and Mn were determined by irradiation for 3 min at a thermal neutron flux of 1.6 × 10 12 n cm −2 s −1 and measured for 15 min. To determine the contents of elements with long-lived isotopes: Na, K, Sc, Cr, Fe, Co, Ni, Zn, As, Br, Rb, Sr, Mo, Sb, Se, Cs, Ba, La, Ce, Sm, Eu, Hf, Nd, Ta, Tb, W, Yb, Zr, Th, and U, the cadmium-screened Channel 1 were used. Samples were irradiated for 4 days at a neutron flux of 1.8 × 10 11 cm −2 s −1 . Gamma spectra of induced activity were obtained after 4 and 20 days using three Canberra HPGe detectors with an efficiency of 40-55% and resolution of 1.8-2.0 keV at 1332 keV 60 Co total-absorption peak.
The analysis of the spectra was performed using the Genie2000 software by Canberra, with peak-fitting verification in interactive mode. Calculation of the concentration was carried out using the software "Concentration" developed in FLNP [34].
Quality control of the analytical measurements was carried out using certified reference materials: NIST SRM 1573-tomato leaves, NIST SRM 1547-peach leaves, NIST SRM 1632c-trace elements in coal (bituminous), NIST SRM 2709a-San Joaquin soil. The difference between determined and certified values was less than 10%.
Elemental Analysis of the Studied Plants
The contents of 36 major and trace elements determined in the plant samples are presented in Tables 1-5, for the species C. sativum, L. angustifolia, S. sclarea, L. officinale, and A. graveolens, respectively. Among the determined elements, 12 are either essential or beneficial to the human organism at certain concentrations (Ca, Cl, Co, Cr, Fe, K, Mg, Mn, Na, Se, V, Zn), 6 are potentially toxic (Al, As, Ba, Rb, Sb, Sr), and the rest of the elements have no biological functions [35].
The obtained results were compared with values introduced by Markert [29,35] for a generalized model of a plant, called the Reference Plant (RP) (Tables 1-5). This model was created with the aim of providing a base system for comparing different analytical data from plant analysis, no matter the type of plant or edaphic and climatic conditions. Data of typical accumulator or rejector plants were not used in Markert's model; therefore, in comparison to the RP, any relatively high concentrations are considered "chemical fingerprints" of the plants under investigation. In our study, any relatively high concentrations might also be considered a factor in explaining the medical properties of the plants. Given that in vascular plants all organs perform distinct physiological functions, the distribution patterns of the elements among the morphological parts differ. It was observed that C. sativum and L. officinale, belonging to the Apiaceae family, bioaccumulated most elements either in inflorescences or roots. In the case of plants of the Lamiaceae family the highest mineral content for almost all elements was found in the leaves and inflorescences. Generally, the mineral content of the stalk was low, which could be explained by the fact that transportation of fluids between the roots and stalk (translocation via the xylem and phloem) is one of the main functions of this organ.
It was determined that among all samples from C. sativum (Table 1), inflorescences had the highest contents of the following elements, in decreasing order of concentration: K > Ca > Mg > Al > Fe > Ti > Mn > Br > V > Ce > Nd > La > Co > Th > Sc > Hf > Sm > Cs > Yb > Sb > Eu > Ta. The roots of this plant contained the largest quantity of the following elements, listed in decreasing order of concentration: Na > Fe > Sr > Ba > Zr > V > Cr > As > Sc > W>U > Yb. However, in roots and inflorescences, the contents of the elements: Al, Co, Cs, Eu, Hf, La, Sb, Sc, Sm, Ta, V, and Yb were almost the same. The contents of Br, Cl, Ni, Rb, and Zn were the highest in the leaves, whereas the elements Sr, As, Ba, Cr, Eu, Na, Ta, U, W, and Yb had the smallest contents in this morphological part.
The stalk was characterized by low contents of almost all of the determined elements, except for As (0.32 mg/kg). It should be noted that the content of this element in inflorescences was almost the same (0.33 mg/kg) but it was the largest in roots (0.42 mg/kg).
By comparing the obtained results for C. sativum to the RP values, it was ascertained that the elements: Al (in inflorescences and roots, the content decreased in the order inflorescences > roots), Br (inflorescences > leaves), Hf (roots > inflorescences > leaves), Na (in roots), Sc (roots and inflorescences, equal amount), Se (in all investigated organs; leaves > inflorescences > stalk = roots), Ta (inflorescences > roots), Th (inflorescences > roots > leaves > stalk), Ti (inflorescences), U (roots), and Zr (roots > inflorescences) had relatively high content, by five times or more than the RP values.
Out of the 36 elements determined in the organs of L. angustifolia (Table 2), 23 were found to have their highest contents in the leaves, listed in decreasing order: K > Al > Fe > Ti > Mn > Zr > Rb > Cr > Ce > La > Nd > Hf > Th > Co > As > Sc > Sm > Yb > Cs > U> Eu > Ta > Tb. The contents of Mg, Na, Ni, Sb, V, and Zn were the highest in roots (in the order Mg > Na > Zn > V > Ni > Sb), and inflorescences had the largest amount of K > Ca > Sr > Ba > Br > W > Se. The stalk of L. angustifolia was characterized by comparatively low quantities of almost all elements, except for Cl (2260 mg/kg); but the content of this element was also high in inflorescences (2080 mg/kg). The contents of Ba, Br, Ca, Sr, in stalks and leaves were similar, whereas for Zn it was almost the same in all aboveground parts of the plant (31 mg/kg in stalks, 38 mg/kg in leaves, and 38.5 mg/kg in inflorescences). In roots and stalks, the content of Tb was on the same level.
The results for S. sclarea (Table 3) showed that, except for Ce and Na, the highest contents of almost all the determined elements were found in the leaves. However, some of the elements were found in almost equal amounts to the leaves in the inflorescences. These were: Br (6.7 mg/kg in inflorescences, 6.4 mg/kg in leaves), Mg (4180 mg/kg in inflorescences, 4120 mg/kg in leaves), Rb (16.8 mg/kg in inflorescences, 18.5 mg/kg in leaves), Se (0.294 mg/kg in inflorescences, 0.297 mg/kg in leaves), and Zn (27.3 mg/kg in inflorescences, 23 mg/kg in leaves). The mineral content of the stalk was poor in comparison to that of the other studied morphological parts but for Ba, since its content in the stalk was equal to that in the leaves (77 mg/kg). The contents of Ce and Na were the highest in the roots.
The obtained data for L. officinale ( Table 4) showed that of the 36 determined elements, 15 had their highest contents in the roots (Na > Al > V > Ce > La > As > Co > Sc > Th > U > Cs > Sm > Yb > Ta > Tb), and 9 in inflorescences (K > Mg > Fe > Zn > Rb > Cr > Ni > Sb > Hf). The quantity of Ba in the roots and stalks was similar (12 mg/kg and 13.2 mg/kg, respectively).
The contents of Mg, Mn, Se, and Zn in the inflorescences and leaves were almost the same. A comparison of the contents between all studied morphological parts of L. officinale showed that the leaves were the richest in Ca, Mn, Se, Sr, and Zn.
However, a comparison with the RP revealed that only the content of Se was high in all organs of L. officinale. The comparison with Markert's model revealed that the content of the following elements was rather high: Al (in roots > inflorescences), Br (stalk > inflorescences > leaves), Fe (inflorescences), Hf (inflorescences > roots > leaves), Na (roots), Sc (roots), Se (leaves > inflorescences > roots > stalk), Ta (roots > inflorescences > stalk), Th (roots > inflorescences > leaves > stalk), and U (in roots). The contents of K > Ca > Mg > Cl > Fe > Na > Sr > Ni in the leaves were two times higher than in the RP. Compared to the other organs of the plants, the stalk was characterized by the highest contents of Ba, Br, and Cl (in the order Cl > Br > Ba). The content of Br in the stalk was 35 times higher than in the RP.
It was observed that among the studied organs of the plant A. graveolens (Table 5), roots had the highest mineral content of the following elements, listed in decreasing order: Al > Fe > Ti > Cr > V > Ce > La > As > Co > Th > Hf > Sc > Sm > Cs > Yb > Sb > Ta > Tb > Eu. The highest contents of these elements were determined in the leaves: Ca > Na > Mg > Cl > Sr > Mn > Ba. For the element Se, the content in leaves and inflorescences was almost the same (0.33 mg/kg and 0.35 mg/kg, respectively).
By comparing the determined concentrations in the roots to the RP, it was ascertained that the content of the following elements: As, Br, Fe, Hf, Na, Sc, Se, Ta, Th, Ti, and V was at least five times higher. It should be noted that the contents of Br, Na, Se, and Th were high in all the studied organs of A. graveolens. The distribution order was as follows: for Br-inflorescences > leaves > stalks > roots; Na-leaves > stalk > roots > inflorescences; Se-leaves = inflorescences > roots > stalk; Th-roots > leaves > inflorescences > stalk. In addition, it was observed that in the leaves of A. graveolens, the contents of Hf and Ta were higher than in the RP by 8 and 20 times, respectively.
The studied members of the Lamiaceae family (L. angustifolia and S. sclarea) were characterized by the following common "chemical fingerprints": As, Eu, Fe, La, Nd, Ta, Th, Sm, Tb, U, V, Yb, Zr. As previously mentioned, C. sativum and L. officinale bioaccumulated most elements either in inflorescences or roots. Zinicovscaia et al. [23] presented the elemental composition of 45 species of medicinal plants of the Lamiaceae family and summarized available literature data on the contents of major, micro-, and rare earth elements. In the referenced study, homogenized samples of the areal parts of the plants were used, collected at the flowering stage. For the aboveground organs of L. angustifolia Mill. and S. sclarea L., it was ascertained that only the content of Sb was similar to the data reported by Zinicovscaia et al. [23] and the rest of the elements were determined to have a greater content in the aboveground organs. The contents of Zn, Ba, Rb, and Cs in the herbs of the Lamiaceae family collected in Bulgaria, determined by ICP-MS, were comparable with the obtained data; the contents of Al, Fe, Ni, and Cr were higher in the plants analyzed in the present study, while the Mn content was greater in plants collected in Bulgaria [36].
The contents of Ca, Fe, Mn, Zn, and Ni determined in the present study fell within the ranges of concentrations determined in plants of the Lamiaceae family collected in Morocco, while the Co content was lower in the Moroccan herbs, and the contents of K, Mg, Cr, Se, As, and V in the Moroccan plants were determined to be higher than those of the plants from the Republic of Moldova [37].
For the Apiaceae (Umbelliferae) family, the following "chemical fingerprints" were characteristic of the three studied members (species): Br, Hf, Se, Sc, Ta, and Th; and the highest mineral contents were found in the leaves and inflorescences. Tunçtürk & Özgökçe [38] performed atomic absorption spectrometry on herbs from the Apiaceae (Umbelliferae) family. The content of Na in the inflorescences of A. graveolens in the present study was similar to the data reported for the same medicinal plant species in the referenced work (1.26 ± 0.05 g/kg); however, the content of Mg (4.53 ± 0.15), K (27.4 ± 0.32), and Ca (20.0 ± 1.21) in the aboveground parts of the plant was higher by a factor of 2, 3, and 2, respectively. Zaidi et al. [39] studied the contents of trace elements in store-bought food spices by means of NAA. It was observed that the elemental content of leaves of coriander in the present study was similar to the reported data for the elements Cl, Hf, and Mn. Similarities to the reported values were observed for the elemental contents of Co, Sc, Zn, and Na in the inflorescences, and Sc, Se, and Sb in the stalks. The reported values for As and K were about three times lower than those determined in the present study. The contents of As, Cs, Co, Ni, and Se in Anethum Sowa L. roots determined by ICP-MS and of Al, Fe, and Na determined by AAS were lower than those ascertained in the present study, while the contents of Ca, Mg, and K were much greater [40].
Data on the effects of chromium, copper, iodine, iron, manganese, molybdenum, selenium, and zinc on the human organism was reviewed in [41]. Thorough data on typical contents of numerous elements in various plants and in the soil has been summarized by Kabata-Pendias and Pendias [42], as well as by Markert et al. [35].
Over the years, international organizations and numerous national regulatory agencies in various countries have imposed or recommended guideline values in order to limit the consumption of potentially toxic elements in food, feed, and in drinking water.
In 1999, the World Health Organization set the maximal permissible level of arsenic in raw plant materials to 1.0 mg/kg [43,44]. This would imply that the leaves and inflorescences of L. angustifolia (Table 2) had an As content almost equal to the obsolete guideline maximal value. In the updated Guidelines for Assessing Quality of Herbal Medicines with Reference to Contaminants and Residues, such maximal levels are no longer prescribed [45]. Instead, provisional tolerable intake (PTI) values established on a regional or national basis are cited and a recommendation for harmonizing the limits and standards for toxic metals is given. Previously set limit values prescribed by the WHO, still applicable for herbal medications, are available only for the elements lead and cadmium: 10 mg/kg and 0.3 mg/kg, respectively.
For other herbal preparations, the National Sanitation Foundation draft proposal (raw dietary supplements) suggests a limit of 5 mg/kg for As, 10 mg/kg for Pb, 0.3 mg/kg for Cd, and 2 mg/kg for Cr [46]. As such, the content of arsenic determined in the studied plants did not exceed the recommended limit value. However, the content of Cr in different morphological parts of four of the studied plants did exceed the limit value of 2 mg/kg. This was the case in roots, stalk, leaves, and inflorescences of L. angustifolia (Table 2), in leaves of S. sclarea (Table 3), in inflorescences of L. officinale (Table 4), and in roots of A. graveolens (Table 5).
Currently, there are no maximum levels established for arsenic in food at EU level, despite the fact that such values have been laid down in national legislation in some Member States. For water intended for human consumption [47,48] a parametric value of 10 µg/L is established. Hajeb et al. [49] summarized that terrestrial foods typically contain low levels of arsenic, less than 0.05 µg/g dry matter, except for rice and other grains, in which As content is often reported between 0.03 to 1 µg/g. [50] lays down maximum levels for contaminants in foodstuff. As regards arsenic, however, there are no statutory limits for its content in food, and instead, a range of benchmark-dose lower confidence limit (BMDL01) values between 0.3 and 8 µg/kg b.w. per day was identified for cancers of the lung, skin, and bladder, as well as skin lesions [51]. Work on this topic is ongoing and it is anticipated that limits will be set for other potentially toxic elements in the near future as the methodology for the quantification improves.
K:Na Ratio
Among the five studied plant species, all except coriander have been reported to have diuretic properties [52][53][54][55]. To assess the diuretic activity of the plants, the K/Na ratio could be used. It was presented graphically in Figure 1. The values ranged between 1.1:1 for the roots of A. graveolens and 486:1 in inflorescences of S. sclarea, which is in good agreement with the findings reported by Zinicovscaia et al. [23] and Szentmihályi et al. [56].
Agronomy 2021, 11, x FOR PEER REVIEW 12 of 20 [51]. Work on this topic is ongoing and it is anticipated that limits will be set for other potentially toxic elements in the near future as the methodology for the quantification improves.
K:Na Ratio
Among the five studied plant species, all except coriander have been reported to have diuretic properties [52][53][54][55]. To assess the diuretic activity of the plants, the K/Na ratio could be used. It was presented graphically in Figure 1. The values ranged between 1.1:1 for the roots of A. graveolens and 486:1 in inflorescences of S. sclarea, which is in good agreement with the findings reported by Zinicovscaia et al. [23] and Szentmihályi et al. [56]. It should be noted that the values for the K/Na ratio determined in all plant organs of C. sativum were greater than those of L. angustifolia, even though the former is not considered a popular herbal diuretic. The results for A. graveolens did not suggest that a substantial diuretic activity could be achieved by ingesting any of the studied organs of the plant.
Biological Transfer Coefficients (BTC)
Biological transfer coefficient (BTC) and translocation factor (TF) could be defined as the ratio of the concentration of a given element in the aboveground part of a plant (leaves, stalk, and inflorescences) and the concentration of the same element in the underground parts (roots) [57]. The calculated values can be found in Appendix A, Table A1.
The results show that in C. sativum, the following elements were translocated to the aboveground parts, with relatively small amounts remaining in the root system: As (BTC = 2. . Data for commercially available herbs from Turkey were inconsistent with the results from our study, as the reported content of Se (23.53 kg/kg) and Cr (5.97 mg/kg) was much higher, while for other elements, it was much lower [58]. In a study conducted on contaminated soils in India, it was suggested that C. sativum could accumulate Mn, Fe, Zn, and It should be noted that the values for the K/Na ratio determined in all plant organs of C. sativum were greater than those of L. angustifolia, even though the former is not considered a popular herbal diuretic. The results for A. graveolens did not suggest that a substantial diuretic activity could be achieved by ingesting any of the studied organs of the plant.
Biological Transfer Coefficients (BTC)
Biological transfer coefficient (BTC) and translocation factor (TF) could be defined as the ratio of the concentration of a given element in the aboveground part of a plant (leaves, stalk, and inflorescences) and the concentration of the same element in the underground parts (roots) [57]. The calculated values can be found in Appendix A, Table A1.
The results show that in C. sativum, the following elements were translocated to the aboveground parts, with relatively small amounts remaining in the root system: As (BTC = 2. . Data for commercially available herbs from Turkey were inconsistent with the results from our study, as the reported content of Se (23.53 kg/kg) and Cr (5.97 mg/kg) was much higher, while for other elements, it was much lower [58]. In a study conducted on contaminated soils in India, it was suggested that C. sativum could accumulate Mn, Fe, Zn, and Cu [59]. Our study suggests that Mn and Zn are accumulated by C. sativum even in the case of unimpacted and unfertilized soils.
As regards L. angustifolia, 30 of the 36 determined elements were characterized by BTC > 2: Al, As, Ba, Br, Ca, Ce, Cl, Co, Cr, Cs, Eu, Fe, Hf, K, La, Mn, Nd, Rb, Sc, Se, Sm, Sr, Ta, Tb, Th, Ti, U, W, Yb, and Zr. The highest values of BTC among all studied plants were observed for the following elements: Al (BTC = 5.5), Ce (BTC = 7), Cs (BTC = 6), Eu (BTC = 5), Hf (BTC = 9), La (BTC = 6), Sc (BTC = 6), Ta (BTC = 9), Th (BTC = 8), Ti (BTC = 7), Yb (BTC = 7), and Zr (BTC = 10). It can be concluded that L. angustifolia has the capability to translocate and deposit a large variety of micronutrients in its shoots, including ultratrace elements. In a study conducted on Lavandula vera L. cultivated on soils contaminated with metals, it was reported that this plant could act as a potential hyperaccumulator of Pb and an accumulator of Cd and Zn [60]. According to Zheljazkov and Astatkie [61] and Angelova et al. [60], the essential oils of Lavandula angustifolia and Lavandula vera L. were not contaminated by heavy metals in the cases of cultivation on lead-enriched soils. Our study demonstrated that Lavandula phytoaccumulates a variety of major and trace elements even when grown on uncontaminated and unfertilized soils.
The BTC values obtained for S. sclarea showed that, in the aboveground mass, the contents of 31 elements were greater than in the roots (BTF > 2): Al, As, Ba, Br, Ca, Cl, Co, Cr, Cs, Eu, Fe, Hf, K, La, Mg, Mn, Ni, Rb, Sb, Se, Sc, Sm, Sr, Ta, Tb, Th, Ti, V, Yb, Zn, and Zr. Among all studied plants, the BTC for the following elements had the greatest values in S. sclarea: As (BTC = 4.1), Ba (BTC = 5), Co (BTC = 5), Fe (BTC = 5), Mn (BTC = 8), Sb (BTC = 3). Angelova et al. [22,62] and Chand et al. [63] reported that under certain soil conditions, clary sage could be a hyperaccumulator of Pb and accumulator of Cd and Zn; therefore, this plant can be utilized for the purposes of phytoremediation of contaminated soils. The same authors ascertained that, in the case of cultivation on polluted soils, the quality and quantity of the extracted essential oils were unaffected.
The data for L. officinale showed that the following elements were accumulated in the aboveground mass of the plant: Ba, Br, Ca, Cl, Co, Cr, Fe, K, Mg, Mn, Ni, Rb, Sb, Se, Sr, and Zn. In comparison to the other studied plants, the rates of accumulation of four of these elements were the highest in L. officinale.: Br (BTC = 27), Cl (BTC = 46), K (BTC = 13), Se (BTC = 6). In addition, the BTC for Rb and Cr were the same as for S. sclarea (BTC = 9 and BTC = 5, respectively).
In a study conducted in Banat region, Romania, it was demonstrated that L. officinale and A. graveolens could be used for phytoextraction of cadmium, as both were characterized by high values for BTC [64]. Our study demonstrated that both plant species did not actively uptake arsenic and could be considered excluders (BTC < 1) [30].
Mineral Content of the Soil
The comparison with the RP revealed that the contents of Al, Hf, Se, Sc, Na, Ta, Th were high in all studied plants, and it could be hypothesized that the mineral content of the soil and/or common plant characteristics could be a factor in explaining this result.
To ascertain the mineral composition of the soil on the experimental field, two soil samples were collected at a distance of 5 km from each other, and NAA allowed for quantification of 38 elements. The data were averaged and presented in Table 6. To express the degree of variability in the determined elemental content, coefficients of variations were calculated. The concentrations of Br, Ca, Cs, Fe, Gd, Hf, Mg, Ni, V, and Zr showed wide variation. Compared to empirical data for European continental-scale soil surveys (GEMAS Ap in Table 6), the contents of trace elements of the soil samples in our study proved to be very similar. Only the content of Ni was found to be twice that of the GEMAS Ap median values [65]. As regards the metal and metalloid content in agricultural topsoils, there was an attempt to harmonize the standards set in member states of the European Union and to define common threshold values, lower, and higher guideline values for As, Cd, Cr, Cu, Hg, Pb, Zn, Sb, V, Co, and Ni [66]. A comparison of these values with the results obtained in this study showed that only the content of Zn exceeded the lower guideline value of 150 mg/kg defined on the basis of ecological risk, so precautionary measures could be necessary under certain conditions. However, the content of Zn was below the higher guideline value of 250 mg/kg, therefore no associated health or food safety risks are implied. The contents of Co, Ni, and V were similar to the threshold values (20 mg/kg, 50 mg/kg, and 100 mg/kg, respectively), while the contents of Cr and Sb were low. The content of As exceeded the proposed threshold value (2 mg/kg) and yet it was much lower than the respective lower guideline value (50 mg/kg).
Local assessment of the soil could be made using Moldavian limit values for metal concentrations in soil set for the elements Cd, Cu, Ni, Pb, Zn, and Hg [67]. The contents of Ni and Zn determined in the soil samples did not exceed the limit values of 75 mg/kg and 300 mg/kg, respectively. As such, it can be concluded that in terms of the mineral content of the soil, the mentioned fields from the country located near the towns of Causeni and Glodeni, could be considered "ecologically clean".
In a study of vineyards in Moldova, the reported values for the elements Cr, Mn, Co, Zn, and As in soils were similar to the results obtained in our work [68]. In another study conducted in Moldova, on the mineral content of Tanacetum corymbosum (L.) Sch. Bip., it was suggested that the high contents of Al, As, Br, Cl, K, Mg, Mo, and Sc determined in the plant could be caused by the composition of local soils, rich in dolomites, limestones, and K-feldspars [69].
Biological Absorption Coefficients (BAC)
The phytoavailability of a given element, or transference from the growth media to the organs of the plant, could be assessed using coefficients known as biological absorption coefficients (BAC) or the index of bioaccumulation (IBA). These coefficients are defined as the ratio of the total content of an element in the plant material to the content of the same element in the associated soils [42]. The BACs were presented in Table A1 alongside the BTCs.
It was observed that the elements characterized by increased phytoavailability in C. sativum were the following: Br (BAC = 5), Ca (BAC = 2.4), Cl (BAC = 127), K (BAC = 9), and Zn (BAC = 2.2). Having in mind that the BTCs for Ca, Cl, K, and Zn were greater than 2, it could be concluded that C. sativum is an accumulator of these four elements in the conditions of cultivation on unpolluted soil and no fertilization. In addition, it was observed that this plant is a rejector (excluder) of U and Zr, since both the BTC and BAC values were smaller than 1, meaning that uptake was limited.
The same set of elements with similar values for BAC were observed for L. angustifolia The BAC for K, which had the value of 5, was the only exception from this observation since in the case of C. sativum it was 9. Additionally, the element Sr was phytoaccumulated by L. angustifolia, BAC = 2.8. As regards S. sclarea, the BACs exceeding or equal to 2, were the following: Ca (BAC = 3), Cl (BAC = 5), K (BAC = 8), and Sr (BAC = 2).
The uptake of elements is influenced by the specific plant-soil interactions and could be improved by root exudates [70]. However, since Br, Ca, Cl, and K were phytoaccumulated in all studied plants, and the BACs for Sr and Zn had rather high values in most, it could be concluded that soils from the experimental field were rich in mobile or phytoavailable forms of these elements (soluble compounds).
Conclusions
By utilizing neutron activation analysis, the number of determined elements was maximized and the quantification of a total of 36 elements was achieved. The leaves, inflorescences, and roots of the studied plants could be considered important sources of nutrients in the food and pharmaceutical industries. The contents of the potentially toxic element Cr in roots, stalk, leaves, and inflorescences of L. angustifolia, in the leaves of S. sclarea, in the inflorescences of L. officinale, and in the roots of A. graveolens exceeded the guideline value (2 mg/kg) laid down by the National Sanitation Foundation International. A comparison with data for Markert's Reference Plant revealed the following "chemical fingerprints": for A. graveolens: As, Br, Fe, Hf, Na, Sc, Se, Ta, Th, Ti, and V; for C. sativum: Al, Na, Br, Hf, Se, Sc, Ta, Th, Ti, U, and Zr; for L. angustifolia: Al, As, Ce, Cr, Eu, Ce, Cr, Fe, Hf, La, Na, Nd, Sc, Se, Sm, Ta, Tb, Th, Ti, U, V, Yb, and Zr; for S. sclarea: Al, As, Co, Eu, Fe, Hf, La, Na, Nd, Sc, Se, Ta, Th, Sm, Tb, Th, U, V, Yb, and Zr; and for L. officinale: Se, Br, Al, Fe, Hf, Na, Sc, Se, Ta, Th, and U. The calculated BAC and BTF revealed that the studied aromatic plants accumulated certain elements when grown on unfertilized soils. These findings concern the biogeochemical properties of the plants and could be used in further in botanical and environmental studies. Table A1. Biological transfer coefficients (BTC), as the ratio between the content of an element in aboveground organs and the roots; and biological accumulation coefficients (BAC), as the ratio between the total content of an element in the plant and the associated soil samples. | v3-fos-license |
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"year": 2022
} | pes2o/s2orc | CD73 controls ocular adenosine levels and protects retina from light-induced phototoxicity
ATP and adenosine have emerged as important signaling molecules involved in vascular remodeling, retinal functioning and neurovascular coupling in the mammalian eye. However, little is known about the regulatory mechanisms of purinergic signaling in the eye. Here, we used three-dimensional multiplexed imaging, in situ enzyme histochemistry, flow cytometric analysis, and single cell transcriptomics to characterize the whole pattern of purine metabolism in mouse and human eyes. This study identified ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2, and ecto-5′-nucleotidase/CD73 as major ocular ecto-nucleotidases, which are selectively expressed in the photoreceptor layer (CD73), optic nerve head, retinal vasculature and microglia (CD39), as well as in neuronal processes and cornea (CD39, NTPDase2). Specifically, microglial cells can create a spatially arranged network in the retinal parenchyma by extending and retracting their branched CD39high/CD73low processes and forming local “purinergic junctions” with CD39low/CD73− neuronal cell bodies and CD39high/CD73− retinal blood vessels. The relevance of the CD73–adenosine pathway was confirmed by flash electroretinography showing that pharmacological inhibition of adenosine production by injection of highly selective CD73 inhibitor PSB-12489 in the vitreous cavity of dark-adapted mouse eyes rendered the animals hypersensitive to prolonged bright light, manifested as decreased a-wave and b-wave amplitudes. The impaired electrical responses of retinal cells in PSB-12489-treated mice were not accompanied by decrease in total thickness of the retina or death of photoreceptors and retinal ganglion cells. Our study thus defines ocular adenosine metabolism as a complex and spatially integrated network and further characterizes the critical role of CD73 in maintaining the functional activity of retinal cells. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-022-04187-4.
Introduction
Extracellular ATP and its metabolites ADP and adenosine (ADO) are important signaling molecules involved in a wide range of (patho)physiological activities in virtually all organs and tissues [1], including the eye [2][3][4][5]. ATP released from damaged neurons, blood vessels, activated microglia, and Müller glial cells triggers diverse proinflammatory, neurodegenerative, and angiogenic processes which are mediated by activation of metabotropic (P2Y) and ligandgated (P2X) nucleotide receptors expressed in the retina and other ocular structures [2,3,6]. Another mechanism of ATP action is conveyed via its ectoenzymatic breakdown into ADO, which in turn binds to adenosine receptors (AR) that function by activating (A 2A R and A 2B R) or inhibiting (A 1 R and A 3 R) adenylyl cyclase [1]. A 2A R and/or A 1 R are especially relevant in terms of ocular physiology by playing a crucial role in pathological retinal angiogenesis [7,8], neuroinflammation [5,9], modulation of the circadian clockwork [10], photoreceptor coupling [11], retinal, choroid and optic nerve blood flow [12,13], and also hyperpolarization of retinal ganglion cells (RGC) and protecting them from apoptosis [6,14].
Along with significant progress in understanding the function of purinergic receptors, recent studies have begun to uncover the complexity of regulatory mechanisms governing the duration and magnitude of purinergic signaling in the eye. Previous research has focused on the expression of key nucleotide-inactivating/ADO-producing enzymes: ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1, also known as CD39), NTPDase2 and ecto-5′-nucleotidase/ CD73 in primate [15][16][17], rodent [3,8,[17][18][19], and zebrafish [19,20] retinas in terms of their role in control of angiogenesis, diabetic retinopathy, intraocular pressure, and neurovascular coupling. In addition, CD73 has been widely employed as a cell surface marker for the enrichment of pluripotent stem cell-derived photoreceptor populations and the isolation of photoreceptors from retinal organoids [21,22]. Soluble forms of CD73, adenosine deaminase (ADA), adenylate kinase-1 and other enzymes were also identified in the human vitreous fluid, where they coordinately regulate ocular ATP and ADO levels via two counteracting, purineinactivating and ATP-regenerating, pathways [16,23]. Multiple human disorders have been linked to abnormalities in purine metabolism, including cancer [24,25], cardiovascular diseases [26], and ocular diseases [9,16]. Several potent small-molecule inhibitors and antibodies directed against CD39 and CD73 were developed recently and tested in clinical trials as potential anti-cancer drugs [25,27,28]. However, one of the obstacles preventing translation of purinergic enzymes to the clinic is the lack of consideration of redundant pathways controlling ATP and ADO levels in a certain synergistic, counteracting or compensatory manner [29,30].
The complexity of the architecture and function of the mammalian eye require the development of advanced tools to study the extracellular space in heterogeneous retinal environment. Conventional histological analyses of protein expression performed using formalin-fixed paraffinembedded tissue sections or cryo-embedded sections can offer high-resolution images, but the limited thickness of slices hampers the acquisition of more information on the z-axis. Recent development of advanced platforms such as clearing-enhanced three-dimensional (3D) and other volumetric imaging techniques permits cell-level analysis of cell positioning in the context of macroscale tissue structure [31,32]. By using a high-resolution 3D multiplexed imaging, in situ enzyme histochemistry and flow cytometric analysis of mouse retina, in combination with single cell transcriptomic data of mouse and human retinal cells, this study was undertaken to assess the whole pattern of purine metabolism in the mammalian eye. Furthermore, we tested pharmacological intervention aimed at reducing intraocular ADO levels by using a novel highly potent and metabolically stable CD73 inhibitor PSB-12489 [27], and demonstrated the essential role of CD73 in protecting the mouse retina from light-induced phototoxicity.
Animals
Female and male C57BL/6N mice were obtained from Janvier Labs (France). The animals were maintained at Central Animal Laboratory of the University of Turku (Turku, Finland) and used for histochemical analysis of the eye, flow cytometry and blood serum preparation. The experimental procedures were reviewed by the local Ethics Committee on Animal Experimentation of the University of Turku and approved by the Provincial State Office of Western Finland with the license ID ESAVI/5762/04.10.07/2017. For intravitreal treatment and BL exposure, male C57BL/6JrJ and BALB/c mice (obtained from Janvier Labs, France, and the Laboratory Animal Centre, University of Tartu, Tartu, Estonia, respectively) were maintained at Experimentica Ltd. Laboratory Animal Center (Kuopio, Finland). The animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the EC Directive 86/609/EEC for animal experiments, using protocols approved and monitored by the Animal Experiment Board of Finland (Experimentica Ltd. license ID: ESAVI-004139-2017/ESAVI-10815-2020). All mice were 3-4 months of age, with a body weight of 22-25 g. The animals were raised in pathogen-free conditions, housed at a constant temperature (22 ± 1 °C) in a light-controlled environment (lights on from 7 am to 7 pm), and provided with food and water ad libitum.
Intravitreal treatment and bright light exposure
C57BL/6JrJ and BALB/c mice (4 animals per group) were used in the first study. Aliquots of PSB-12489 (2 μL of 2 mM) and equal volumes of PBS were administered by intravitreal injections into the right (OD) and left (OS) eyes, respectively. Mice were kept in the dark for 12 h before fERG recording, except for the acute action studies of CD73 inhibitor, where treated mice were dark adapted for only 6 h. In the second study, a total of 24 BALB/c mice were divided into six groups (3-5 animals per group). The animals were kept in transparent plastic cages and subjected to different experimental settings, as outlined below. White light lamps (URZ3372, 6400K, Kemot, Poland) were directed to the cages (one from the bottom and one from the top). Mice were exposed to 9500 lx bright light (BL) for 14 h, starting from 19.00 on day 0 until 9.00 on day 1. Five hours before BL induction, 2 μL aliquots of PSB-12489 (2 mM) were administered by bilateral intravitreal injections to both eyes. Equal volumes of PBS were administered to the eyes of vehicle-treated mice. The animals were returned to a normal facility light/dark cycle of 12 h/12 h and used in experiments 7 days after the treatments, as described below.
Flash electroretinography (fERG)
Mice were dark adapted for 12 h unless otherwise specified. All procedures for fERG recordings were performed under dim red light. The animals were anesthetized with a mixture of ketaminol (37.5 mg/kg) and medetomidine (0.45 mg/kg) administered intraperitoneally. Body temperature was maintained through the use of a heating pad (set to 38 °C). The pupils were dilated by administering tropicamide, and 3 min later phenylephrine hydrochloride (100 mg/mL). To improve conductivity between eye and electrode, one drop of 0.9% saline was applied to both eyes. The reference electrode needle was inserted in between the eyes and the common grounding electrode was inserted into the base of the tail. Signals were recorded using the Celeris-Diagnosys system (Diagnosys LLC, Massachusetts, USA). To evaluate the functional response from rods and cones, a flash of various intensities (0.003-10 cd s/m 2 ) was used (Table S2). General anesthesia via medetomidine was immediately reversed by an α2-antagonist atipamezole (0.5 mg/kg sc.) The following parameters were analyzed and reported: amplitude and latency of the a-waves (first negative fERG component) and amplitude and latency of the b-waves (first positive fERG component). All parameters are provided as raw data.
Optical coherence tomography (OCT)
High-resolution spectral domain OCT was performed on the baseline and 7 days after exposure of the BALB/c mice to PSB-12489 and/or BL, as described elsewhere [33]. All measurements were performed under general anesthesia. The pupils of both eyes were dilated after application of 5 mg/mL solution of tropicamide. To prevent corneal drying, a Systane Ultra Eye gel (Systane ® Ultra, Norbrook, England) was applied on the cornea. The mice were fixed in the holder and ten series of 100 b-measurements were carried out (each b-measurement had 1000 a-measurements). The data obtained were aligned, averaged and a 3D image was created. Photoreceptor layer thickness was measured at 25 different points, which were selected using InVivoVueDiver (Bioptigen, JAV) software. The central point was targeted at the center of the optic nerve. The photoreceptor layer thickness was estimated by measuring the distance between the outer plexiform layer and the external limiting membrane.
Sample collection and processing
Mice were killed by carbon dioxide and the eyeballs were immediately enucleated and processed for further analyses in the following ways. For enzyme histochemistry, the eyeballs were embedded in the cryo-mold with Tissue-Tek ® optimal cutting temperature compound (Sakura Finetek Europe B.V., the Netherlands), cut at 10 μm onto Superfrost ® Plus slides (ThermoFischer Life Technologies) using a Leica CM 3050S cryostat, air-dried and stored at − 80 °C. For immunofluorescence staining, the eyes were fixed for 2 h at room temperature (RT) with PBS containing 4% paraformaldehyde (PFA) and embedded in the mold with 4% solution of LMA dissolved in PBS and pre-heated in the microwave oven. LMAembedded eyes were sectioned at 100 μm thickness using a Leica VT1200S vibrating microtome, additionally fixed for 30 min with 4% PFA, stored in PBS at 4 °C, and processed for 3D immunofluorescence staining within 1 week of preparation. For flow cytometry analysis, the retinas were dissected from the eyecups and digested into the single cell suspension, as described below.
In situ enzyme histochemistry
For localization of ecto-nucleotidase and TNAP activities, the combined histochemical approach was employed [34]. In brief, tissue cryosections were thawed, fixed for 5 min with 4% PFA, and pre-incubated for 45 min at RT in Trizma-maleate sucrose buffer (TMSB) [40 mmol/L Trizmamaleate, 0.25 mol/L sucrose, pH 7.4] supplemented with the TNAP inhibitor tetramisole (2 mM). The slides were subsequently incubated for 1 h at RT in a mixture containing TMSB (pH 7.4), 2 mM tetramisole, 2 mM Pb(NO 3 ) 2 , 0.5 mM CaCl 2 and one of the following nucleotide substrates: ATP (300 µmol/L), ADP (300 µmol/L) and AMP (1 mmol/L). In blank specimens, the substrate was omitted from the incubation solution. The lead orthophosphate precipitated in the course of nucleotidase activity was visualized as a brown deposit by incubating the sections for 15 s in 0.5% (NH 4 ) 2 S. TNAP activity was additionally evaluated by measuring the intensity of dark purple precipitate after incubating the tissues for 20 min at RT in a mixture containing TMSB (pH 9.3), 5 mM MgSO 4 and artificial enzyme substrates BCIP and NBT (2 mmol/L each). Tissue sections were also stained with hematoxylin and eosin (H&E). Whole tissue section imaging was performed using Pannoramic-250 Flash slide scanner (3DHistech Ltd., Budapest, Hungary) with a 20 × objective.
Immunofluorescence staining
LMA-embedded vibratome-cut eye sections (100-150 µm thickness) were incubated for 1 h at RT in 300 μL PBS containing 2% bovine serum albumin (BSA) and 0.5% (vol/vol) Triton X-100 (blocking buffer) and subsequently incubated overnight at 4 °C with biotin-conjugated IB4 and primary antibodies diluted in 300 μL of blocking buffer, as specified in Table S1. To avoid off-target background signal during staining of mouse eyes with mouse anti-rhodopsin and anti-NeuN antibodies, endogenous immunoglobulins were blocked by pretreating the samples for 2 h with unconjugated Fab fragment of donkey anti-mouse IgG (20 μg/mL). Negative control staining was also performed in which eye sections were incubated with isotype-matched pre-immune sera from rabbit and guinea pig used at the same dilutions as the primary anti-CD73 and anti-CD39 antibodies. The samples were incubated overnight at 4 °C with the appropriate fluorochrome-conjugated secondary antibodies and Fluor ® 647-streptavidin diluted in blocking buffer at ~ 1:800. Alexa Fluor ® 488-conjugated anti-CD31, NL493-conjugated anti-tubulin-βIII, and Cy3™-conjugated anti-smooth muscle actin-α (SMA-α) antibodies and Alexa Fluor ® 546-Phalloidin were added during the incubation with secondary antibodies for labeling the vascular endothelial cells, neuronal filaments, perivascular cells, and F-actin filaments, respectively. All staining procedures were performed in a 24-well plate under 60 rpm orbital rotation, by washing the wells after each treatment with 300 μL of blocking buffer (3 × 30 min). Stained eye sections were additionally washed for 10 min in 500 μL PBS, transferred onto the microscope slide, aligned using forceps under stereomicroscope, and mounted with ProLong ® medium with glass spacers inserted between the slide and the coverslip. Imaging was performed using 3i CSU-W1 spinning disk confocal microscope (Intelligent Imaging Innovations, Inc.) equipped with Hamamatsu ORCA Flash 4 sCMOS camera (Hamamatsu Photonics, Hamamatsu, Japan) and Slidebook 6.0 software. Z-stacks of the medial retina and other eye structures were captured using the following objectives: Plan-Apochromat 10 ×/0.45, Plan-Apochromat 20 ×/0.8, LD C-Apochromat 40 ×/1.1, and Plan-Neofluar oil 63 ×/1.4. Maximum intensity projections and 3D reconstructed images were prepared using Imaris 8.4 software (Bitplane). 3D datasets were rendered into movies using Imaris Animation technology and exported to mp4 format.
To study the effect of CD73 inhibitor and BL exposure on photoreceptor CD73 expression, eye tissue cryosections were incubated for 2 h at RT with anti-CD73 and antirhodopsin antibodies (diluted at 1:300 and 1:1000, respectively), and subsequently incubated for one hour with the appropriate fluorochrome-conjugated secondary antibodies. Stained eye sections were mounted with ProLong ® medium with DAPI and imaged using Pannoramic Midi Fluoresence slide scanner (3DHistech Ltd., Budapest, Hungary).
Flow cytometry
Eyes were gently enucleated from C57BL/6N mice. The cornea and lens were removed and retina was carefully dissected from the eyecup using a dissection microscope, fine forceps and surgical microscissors. Retinas from two mice were pooled and digested for 1 h at 37 °C with RPMI-1640 medium containing 0.2 mg/mL collagenase P and 0.1 mg/ mL DNase. The cells were passed through a 100 µm filter and blocked for 15 min with purified rat anti-mouse CD16/ CD32 (Mouse BD Fc Block™). The single cell suspension was subsequently incubated for 20 min with anti-CD73 and anti-CD39 antibodies (or isotype-matched immunoglobulins), together with biotin-conjugated IB4 and fluorescentlylabeled antibodies against CD45, CD11b, P2Y 12 R, and CD31 (see Table S1). After washing, secondary antibodies including Cy3™-or Alexa Fluor ® 633-conjugated donkey anti-guinea pig IgG, Alexa Fluor ® 488-conjugated goat anti-rabbit IgG, and a BV421™-conjugated streptavidin were added. Stained cells were washed and fixed with 2% paraformaldehyde for 10 min. Flow cytometry analyses were performed using BD LSRFortessa (BD Biosciences) and analyzed using FlowJo software (TreeStar Inc).
Competitive CD73 assays and analysis of photoreceptor AMPase activity
Soluble CD73 activity was determined by thin layer chromatography (TLC) using human and mouse sera as enzyme sources and [ 3 H]AMP as preferred enzyme substrate [27]. The effect of CD73 inhibitors on photoreceptor AMPase activity was also evaluated in situ by using lead nitrate-based enzyme histochemistry [34,35]. Noteworthy, both AMPCP and PSB-12489 act as reversible CD73 inhibitors. Therefore, it was necessary to maintain these compounds in the assay medium during both the pre-treatment step (30 min at room temperature) and subsequent 1-h incubation of the mouse eye cryosections with the AMP substrate. AMPase activity was determined by measuring AMP-specific brown staining intensities from the whole slide images using QuPath v.0.2.3 software [36]. Shortly, a project including all images was created. Tissue areas were detected using the threshold classifier. The classifier was run with full resolution (240 nm/ pixel) for the whole project. The DAB (3,3′-diaminobenzidine) channel and Gaussian pre-filtering were selected. The threshold was set to 1.15 without smoothing and areas above the threshold were classified as "positive". Representative areas of the OPL, ONL and OS of the photoreceptor layer were manually selected, and the average DAB intensity level was used for AMPase intensity quantification. The script for the analysis is shown in Table S3.
Quantification of cell nuclei in the retinal layers
The images of transverse eye sections stained with H&E were captured using slide scanner, as described above. Cell nuclei in the major retinal layers (ONL, INL, and GCL) were quantified using the Stardist deep learning platform with Fiji-ImageJ. First, the mrxs files were converted into tiff format. The Stardist Versatile ("H&E Nuclei") pre-trained model was used in the analysis. The model was originally trained on images from the MoNuSeg 2018 training data and the TCGA archive [37]. For all retinal layers, images were normalized and the lower and upper percentiles were selected as 1 and 99.8, respectively. For GCL, probability/ score threshold value was set to 0.5, and the overlap threshold was set to 0.4. For INL and ONL, the values were 0.1 and 0.6, respectively. After Stardist analysis, two representative 1000 µm long areas from both sides of the optic nerve head were chosen and the number of nuclei per area unit was calculated. The drawing of the regions started approximately 300 µm from the optic nerve head.
TUNEL assay
Eye cryosections were fixed with 4% PFA for 15 min at RT. Then sections were treated with PBS containing 0.25% Triton X-100 and 3% BSA for 20 min at RT. To detect fragmented DNA in apoptotic cells within the retinal layers, "Click-iT™ Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor™ 488 dye" (Invitrogen) was used in accordance with manufacturer's protocol. A positive control sample was treated beforehand with 100 μg/mL of DNase-I (Sigma) to induce DNA strand breaks. After the TUNEL reaction, samples were mounted with ProLong ® medium with DAPI and imaged using Pannoramic Midi Fluoresence slide scanner (3DHistech Ltd., Budapest, Hungary). Apoptotic (TUNEL-positive) cells in the ganglion cell layer were counted manually using QuPath v.0.3.0 software [36]. Total ganglion cell numbers were analyzed using Stardist deep learning platform via Fiji-ImageJ [37]. The Versatile (fluorescent nuclei) model was used. Images were normalized, and lower percentile was set to 1.0 and upper percentile to 99.8. Probability threshold was 0.50 and Overlap threshold 0.40.
Statistical analysis
Statistical significance was determined by using two-tailed Student's t test and Mann-Whitney U test. In the case of fERG study, the difference between the control and treated groups was evaluated by multiple t test grouped analysis using the Holm-Sidak method. The levels of statistical significance were denoted as *P < 0.05 and **P < 0.01. For competitive analysis, concentration-inhibition curves were generated in three to five separate experiments, and the IC 50 values were calculated from one-site competition curves constructed using nonlinear least-squares curve fitting. All results were analyzed with Prism GraphPad 7 software (GraphPad, San Diego, CA, USA).
CD73 is selectively compartmentalized in the photoreceptor layer of the mouse retina, while CD39 is highly expressed in the eye vasculature, retinal microglia and cornea
The first part of this study was designed to assess the tissuespecific distribution of nucleotide-inactivating enzymes in the naïve mouse eye. The use of sections of whole eyeball dissected from C57BL/6N mice and embedded in low melting point agarose (LMA) and their sequential incubation with antibodies against CD73, CD39 and NTPDase2 in combination with a wide range of molecular markers allowed us to characterize the phenotypic identity and spatial localization of key ecto-nucleotidases in a relatively thick (~ 100 μm) tissue volume. Staining of the eye with anti-CD73 antibody (Fig. 1a, b), but not with isotype-matched rabbit pre-immune serum (Fig. 1c), revealed selective compartmentalization of CD73 in the photoreceptor layer. The highest CD73 immunoreactivity was associated with the outer segments (OS) of photoreceptor cells, where it is co-localized with a lightsensitive receptor protein rhodopsin (a marker of rod cells) (Fig. 1b). Another nucleotide-inactivating enzyme CD39 is highly expressed in the retinal vasculature, including the central retinal artery and vein, which enter the optic nerve head and further bifurcate into smaller arterioles, venules and capillaries extensively branching throughout the inner (superficial) plexus and deeper capillary plexus, as well as in the choroid layer (choriocapillaris) and extraocular blood vessels (Fig. 1a). Co-staining of the eyes with anti-CD39 antibody and different vascular markers demonstrated the presence of CD39 on all components of the vessel wall, including CD31 + /IB4 + vascular endothelial cells which share their basement membranes with adjacent NG2 + /Phalloidin + pericytes, and also contractile SMA-α + /Phalloidin + smooth muscle cells (SMC) wrapped in a circumferential pattern around larger arterioles ( Fig. 2a and Fig. S1). Interestingly, the close-up view of the deep and intermediate plexuses of the mouse retina validated recent data on the presence of so-called "interpericyte tunnelling nanotubes" that connect two bona fide pericytes on separate capillary systems and regulate neurovascular coupling in the living retina [41], and further extend these observations by showing that these fine structures do not express CD39 and as a consequence are unable to metabolize ATP (Fig. 2a, inset). CD39 is also expressed, albeit faintly compared to blood vessels, on other ocular structures, including rhodopsin + OS of photoreceptor cells (Fig. 1b), NeuN + neuronal cell bodies located in the ganglion cell layer (GCL), as well as P2Y 12 R + microglial cells, which mainly reside in two synaptic compartments of the neural parenchyma: the outer plexiform layer (OPL) and the inner plexiform layer (IPL), and in the optic nerve head (Fig. 2b). Furthermore, CD39 is co-localized with another member of the NTPDase family, NTPDase2, on tubulin-βIII + neuronal processes lining the innermost margin of the retina and cornea, as well as corneal IB4 + /Phalloidin + epithelial cells, and stromal keratocytes ( Fig. 2c and Fig. S1). The specificity of CD39 staining was further confirmed by the absence of any fluorescence signal in the negative control sample incubated with guinea pig pre-immune serum used at the same dilution as the primary anti-CD39 antibody (Fig. 1c).
The advantage of our workflow is that it provides additional information on high-resolution 3D mapping of cell positioning in the context of macroscale tissue. Given that the commonly used 2D immunofluorescence images or maximum intensity projections of 3D images significantly underestimate microglial cell motility [31,42], such volumetric approach may be particularly relevant for evaluation of stereoscopic morphology of retinal microglial processes and their heterotypic interactions with other components of the neurovascular unit. The 3D reconstructed images enabled visualisation of extensively branched microglial cell processes that co-express two important components of the purinergic machinery, P2Y 12 R and CD39, and form direct contacts with exterior walls of CD39 + retinal blood vessels (Fig. 3a, and Movie 1), as well as with neuronal cell bodies which express CD39 at relatively low levels (Fig. 3b, and Movie 2).
In situ enzyme histochemistry and flow cytometric assays confirm cell type-and tissue-specific localization of ecto-nucleotidases in the mouse retina
In a different set of experiments, the activities of ecto-nucleotidases were measured in the mouse eye cryosections by using lead nitrate-based enzyme histochemistry assay [34]. Additional staining of the samples with haematoxylin and eosin (H&E) (Fig. 4a) enabled the visualization of the main retinal layers and other ocular structures. The presence of dark light-absorbing melanin granules in the exterior retinal pigmented epithelium (RPE) partially interferes with enzyme histochemistry of the eye. Nevertheless, there were clear-cut differences in staining intensities between the samples incubated without (Fig. 4b) and with (Fig. 4c-e) exogenous nucleotides. High ATPase (Fig. 4c) and ADPase (Fig. 4d) activities were detected in the retinal vessels, OS of photoreceptor cells, outer limiting membrane, and neuronal bodies, while AMP-specific staining was mainly confined within the photoreceptor layer (Fig. 4e). High ATPase and ADPase (but not AMPase) activities were also detected in the stromal keratocytes and basal epithelial layer of the cornea (Fig. 4c-e). Notably, similar staining patterns were observed when eye cryosections were incubated with were captured using a spinning disk confocal microscope. c In the negative control staining the eye sections were incubated with CD39-PI (mN1-2 C PI) and CD73-PI (rNu9L-PI) pre-immune sera used at the same dilutions as in the b staining with primary anti-CD39 and anti-CD73 antibodies. Maximum intensity projections for each channel are shown in grayscale, with the right panels displaying merged images with nuclei counterstained with DAPI. CBV choroid blood vessels, CRV central retinal vessels, EBV extraocular blood vessels, EM extraocular muscles, Mü Müller cells, NS nonspecific staining (caused by binding of mouse anti-mouse rhodopsin antibody to endogenous immunoglobulins in the blood vessel), ONL outer nuclear layer, OPL outer plexiform layer, OS outer segments of photoreceptors, PI pre-immune serum, VC vitreous cavity. Scale bars: 300 μm (a), 100 μm (b), and 50 μm (c) nucleotide substrates in the presence (Fig. 4c-e) and absence (data not shown) of the inhibitor of tissue-nonspecific alkaline phosphatase (TNAP) tetramisole. On the other hand, the use of the artificial chromogenic substrates of TNAP, BCIP and NBT revealed the development of specific dark blue staining in the inner and outer plexiform layers of retina, as well as in the superficial corneal epithelial cell layer (Fig. 4f), which disappeared after pretreating the samples with tetramisole (data not shown). These data suggest that despite the selective expression of TNAP in certain eye structures, this broad substrate-specificity ectoenzyme is not implicated in the metabolism of ocular ATP and other nucleotides. Collectively, in situ enzyme histochemistry, together with the multiplexed imaging data described above, identified CD39 as the predominant ATP-and ADP-inactivating enzyme in the mouse eye which is expressed to varying degrees among vascular, immune, neural and stromal cells. The downstream step of hydrolysis of ATP/ADP-derived AMP into ADO is mediated through ecto-5′-nucleotidase/ CD73 activity, which is mainly localized in the photoreceptor layer. Flow cytometric analysis of isolated mouse retinal cells provided independent line of evidence for the presence of CD39 on CD45 + /CD11b + /P2Y 12 R + microglial cells (Fig. 4g) and CD45 − /IB4 + /CD31 + vascular endothelial cells (Fig. 4h). CD73 is also weakly expressed on retinal microglial cells, but not in the blood vessels (Fig. 4g, h).
Single cell transcriptomic analysis of mouse and human retinal cells reveals relatively conserved purinergic signatures between the species
The expression profiles of genes encoding major purineinactivating enzymes and ARs were also characterized at a single cell resolution by using publicly available scRNAseq data of mouse retinal cells [38]. Single cell transcriptomic analysis demonstrated specific distribution of ectoenzymes in mouse vascular endothelial cells (Entpd1/CD39 high , Alpl/ TNAP high ), perivascular cells (Entpd1/CD39 low , Enpp1/ ENPP1 low ), retinal microglia (Entpd1/CD39 low ), rod photoreceptors (Nt5e/CD73 high , Alpl/TNAP low ), horizontal cells (Nt5e/CD73 high ), RGC (Entpd2/NTPDase2 low ), Müller glia and astrocytes (Entpd2/NTPDase2 high ) (Fig. 5a). In contrast to our multiplex imaging data showing the presence of CD39 immunoreactivity (Fig. 1b) and ATP/ADPinactivating activity (Fig. 4c, d) in the OS of photoreceptor cells, transcriptomic approach did not reveal CD39-encoding gene in rod cells at mRNA level (Fig. 5a). Although the expression of Entpd1 on microglial cells was very low in this study, the use of another scRNAseq dataset of sorted Cx3cr1 + mouse retinal cells [39] revealed that Entpd1/CD39 is highly expressed on two major populations of P2ry12 + and Hmox1 + microglial cells, and additionally demonstrated the presence of other enzyme of the purine catabolic chain, ADA, on retinal Hmox1 + microglial cells and perivascular macrophages (Fig. 5b). While a detailed characterization of signal transduction pathways mediating biological effects of ADO lies beyond the scope of this study, we also analyzed the expression profiles of major AR subtypes. ARs are selectively expressed on various mouse retinal cells, including vascular endothelial (Adora2a/A 2A R low ) and perivascular (Adora2a/A 2A R high ) cells, RGC (Adora1/A 1 R high ), Müller glia and astrocytes (Adora1/A 1 R low ), P2ry12 + microglial cells and Rorb + macrophages (Adora3/A 3 R high ) (Fig. 5a, b). Notably, data on highly selective expression of Nt5e/ CD73 on the latter subset of Adora3 + /Rorb + macrophages suggest that ADO metabolism may be relevant in controlling adenosinergic signaling and function in this relatively small population of mouse retinal myeloid cells (Fig. 5b).
To further identify the similarities and differences in the purinergic signatures between rodent and human eyes, we utilized single cell transcriptomic atlas of the human retina [40]. Ecto-nucleotidases and TNAP are selectively expressed on the human retinal endothelial cells (ENTPD1/CD39 low , ALPL/TNAP high ), microglial cells (ENTPD1/CD39 high , NT5E/CD73 low ), rod photoreceptors (NT5E/CD73 high , ALPL/TNAP low ), amacrine cells (ENPP1/ENPP1 low ), and Müller glia and astrocytes (ENTPD2/NTPDase2 low ), while the expression of ADO-inactivating enzyme ADA was maintained at very low or undetectable levels in all human retinal cells (Fig. 5c). These human transcriptomic data are consistent with our recent in situ enzyme histochemistry and immunofluorescence imaging data showing tissue-specific distribution of key ecto-nucleotidases (CD39, NTPDase2, CD73) and TNAP in the human sensory neuroretina and optic nerve head [16]. Additional scRNAseq analysis of adenosinergic signaling pathways revealed that, similar to mouse retina, human retinal microglial cells and Müller glia and astrocytes express high levels of A 3 R (ADORA3) and A 1 R (ADORA1), respectively. However, unlike mouse blood vessels which express Adora2a/A 2A R, human retinal endothelial cells express another A 2 R subtype, ADORA2B/ A 2B R. Other human retinal cell subsets do not appear to express either AR subtype (Fig. 5c).
Overall, despite some species-specific variations, the expression profiles of key purinergic enzymes and ARs appear to remain relatively conserved between the mouse and human eyes. In particular, data on selective compartmentalization of CD73 in both mouse and human photoreceptors provide a solid background for more thorough investigation of the role of this ectoenzyme in retinal function under various challenging and noxious conditions and further translation of these experimental data to clinic.
Pharmacological inhibition of ocular CD73 impairs retinal activity in dark-adapted mice exposed to bright light
Taking into account data on direct involvement of adenosinergic signaling in the modulation of light-evoked responses of retina [43][44][45], we hypothesized that pharmacological inhibition of the CD73-ADO axis may affect retinal function. Several novel CD73 inhibitors have been designed and synthesized recently in our laboratories based on N 6 -benzylα,β-methylene-ADP (PSB-12379) as a lead structure, which are characterized by exceptionally high selectivity, nanomolar inhibitory potency toward human, rat and mouse CD73, and high metabolic stability in human plasma and in rat liver microsomes [27]. Studies with fluorescein-conjugated CD73 inhibitors additionally confirmed the utility of these compounds as fluorescent probes capable of binding directly to CD73 on various cells and tissues, including mouse CD73 + photoreceptor cells [46]. The most potent CD73 inhibitor, PSB-12489 (Fig. 6a) [27], was chosen as a suitable drug for further examination in our competitive and functional assays. Radio-TLC enzymatic assays confirmed the ability of PSB-12489 to inhibit the hydrolysis of [ 3 H]AMP by human and mouse sera in a concentration-dependent manner with the IC 50 values in the low nanomolar range, whereas the classical CD73 inhibitor AMPCP exerted inhibitory effects at ~ 100 times higher concentrations (Fig. S2). This conclusion was independently ascertained by in situ enzyme histochemistry showing that treatment of mouse eye cryosections with increasing concentrations of PSB-12489 (0.1-1 μM), but not with equimolar concentrations of AMPCP, progressively reduced the intensity of AMP-specific staining in the photoreceptor layer (Fig. 6b).
The effect of CD73 inhibitor on the retinal function was assessed in vivo. Electrophysiological analysis of the retina was performed by recording fERG responses from darkadapted (scotopic) eyes stimulated with increments of light intensity from 0.003 to 10 cd s/m 2 . Figure 6c shows representative electroretinograms, which can be divided into the following components: the first a-wave that appears as a negative amplitude change, and the b-wave that appears as a large positive amplitude change immediately after the a-wave. The a-wave of the electroretinogram reflects the functional activity and integrity of the photoreceptors, whereas the b-wave originates in retinal cells that are postsynaptic to the photoreceptors, including inner retinal cells (bipolar and amacrine cells) and RGC [43,47,48]. Notably, C57BL/6N mice are known to be homozygous for the rd8 mutation in Crumbs homolog 1 (Crb1) gene, which may lead to severe retinal dysplasia in the inferior retina and other ocular abnormalities [49]. These lesions appear as white to yellow flecks on fundus examination, and the phenotype is worsened by exposure of C57BL/6N mice to BL [50]. Therefore, we first compared the effects of CD73 inhibitor on retinal electrical activity in different strains of mice. To achieve sufficient inhibitory effect, PSB-12489 was injected locally into the vitreous cavity at a relatively high dose, with a final concentration of ~ 200 µM in the eye. Measurement of fERG responses in C57BL/6JrJ mice showed no differences in a-wave and b-wave amplitudes between the groups that received CD73 inhibitor or PBS for 6 or 48 h. On the other hand, treatment of BALB/c mice with PSB-12489 for 48 h was accompanied by significant decreases in the b-wave amplitudes recorded at light intensities from 0.01 to 1 cd s/ m 2 (Fig. 6d). Based on these observations, BALB/c mice were chosen as an appropriate model for further investigation of the role of ADO metabolism in retinal function and BL-induced phototoxicity.
To study the role of CD73-generated ADO in the maintainance of retinal activity, BALB/c mice remained untreated or received a single intravitreal injection of PSB-12489 or vehicle (PBS), and subsequently exposed to continuous illumination for 14 h, as schematically illustrated in Fig. 7a. Electrical activity of retina was examined in live animals at baseline and 7 days after the treatment. Measurement of basal fERG values before treatment did not detect any differences in the a-wave and b-wave amplitudes in the study groups (Fig. S3a). However, when fERG was repeated on day 7 post-treatment, relatively moderate but significant decreases in the b-wave amplitudes were found in the PSB-12489-treated eyes (group G2), when compared to vehicle-treated (G3) and non-treated control (G1) groups (Fig. 7b). These differences became even more substantial after exposing the PSB-12489-treated animals to BL (group G5). These mice were characterized by ~ 40-50% decrease in the b-wave amplitudes at all stimulus levels of the light intensity tested and also showed a decrease in the a-wave amplitude recorded at high light intensities (1-10 cd s/m 2 ), when compared to vehicle-treated (G6) and non-treated (G4) groups exposed to BL (Fig. 7c). Notably, in contrast to the conventional experimental model of BL-induced retinal damage induced by extending the period of dark adaptation up to 24 h and characterized by markedly impaired scotopic responses (our unpublished observations), the combination of dark adaptation and light illumination parameters used in this work did not by itself cause any adverse effects on retinal electrical activity (Fig. S3b).
Impaired functional responses of retinal cells in PSB-12489-treated mice were not accompanied by decrease in total thickness of the retina or death of retinal ganglion cells
The thickness of the retina was determined in live animals immediately after fERG recording by using high-resolution spectral domain optical coherence tomography (OCT). It was measured in superior temporal area of the retina, which is the most sensitive to the retinal damage. No significant changes in total retinal thickness were observed between the groups studied (Fig. 7c). The expression levels and activity of CD73 in the treated retina were also interrogated at the Fig. 3 3D imaging identifies specific "purinergic junctions" in the mouse eye formed via direct interactions between microglial processes, retinal blood vessels and neuronal cell bodies. LMA-embedded sections of the mouse eye were co-stained with anti-CD39 antibody and molecular markers of blood endothelial cells (CD31), microglial cells (P2Y 12 R), and neuronal cell bodies (NeuN), as indicated. Z-stacks of the medial retina were captured using a spinning disk confocal microscope, and presented as reconstructed 3D images. Single channels are shown in grayscale and the right-hand panels display merged images with nuclei counterstained with DAPI. The insets display 3D images of representative areas at a higher magnification.
The bottom inset of a shows a close-up of retinal arteriole, cropped and rotated 90° to visualize the lumen of the vessel. (inset in b). 3D reconstructed datasets from a and b were also rendered as movie sequences and are presented in Supplementary data as Movie 1.mp4 and Movie 2.mp4, respectively ◂ histological level. Given the uneven distribution of CD73 in the mouse photoreceptor layer, AMP-specific staining intensities were determined in three different regions, including highly CD73-positive OS of photoreceptor cells, as well as ONL and OPL characterized by intermediate enzyme expression (Fig. S4a). Quantitative analysis did not detect any down-regulation of AMPase activity in the eyes receiving PSB-12489 (Fig. S4b). This conclusion was independently ascertained by immunofluorescence assays showing a similar pattern of CD73 staining in rhodopsin + photoreceptor cells in the control (Fig. S5a) and PSB-12489-treated (Fig. S5b) eyes. Notably, PSB-12489 acts as a reversible small-molecule inhibitor of CD73 and therefore, it can be washed out during preparation of eye cryosections and their subsequent incubation with exogenous AMP. Therefore, in situ enzyme histochemistry data on comparable AMPase activity in all groups do not rule out the possibility that PSB-12489 prevents intraocular adenosine production in live mice via temporal and reversible inhibition of CD73 throughout the whole period of treatment.
The numbers of nuclei in the retinal layers were quantified to provide a further assessment of cell survival in the treated eyes. Mice receiving PSB-12489 and exposed to BL did not show any significant changes in the total number of retinal neurons in the ONL, INL, or GCL (Fig. S6). To further assess potential harmful cytotoxic effects of the drug, we determined the number of apoptotic cells in the innermost retinal ganglion layer by using TUNEL Assay. Combined exposure of the mice to BL and PSB-1249 did not trigger any additional apoptosis in the treated retina, and even caused significant decrease in the number of TUNELpositive RGC (Fig. S7).
Discussion
By investigating the combined features of ocular purine homeostasis and electrical activity of the retina, we have identified a link between these different but apparently interrelated processes. The major findings are summarized as follows: (i) this work identified the presence of an extensive and spatially arranged network of ectoenzymes in the mouse and human eyes where they coordinately control ATP and ADO levels; (ii) the role of the CD73-generated ADO was ascertained in functional in vivo assays showing that temporary inhibition of ocular CD73 activity in dark-adapted mice prior to their transition from darkness to light caused a decrease in fERG responses. These findings are summarized in Fig. 8 which schematically illustrates cell-and tissue-specific distribution of ecto-nucleotidases in the mammalian eye (panel a), and further highlights the role of the ATP-ADO axis in retinal functioning (panel b).
To our knowledge, this is the first study providing a holistic view of ocular purine metabolism and signaling as a complex and spatially integrated network. By using two independent and complementary approaches, in situ enzyme histochemistry and multiplexed imaging, we were able to pinpoint both the catalytic activities and the expression levels of major purinergic ectoenzymes in the mouse neuroretina, optic nerve head and cornea. These imaging data, in combination with dissociation-based flow cytometric and scRNAseq analyses of mouse and human retinal cells, provide sufficient justification for re-evaluating the existing models of ocular purine metabolism and its role in retinal functioning. Similar to ubiquitous expression of CD39 in the systemic circulation where it controls hemostasis through termination of prothrombotic, proinflammatory and vasoactive effects of circulating ATP and ADP [26,51], CD39 was also shown to be highly expressed on blood vessels of various caliber located in the optic nerve head and retinal and choroid layers. On the other hand, contrary to the previous reports showing high CD73 expression on endothelial cells lining the lumen of large blood vessels, such as human and rodent aorta, carotid and coronary arteries [26,52], and also central retinal vessels of human optic nerve head [16], we did not detect any CD73 immunoreactivity, AMPase activity, or Nt5e/CD73 gene expression in the mouse retinal vasculature. This observation contrasts with the current view of ADO as a key regulator of ocular blood flow and vascular tone which elicits its vasoactive effects through binding to Fig. 4 In situ enzyme histochemistry and flow cytometric analysis of the expression of ecto-nucleotidases in the mouse eye. a Eyeballs were enucleated from C57BL/6N mice and major retinal layers and other ocular structures were visualized by hematoxylin and eosin (H&E) staining. b-e Ecto-nucleotidase activities were assayed in situ by incubating eye cryosections for 30 min with Pb(NO 3 ) 2 in the absence ("Blank", b) and presence of ATP (c), ADP (d), and AMP (e) followed by microscopic detection of the nucleotide-derived inorganic phosphate (P i ) as a brown precipitate. f The activity of tissuenonspecific alkaline phosphatase (TNAP) was measured by using the artificial chromogenic substrates BCIP and NBT and subsequent monitoring of the development of the blue color reaction. All images were captured by using Pannoramic 250 slide scanner. BV blood vessels, EM extraocular muscles, EnC endothelial cells, EpC epithelial cells, GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, N neurons, NFL nerve fiber layer, OLM outer limiting membrane, ONH optic nerve head, ONL outer nuclear layer, OPL outer plexiform layer, OS outer segments of photoreceptor cells, RPE retinal pigmented epithelium, VF vitreous fluid. Scale bars: 1 mm (left) and 80 μm ("inside the eyeball" and right-hand insets). g, h Flow cytometric analysis of CD39 and CD73 expression in the mouse retina. Single-cell suspension of freshly isolated retinal cells was incubated with anti-CD73 and anti-CD39 antibodies, together with fluorescently-labeled antibodies against CD45, CD11b, P2Y 12 R, and CD31, and also biotin-conjugated Isolectin B4 (IB4), as indicated. The right-hand panels show histograms of CD45 + /P2Y 12 R + /CD11b + microglial cells (g) and CD45 − /IB4 + /CD31 + vascular endothelial cells (h) stained with anti-CD39 and anti-CD73 antibodies, as well as isotype-matched control immunoglobulins (Neg Co). Data are representative of three independent experiments Left panels show uniform manifold approximation and projection (UMAP) of cell types in the retina. Known markers of retinal cells were used to identify each cell type (see also "Materials and methods"). Dot plots (right panels) show the expression of key enzymes of ADO metabolism, NTPDase1/CD39 (mouse and human gene names Entpd1 and ENTPD1, respectively), NTPDase2 (Entpd2 and ENTPD2), ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (Enpp1 and ENPP1), ecto-5′-nucleotidase/CD73 (Nt5e and NT5E), TNAP (Alpl and ALPL), adenosine deaminase (Ada and ADA), as well as ADO receptor subtypes A 1 R (Adora1 and ADORA1), A2AR (Adora2a), A 2B R (Adora2b and ADORA2B) and A 3 R (Adora3 and ADORA3) in mouse (a, b) and human (c) retinal cell subsets identified in UMAP plots. The relative expression levels of the indicated genes are shown on a pseudocolor scale (log2(FC)), with the size of the dot representing the percentage of cells in a subset where the gene is detected. Markers used to phenotype different subsets of mouse retinal Cx3cr1 YFP+ cells included P2ry12, P2ry13 (P2ry12 + microglia), Hmox1, Ifrd1, Il1a (Hmox1 + microglia), Mrc1, Cxcl2 (perivascular macrophages), Rorb, Rora (Rorb + macrophages). NTPDase2 (Entpd2) and A 2B R (Adora2b) were not found in the mouse retinal microglial cells (b), and A 2A R (ADORA2A) was not found in the human dataset (c) A 2A R/A 2B Rs expressed on retinal endothelial and perivascular cells ( [12], also Fig. 5). This apparent discrepancy can be explained by the existence of alternative pathways which ensure local ADO supply to the retinal vessels. These mechanisms might particularly include the direct release of endogenous ADO by vascular and neuronal cells via bidirectional equilibrative nucleoside transporters [29,53], ADO formation at the vitreoretinal interface through soluble intravitreal CD73 activity [16,23], as well as metabolism of AMP into ADO by neighboring CD73 + microglial cells located in close vicinity to the vessel wall (current study).
Microglia are the main resident macrophages in the central nervous system. They maintain brain homeostasis by monitoring and scavenging dying cells, engulfing synaptic material through a pruning process, and also responding to pathogenic stimuli by the release of IL-1β, TNF-α and other proinflammatory cytokines [31,54,55]. Recent studies have also demonstrated a key role for the ATP-ADO axis in microglia-driven inhibition of neuronal activity in Fig. 6 The effect of CD73 inhibitors on AMPase activity and electrical activity of the mouse retina. a Chemical structures of the standard CD73 inhibitor, AMPCP, and the new inhibitor PSB-12489 (12489). b The effect of CD73 inhibitors on retinal AMPase activity was determined in situ by incubating mouse eye cryosections with 1 mM AMP and 2 mM Pb(NO 3 ) 2 in the absence (control) and presence of the indicated concentrations of inhibitory compounds. Mean pixel intensities of AMP-specific brown staining were quantified in the selected regions of photoreceptor layer using QuPath v.0.2.0 software, and expressed as a percentage of control activity (mean ± SEM; n = 3). *P < 0.05 compared with control, determined by Student's t test (paired, two-tailed). c Electrical activity of retina was examined in live animals using fERG. The upper panel shows representative fERG waveforms recorded from scotopic retinas stimulated at light intensity increments from 0.003 to 10 cd s/m 2 . The amplitude of the a-wave was measured from the baseline to the lowest point of the wave, while the b-wave was measured from the trough of the a-wave to the highest point, as indicated in the lower inset. d PSB-12489 (12489) and PBS were injected into the vitreous cavity of C57BL/6JrJ (C57BL/6) and BALB/c mice. fERG responses were measured in the dark-adapted eyes 6 or 48 h after the treatment. The graphs show the amplitudes of the a-waves (upper panels) and b-waves (lower panels) versus luminance intensity (mean ± SEM; n = 4). *P < 0.05, determined by multiple t test grouped analysis Fig. 7 Inhibition of CD73 in dark-adapted mice before exposure to bright light impairs fERG responses, but has no effect on total retinal thickness. a Experimental design for analysis of the effects of CD73 inhibitor on the retinal function and structure. BALB/c mice were divided into six groups, which either remained untreated (G1 and G4) or received a single bilateral injection of PSB-12489 (12489; G2 and G5) and vehicle (PBS; G3 and G6). The animals were kept in transparent plastic cages without any treatment (G1-G3) or additionally exposed to continuous bright light (BL; G4-G6). Electrical responses of the retina and retinal thicknesses were examined in live mice both at baseline and at the end of the experiment. b fERG responses were measured in the dark-adapted eyes 7 days after the treatments. The graphs show the amplitudes of the a-waves (upper panels) and b-waves (lowe panels) versus luminance intensity (mean ± SEM; n = 6-10). *P < 0.05 and **P < 0.01, determined by multiple t test grouped analysis. c The thickness of outer nuclear layer was determined by high-resolution spectral domain optical coherence tomography. The right-hand image depicts retina fundus with superior (S), inferior (I), temporal (T), and nasal (N) parts of the retina. Twentyfive spots were selected for retinal thickness analysis, with the central point targeted at the centre of the optic nerve. Total retinal thickness was measured from inner plexiform layer to external limiting membrane, as shown in the lower cross-sectional image of retina. The left panel displays the outer nuclear layer thickness determined in the most superior temporal area of the retina (top left cell) of the treated mice (mean ± SEM; n = 6). The inset shows the cell-specific compartmentalization of ecto-nucleotidases in major retinal layers. CRV central retinal vessels, GCL ganglion cell layer, NFL nerve fiber layer, IPL inner plexiform layer, INL inner nuclear layer, ONL outer nuclear layer, OPL outer plexiform layer, OS outer segments of photoreceptor cells, RPE retinal pigmented epithelium. b Cellular purine turnover depends on interactions between extracellular ATP release, binding to nucleotide (P2YR and P2XR) and adenosine (AR) selective receptors, inactivation of nucleotides through ectoenzymes CD39 and CD73, cellu-lar uptake of nucleotide-derived ADO via equilibrative nucleoside transporters (ENT) and its phosphorylation into intracellular ATP through complex phosphotransfer reactions. Site of directional inhibition of this metabolic cascade by selective CD73 inhibitor PSB-12489 is pointed by T-shaped arrow pointer. Potential mechanisms underlying the effects of CD73 inhibitor on retinal function can fall into three main categories: (i) the impaired activation of ARs due to the insufficient generation of ADO; (ii) a simultaneous shift in purine homeostasis from the generation of anti-inflammatory and vasoactive ADO toward a proinflammatory and cytotoxic ATP-regenerating phenotype; and (iii) a role that is linked to the reduced cellular uptake of ADO with subsequent deregulation of cellular bioenergetics and related signaling pathways mouse and human brains [54,55], which mainly occurs via so-called "somatic microglia-neuron junctions" characterized by a highly specialized nanoarchitecture optimized for purinergic signaling [56]. While much of our knowledge concerning microglia-neuron interaction has been derived from brain research, these results are not directly transferrable to the retinal microglial cells which differ substantially in terms of morphological features and functional properties [57], and may also undergo dramatic transcriptomic alterations and differentiate into a plethora of subsets during retinal homeostasis and degeneration [39,42]. Here, we showed that retinal microglia express several key purinergic receptors (P2Y 12 R, P2Y 13 R, and A 3 R) (Fig. 5b) and in addition, create an intricate and spatially arranged network in the retinal parenchyma by extending and retracting their extremely branched and motile CD39 high /CD73 low processes and forming local "purinergic junctions" with CD39 low /CD73 − neuronal cell bodies and CD39 high /CD73 − blood vessels (Fig. 3, Movies 1 and 2). With this knowledge in mind, and knowing that extracellular ATP acts as a local chemoattractant that leads to the targeted recruitment of microglial protrusions to activated synapses [54,55], while ATP-derived ADO plays a counteracting role in protecting retinal neurons from hyperexcitation [3,58,59], it may be reasonably suggested that retinal microglial cells play a pivotal role in the regulation of functional hyperemia and neurovascular coupling in the eye via coordinated control of local ATP and ADO levels.
Along with CD39, another member of this family, NTP-Dase2, contributes to the metabolism of ATP in the eye. This study, when analyzed together with previous data on human, rodent and zebrafish eyes, provides evidence for selective localization of NTPDase2 in the optic nerve bundles [16], Müller glia ( [4,15,18]; Fig. 5a), as well as tubulin-βIII + neuronal filaments and corneal keratocytes (Fig. 2b). Due to the high preference of NTPDase2 for the hydrolysis of ATP over ADP [51,60], this ectoenzyme presumably has functionality in the rapid scavenging of ATP in a neuronal environment, while the subsequent degradation of ATPderived ADP will occur with a considerable delay. We have also identified the presence of yet another enzyme, TNAP, in the mouse RGC, blood vessels and corneal epithelium (Figs. 4f and 5), as well as in the human sensory neuroretina, optic nerve head and vitreous fluid [16]. Although TNAP does not appear to contribute significantly to the metabolism of ocular adenine nucleotides, due to its surprisingly broad substrate specificity, this ectoenzyme can regulate blood clotting, bone mineralization, cartilage formation, and other cellular functions by degrading other phosphate-containing compounds, such as pyrophosphate (PP i ) and inorganic polyphosphates [26,30,60]. Since TNAP has been identified among the top calcification-related genes overexpressed in the human trabecular meshwork [61], and as it is also expressed in pathological neofibrovascular tissues surgically excised from eyes with diabetic retinopathy [16], it would be interesting to evaluate the distribution of this enzyme in the eyes with pathological neovascularization and ectopic mineralization.
This work also points to the need of more careful evaluation of similarities and differences in purinergic signatures across species, which should be taken into account during studying ADO homeostasis in rodent eyes and further translating these experimental data to humans. Similar patterns of high expression of CD73 both in the human [16,21] and rodent [22,62] (current study) photoreceptors suggest an equally important role for this ecto-nucleotidase in governing adenosinergic signaling along the sensory retina and hence, in the development of electrical excitation in all mammalian eyes. A salient finding of this work is that pharmacological inhibition of CD73 has a fairly moderate effect on the fERG responses in dark-adapted eyes, but rendered animals became hypersensitive to continuous exposure to BL at levels that by themselves would not normally cause any adverse shifts in visual cycle or retinal structure. Taking into account data on a crucial role of ADO in regulating photoreceptor coupling [11], light and sleep signaling [10], and functional hyperemia [12] in dark-adapted eyes, it is tempting to speculate that CD73-generated ADO confers endogenous protection against light-induced phototoxicity to the retina. According to this scenario, moderate but significant decrease in b-wave waveforms in PSB-12489-treated eyes could reflect the reduced activity of photoreceptor CD73 and/or intravitreal soluble CD73 and as a consequence, insufficient activation of ARs localized on the INL and RGC facing the vitreous lumen. This impact was amplified by continuous illumination of the treated eyes, as ascertained by marked reduction of both a-wave and b-wave amplitudes (Fig. 7b). Further studies would be required to elucidate the exact mechanism(s) underlying the effects of PSB-12489 on retinal function. Taking into account the complexity of purine homeostasis in the mammalian eye (Fig. 8a), and the important role of adenosinergic signaling in retinal functioning during transition from darkness to light [11,12], it is reasonable to conclude that inhibition of the CD73-ADO axis in dark-adapted mice could shift the balance between ocular ATP and ADO levels. Potential consequences of blocking this metabolic chain are highlighted in Fig. 8b and may particularly include the impaired hyperemic and neuroinflammatory responses mediated via activation of ARs, as well as the simultaneous accumulation of proinflammatory and cytotoxic ATP in the retinal environment.
While most of the effects of extracellular ATP and ADO are thought to be mediated via canonical signal transduction pathways, the alternative receptor-independent mechanism that is linked to the cellular uptake of ADO and its phosphorylation into ATP may play an equally important yet understudied role. Previous data demonstrated potentiation of retinal hyperemia, post-ischemic recovery, and both spontaneous and light-evoked activities of retinal neuronal cells after prevention of endogenous ADO transport and metabolism in the presence of the inhibitors of nucleoside transporters (NBTI and dipyridamole) or adenosine deaminase (EHNA) [58,63]. Increasing the metabolic clearance of intracellular ADO in the eyes of transgenic mice overexpressing human adenosine kinase also affected circadian rhythm, manifested in the reduced slow-wave activity after sleep deprivation [10]. This intracellular purine salvage pathway might be especially relevant for RGC, because of their high ATP turnover rate and great energy demands [64]. In fact, these cells are capable of accumulating intravitreally injected [ 3 H]ADO in their cellular body [43], and are particularly sensitive to the light-induced effects of ADO [44]. However, combined exposure of the mice to PSB-12489 and BL did not trigger additional apoptosis in the innermost retinal layer (Fig. S7). These observations, together with data on rather minor contribution of RGC to the mouse electroretinogram [65], allow concluding that the revealed exacerbated effects of PSB-12489 on retinal cell function were not associated with RGC death or other adverse shifts in cellular energetics in this high oxygen consumption layer.
In conclusion, these data point out the need for a more careful evaluation of the entire purinome in the mammalian eye by taking into account the complexity and redundancy of metabolic and signaling pathways involved in biological effects of ATP and ADO. Our 3D imaging workflow also provides novel insights into spatial relationships and heterotypic interactions between different cell types in the retinal environment and on this basis, suggests the important and hitherto unrecognized role of retinal microglia in the purinergic control of retinal blood flow and neuronal activity. Furthermore, data on impaired fERG responses in the mouse eyes treated with CD73 inhibitor provide evidence for a crucial role of the CD73-ADO axis in the maintenance of retinal integrity and function in "steady-state" and especially under challenging conditions induced by prolonged light illumination. As a consequence, a new enzyme-based strategy could be used to restore ADO levels and photoreceptor function in the injured retina. There is also a paucity of knowledge regarding the relationship between nucleotideinactivating/ADO-producing and counteracting ATP-regenerating ectoenzymes, as well as intracellular purine salvage pathways in the eye. Improving our knowledge in this field may be useful for understanding the role of purine homeostasis in ocular (dys)functions and on this basis, developing effective new strategies for the treatment of retinal degeneration and other vitreoretinal diseases.
Author contributions KL collected the samples, performed imaging experiments, and analyzed and interpreted the results. AT conducted flow cytometric experiments, analyzed scRNAseq dataset, and contributed to writing the manuscript. CCS, GR, and CEM provided CD73 inhibitor PSB-12489 and contributed to data analysis and interpretation. MC-G, MV, SR, SK, and GK designed and performed in vivo mouse experiments, and measured retinal thickness and fERG responses in the treated eyes. MLP helped with several experiments and contributed to writing the manuscript. JS contributed to image analysis, data acquisition and manuscript preparation. SJ contributed to project administration, funding acquisition, and manuscript preparation. GGY designed experiments, supervised the research, and prepared the manuscript. All authors reviewed and approved the manuscript.
Funding Open Access funding provided by University of Turku (UTU) including Turku University Central Hospital. K. Losenkova was supported by Orion Research Foundation sr, and Ida Montinin Säätiö. A. Takeda and S. Jalkanen were supported by Academy of Finland. Open access pubishing was provided by the University of Turku, Finland.
Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request.
Conflict of interest
The authors disclose no conflicts of interests.
Consent for publication All authors have read and approved the final manuscript
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. | v3-fos-license |
2018-10-03T11:14:13.696Z | 2008-09-05T00:00:00.000 | 53076894 | {
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} | pes2o/s2orc | Catalytic interaction of 1,3-diheteracycloalkanes with diazo compounds
The results are presented of research on the catalytic interactions of 1,3-diheteracycloalkanes with diazocompounds (N 2 CH 2 , N 2 CHCO 2 Me), the influence of the nature of the catalyst and structure of the starting heterocycles on the yield and structure of products formed.
In the reactions of 3-ethyl-2-phenyl-and 2,3-diphenyl-oxazolidines with methoxycarbonylcarbene, which is generated by thermocatalytic decomposition of methyl diazoacetate in the presence of copper bronze, 16 insertion occurred predominantly into the C-N bond of the oxazolidine ring to give morpholine-3-carboxylic acid esters.It was also noted 16 that in the presence of Rh 2 (OAc) 4 neither insertion products of carbene into the C-N bond nor into the C-O bond are formed.
ARKAT USA, Inc.The morpholine derivatives 4a,b are generated apparently through the attack of methoxycarbonylcarbene on the oxygen atom of oxazolidines 3a,b to form O-ylides, which undergo the Stevens rearrangement leading to ring enlargement. 20It should be noted that thermocatalytic decomposition of N 2 CHCO 2 Me (120 °С, copper bronze) 16 with oxazolidine 1a affords a complex mixture of compounds, in which the percentage of morpholine 4a is lower than 13%.
The stereochemical compositions of compounds 4a,b were determined by analyzing the chemical shifts and spin-spin coupling constants in the 1 H NMR spectra.The 1 H NMR spectrum of compound 4a shows doublets at δ 3.36 and 3.95 ( 3 J 2,3 = 8.9 Hz) corresponding to the methine protons at the C(3) and C(2) atoms, respectively, of the morpholine ring.The spin-spin coupling constant is indicative of the trans arrangement of the substituents at the adjacent carbon atoms.The COLOC 2D NMR spectrum of ester 4a shows a cross-peak between the signal for the carbonyl carbon atom (δ 169.6) and a low-field signal for the methine proton at the C(2) atom (δ 3.95), which confirms that methoxycarbonylcarbene is inserted into the C(2)-O bond of oxazolidine 3a.
Scheme 3
To reveal the relationships between the structure of 1,3-diheteracyclopentanes and the rate of insertion of methoxycarbonylcarbene into the carbon-heteroatom bond, the reactions of compounds 1b, 3a, 3c with N 2 CHCO 2 Me in the presence of Rh 2 (OAc) 4 were studied by the competitive reaction method (Scheme 4).The relative reactivity was determined at 40 °С by adding a solution of N 2 CHCO 2 Me in dichloromethane to a mixture containing dioxolane 1b and its heteroanalog 3a or 3c in a molar ratio 1b: 3a (or 3c): N 2 CHCO 2 Me: Rh 2 (OAc) 4 = 250: 250: 100: 1.
Scheme 4
As expected, 2-phenyl-1,3-oxathiolane 3c showed the highest reactivity (k rel (3c/1b) = 9.8), whereas oxazolidine 1а appeared to be only just slightly more reactive than 1,3-dioxolane 1b (k rel 3a/1b) = 1.7) although it is characterized by the insertion of the carbene fragment into the C-O bond rather than into the C-N bond.This fact is apparently attributed to the additional replacement at the nitrogen atom, which hinders the intermediate formation of N-ylide.
Scheme 5
The possible mechanism of the reaction can include the generation of ylide followed by 1,2anionic rearrangement (the Stevens rearrangement). 1,20Apparently, the O(1) atom is involved in the formation of ylide; this is confirmed by the selective formation of products of formal insertion of methoxycarbonylcarbene into O(1)-C(2) bond.Successful reaction of methyl diazoacetate with benzaldehyde derivatives correlates well with the mechanism of 1,2-anionic rearrangement. 20According to this mechanism, the migrating group in its transition state is a free radical stabilized by conjugation in its substituents, and thus the process occurs more easily.
Catalytic interaction of N 2 CH 2 and N 2 CHCO 2 Me with unsaturated 1,3-diheteracycloalkanes.It has been demonstrated 24 that the introduction of the oxazolidine or boronate group into unsaturated compounds leads to an increase in both the yields of cyclopropanation products compared to those obtained in reactions with unfunctionalized molecules and the regioselectivity of cyclopropanation of dienes with N 2 CH 2 in the presence of Pd 2 (OAc) 2 .The influence of the characteristics of the acetal substituents in olefins on catalytic reactions of the latter with N 2 CH 2 has not been previously examined.In the present study, we examined the influence of the nature of the acetal group and the catalyst on the catalytic cyclopropanation with diazomethane of a series of unsaturated compounds, derived from trans-crotonaldehyde (8a,d), trans-cinnamaldehyde (8b,e) and hex-5-en-2-one (8c,f) (Scheme 6).
Cyclopropanation was carried out at 5-10 °C by adding a solution of N 2 CH 2 in Et 2 O or CH 2 Cl 2 to an unsaturated compound in the presence of a catalyst, in the molar ratio of 50: 150: 1 of olefin: N 2 CH 2 : catalyst, for 30 min.Investigation of cyclopropanation of dioxolane 8a with the use of Pd(OAc) 2 , PdCl 2 , Pd(acac) 2 , CuCl, [CuOTf] 2 •C 6 H 6 , Cu(acac) 2 , and Cu(OTf) 2 , as the catalysts demonstrated that Pd(acac) 2 and Cu(OTf) 2 are the most efficient palladium and copper catalysts, respectively, under the reaction conditions used.Cyclopropanation catalyzed by Pd(acac) 2 or Cu(OTf) 2 afforded products in 99 and 49% yields, respectively.Hence, all further reactions were carried out with the use of these two catalysts.The resulting cyclopropanes were isolated by preparative TLC and characterized by 1 H-and 13 C-NMR spectroscopy.
Compound 8a reacts with N 2 CH 2 in the presence of Pd(acac) 2 or Cu(OTf) 2 to give a complex mixture of products.By contrast, cyclic acetal 8d containing two electron-withdrawing butoxycarbonyl groups at positions 4 and 5 of the dioxolane fragment is readily subjected to cycloprotonation in the presence of Pd(acac) 2 to form dibutyl 2-(trans-2-methylcyclopropyl)-1,3dioxolane-trans-4,5-dicarboxylate (8d).Unlike simple crotonaldehyde derivatives 8a, cinnamaldehyde derivatives react with N 2 CH 2 in the presence of Pd(acac) 2 to give the corresponding cyclopropane derivatives 9b,e in high yields.Cyclopropanation of hexenone derivatives 8c,f occurs with a somewhat higher efficiency compared to hexenone and produces cyclopropanes 9c,f in 87-99% yields.
The Cu(OTf) 2 catalyst is less efficient than Pd(acac) 2 in cyclopropanation of cinnamaldehyde derivatives 8b,e or hexenone derivatives 8c,f, and these reactions give the corresponding cyclopropanes in low yields.In the reaction of unsaturated compound 8b, Cu(OTf) 2 catalyzes the acetal deprotection giving rise to the starting cinnamaldehyde, the reaction being typical only of cinnamaldehyde derivatives.
The higher efficiency of Pd compounds in the cyclopropanation of unsaturated acetals is apparently associated with intramolecular stabilization of π -olefin complexes by oxygen atoms.13b The study of catalytic cyclopropanation of 1,2-disubstituted double bonds in unsaturated carbonyl compounds and their acetal (ketal) derivatives with diazomethane provided evidence for higher selectivity of cyclopropanation of the latter compared to the starting unsaturated carbonyl compounds and for the activating effect of the acetal fragments on the reactivity of the C=C bond compared to the cyclopropanation of usual 1,2-disubstituted alkenes.13b The interaction of equimolar quantities of methyl diazoacetate with cyclic acetals 10a,b and 1,3-oxathiolanes 10c,d in the presence Rh 2 (OAc) 4 proceeds selectively and results in products of C-X insertion 11a-c and [2,3]-sigmatropic rearrangement 12a-d (Scheme 7).The absence of cycloaddition products of methoxycarbonylcarbene to the C=C bond in the reaction mixture and arrangement of substituents in the isolated products testifies that reaction proceeds through formation of one ylide.The formation of ylides takes place by the electrophilic addition of carbenoid species generated from methyl diazoacetate to the heteroatom under the action of the catalyst.The selectivity of formation of products of Stevens rearrangement 11a-c and [2,3]-ARKAT USA, Inc. sigmatropic rearrangement 12a-d is defined by the influence of both electronic and steric factors of the substituents. 25,26
Experimental Section
General Procedures.The 1 H-and 13 C-NMR spectra were recorded on a Bruker AM-300 spectrometer (300.13 and 75.47 MHz, respectively) in CDCl 3 with SiMe 4 as the internal standard.The IR spectra were measured on a Specord M82-63 instrument in a thin layer.The mass spectra were obtained on an MX-1320 instrument; the ionizing electron energy was 70 eV; the temperature of the ionization chamber was 50-70 °С.The GLC analysis was carried out on a Chrom-5 chromatograph equipped with a flame ionization detector (with a 1200×5 mm column with 5% SE30 on Inerton N-AW DMCS (0.125-0.160 mm) using helium as the carrier gas.The TLC analysis was performed on Silufol chromatographic plates (Merck).Preparative separation was performed by column chromatography on silica gel Chemapol (60 L, 100/160 µm).Starting 1,3-diheteracycloalkanes were synthesized according to known procedures, 19,27 distilled under a stream of argon, and stored under an inert atmosphere over metallic sodium.The solvents (CH 2 Cl 2 , diethyl ether, benzene, hexane, and petroleum b.p. 40-70 °С) were purified according to standard procedures.
Reactions of 1,3-dioxolanes 1a-f with methyl diazoacetate in the presence BF 3 .OEt 2 (general procedure).Methyl diazoacetate 2.5 g (25 mmol) was added with vigorous stirring at 20 °С over 1 h to a solution of l,3-dioxolane (50 mmol) and BF 3 .OEt 2 0.07 g (0.5 mmol).The reaction mixture was additionally stirred for 1 h.The residue was dissolved in 10 mL of diethyl ether and passed through a thin layer of Al 2 O 3 .All products 2a-f were purified by vacuum distillation. | v3-fos-license |
2017-10-27T11:29:15.590Z | 2017-05-04T00:00:00.000 | 13767762 | {
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} | pes2o/s2orc | Isradipine attenuates MPTP-induced dopamine neuron degeneration by inhibiting up-regulation of L-type calcium channels and iron accumulation in the substantia nigra of mice
The aim of this study is to investigate the effects of L-type calcium channels (LTCCs) on MPTP-induced dopamine (DA) neuron degeneration and iron accumulation in the substantia nigra (SN) of mice. By real-time PCR and western blots, we first quatified expressions of L-type Cav1.2 and Cav1.3 calcium channel α1 subunits in the SN of experimental mice treated with MPTP. We found that the expressions of Cav1.2 and Cav1.3 calcium channel α1 subunits markedly increased after MPTP treatment for 2 and 3 weeks. Secondly, we observed the effects of isradipine, a LTCC antagonist, on MPTP-induced DA neuron degeneration and iron accumulation in the SN. Our results showed that isradipine treatment prevented against MPTP-induced Cav1.2 and Cav1.3 calcium channel α1 subunits up-regulation in the SN. We also found that isradipine prevented against MPTP-induced DA neuron depletion in the SN and partly restored the DA content in the striatum. Moreover, we found that isradipine inhibited the increase of iron positive cells in the SN of the MPTP-treated mice. Finally, we investigated the effects of isradipine on cellular iron accumulation in the dopaminergic MES23.5 cell line. Our studies showed that MPP+ treatment accelerated iron influx in the MES23.5 cells. Treatment with Bayk8644 further aggravated iron accumulation. Treatment with isradipine prevented against MPP+-induced iron influx in the MES23.5 cells. These results suggest that up-regulation of LTCCs may be responsible for the DA neuron degeneration in the MPTP-treated mice, The LTCCs may directly contribute to iron influx into DA neurons, and isradipine may suppress cellular iron accumulation and prevents neurodegeneration.
INTRODUCTION
Parkinson's disease (PD) is one of the slowly progressing neurodegenerative disorders characterized by resting tremors, bradykinesia, and rigidity. The pathogenesis of PD is selective loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) and exhaustion of dopamine in the striatum [1]. Many factors have been implicated in the pathological process of DA neuron degeneration, which include but is not limited to oxidative stress, neuroinflammation, apoptosis, increased iron leave, excito-toxicity, and decreased proteasome function [1][2][3][4].
Increasing evidence has indicated that iron plays a key role in the pathogenesis of DA neuron degeneration. Substantially increased content of iron in the PD brains was observed and demonstrated in previous studies. This iron increase only occured in the SN but not in the ventral tagmental area (VTA) [5][6][7][8][9][10]. The exceedingly increased iron may lead to neuronal death by reacting with oxygen and produces highly reactive hydroxyl radicals. Increased iron content may damage lipids, carbohydrates, proteins and nucleic acids, thereby inducing DA neuron degeneration in the SN [11]. However, the underlying mechanisms of iron selective accumulation in the SN remain unclear.
Research Paper
In addition to the iron selective accumulation in the SN, growing evidence suggests that the L-type calcium channels (LTCCs) may play an important role in the selective DA neuron degeneration in the SN [12][13][14]. The potential linkage of LTCCs to PD is strengthened by epidemiological studies, which showed a decreased risk for PD in humans who were treated for hypertension with dihydropyridines (DHPs) [4,15,16]. L-type Cav1.2 and Cav1.3 calcium channels, especially the Cav1.2 calcium channels are abundant in neurons [16]. Nevertheless, the LTCCs are responsible for mitochondrial oxidant stress; and increased vulnerability in the SN DA neurons is largely attributable to the expression of Cav1.3 calcium channels [17,18]. DA neurons in the SN rely on sodium channels for pacemaking at their juvenile, but rely on L-type Cav1.3 calcium channels to drive autonomous pacemaking at the progress of adulthood; whereas, DA neurons in the VTA rely on sodium channels for pacemaking [19,20]. Previous studies showed that Ca 2+ enter DA neurons via the continuous opening of Cav1.3 calcium channels during pacemaking. This could be a reason why SN DA neurons exhibit vulnerability when stressed [3,21]. Further support for an involvement of LTCCs in the pathogenesis of PD, comes from the finding that Cav1 subtype was elevated in the brain regions affected in PD, resulting in a generally higher level of Cav1.3 subtype expression relative to that of the Cav1.2 subtype [22]. The increased expression and therefore, presumably increased cellular use of Cav1 subtypes could lead to increased metabolic stress on neurons. Yet, to date, the dynamic variations of Cav1 subtypes expression in the pathological process of PD are still unknown.
In the last few years, LTCC was reported to provide a major pathway for iron entry into cardiomyocytes [23,24]. LTCC may also provide an alternative route for iron import to neuronal cells [25]. Previous study in my laboratory demonstrated that LTCC blocker nifedipine attenuated iron deposit in the SN during iron-overload condition [26]. We proposed that LTCC might partly mediate iron selective accumulation in the SN. In the present study, we first quantified the expressions of L-type Cav1.2 and Cav1.3 calcium channel α1 subunits in the SN of experimental mice treated with MPTP. Secondly, we observed the effects of isradipine, a LTCC blocker, on the MPTP-induced neurotoxicity and iron accumulation in the mice.
Isradipine prevented against MPTP-induced motor coordination ability impairment assessed by rotarod test
As shown in Figure 1, after 1-4 weeks of MPTP treatment, the time on the rod of the MPTP treated mice reduced significantly compared with the control mice. The group, which was treated with isradipine followed by MPTP, partly resist this reduction in the 3-and 4-week treated subgroups. Time on the rod of the isradipine treated mice from the 1-and 2-week treatment subgroups also showed an increasing trend, however, no significant changes were observed (*P < 0.05, ***P < 0.001 compared with control; # P < 0.05 compared with MPTP treatment group, n = 10).
Expressions of Cav1.2 and Cav1.3 α1 subunits altered with the progression of PD After 2 and 3 weeks of MPTP treatment, the mRNA expressions of Cav1.2 and Cav1.3 α1 subunits in the SN markedly increased compared with the controls. However, we cannot observe significant changes after MPTP treatment for 1 or 4 weeks compared with the controls. As for the Cav1.2 α1 subunit, the peak of its mRNA expression appeared after MPTP treated for 2 weeks (Figure 2A). While the peak of Cav1.3 α1 subunit mRNA expression appeared after MPTP treatment for 3 weeks ( Figure 2B). Treatment with isradipine conferred significant protection against MPTP-induced up-regulation (**P < 0.01, ***P < 0.001, compared with control; ## P < 0.01, ### P < 0.001, compared with MPTP treatment group; n = 5).
We also investigated the expressions of Cav1.2 and Cav1.3 α1 subunits by western blots. As shown in Figure 3, the same tendency of protein expressions with their mRNA in the SN were observed. No significant changes at 1 and 4 weeks, while a significant up-regulation was observed at 2 and 3 weeks in the MPTP treated group compared with the controls. These results could be partly restored by isradipine (*P < 0.05, **P < 0.01, ***P < 0.001, compared with control; # P < 0.05, ### P < 0.001, compared with MPTP treatment group; n = 5).
Isradipine protected against MPTP-induced decrease of TH positive neurons in the SN of mice
The numbers of TH positive neurons were evaluated by immunohistochemistry. Figure 4A showed the fluorescence pictures of the whole SN. The summarized data of TH positive neurons were shown in Figure 4B. Compared with the controls, the TH positive neurons exhibited a progressive loss in the SN in the MPTP treated group. This effect was partly restored in the isradipine treatment group (*P < 0.05, ***P < 0.001, compared with control; # P < 0.05, ### P < 0.001, compared with MPTP treatment group; n = 5).
Isradipine protected against MPTP-induced decrease of DA content in the striatum of mice
As shown in Figure 5, the mean contents of DA and its metabolites DOPAC and HVA in the striatum were significantly decreased in the MPTP treatment group compared with the controls. Co-treatment with isradipine partly restored the DA content after MPTP treated for 1 and 2 weeks. Although we found a tendency of increase in the DA content after isradipine treated for 3 and 4 weeks, no significance was observed. The DOPAC and HVA contents were also partly restored by isradipine, however, no significance was observed (*P < 0.05, **P < 0.01, ***P < 0.001, compared with control; # P < 0.05, compared with MPTP treatment group; n = 5).
Isradipine inhibited MPTP-induced increase of iron-staining cells in the SN of mice
The iron staining cells in the SN were detected by Perls' iron staining. As shown in Figure 6, No changes in the numbers of iron staining cells were observed in the 4 subgroups of control group. However, a marked and time-dependent increase in the numbers of ironstaining cells were detected after MPTP treated for 2 to 4 weeks. A significant decrease of the numbers in ironstaining cells were observed in isradipine co-treatment group compared with that of MPTP-treatment group (*P < 0.05, ***P < 0.001, compared with control; # P < 0.05, ### P < 0.001, compared with MPTP treatment group; n = 5).
Isradipine inhibited MPTP-induced iron influx in the MES23.5 cells
The intracellular iron contents in MES23.5 cells were measured by fluorescence dye calcein. Fluorescence quenching indicates that extracellular iron was transported into cells. As shown in Figure 7, there was a timedependent intracellular fluorescence quenching with 1 mmol/L ferrous iron perfusion (control), indicating the increased intracellular iron level. When cells were treated with MPP + for 24 h, a rapid fluorescence quenching were detected (MPP + treatment group). The fluorescence quenching further accelerated when cells were perfused with 0.01 mmol/L Bayk8644 compared with MPP + treatment group, indicating an further iron influx in these cells. Fluorescence quenching in the MPP + treatment group was blocked by 0.02 mmol/L isradipine perfusion (*P < 0.05, **P < 0.01, compared with control; # P < 0.05, compared with MPP + treatment; ^P < 0.05, ^^P < 0.01, ^^^P < 0.001, compared with MPP + treatment; n = 6).
DISCUSSION
In the present study, we found out that isradipine, the LTCC antagonist, was able to improve motor coordinate deficit, to protect against DA neuron degeneration in the SN, and to resist against the decrease of DA content in the striatum induced by MPTP in the mice. These findings confirm the critical role of LTCCs played in the pathogenesis of DA neuron degeneration. Our results for the first time clearly showed the up-regulation of Cav1.2 and Cav1.3 α1 subunits in the SN of MPTP-induced PD mice. It has been shown that L-type Cav1.2 calcium channels were widely expressed in mouse brain (about 76.5% of Cav1.2 in total L-type α1 subunit mRNA) [27], meanwhile, a relatively lower expression of Cav1.3 calcium channels (23%) and an even lower expression of Cav1.1 and Cav1.4 calcium channels (0.5% together) were observed [27]. The DA neurons in the SN are inhabited by both Cav1.2 and Cav1.3 calcium channels α1 subunit isoforms. Recently, the Cav1.3 calcium channel's functions were identified, including pacemaking and making SN MPTP for 1-4 weeks, the time on the rod of the mice significantly reduced compared with the controls. This effect was partly restored by co-treatment with isradipine (*P < 0.05, ***P < 0.001, compared with control; # P < 0.05, compared with MPTP treatment group; n = 10). www.impactjournals.com/oncotarget DA neurons more vulnerable to toxins [21]. Research suggested that Ca1.3 calcium channels were the cause of Ca 2+ overload in SN DA neurons [19]. There is also evidence shows that, disregulated calcium homeostasis is one of the major factors that implicated the pathogenesis of PD [4,7,22,28]. In our acquired results, Cav1.2 and Cav1.3 calcium channels increased with the progression of PD. Both type of channels are up-regulated in the SN. Therefore, with the increase of these two channels, Ca 2+ concentration also increases in the cytoplasm, this proportional alteration results in a series of reactions accompanied by the Ca 2+ overload. It was worth noting that the up-regulation of both channels decreased after 4-week MPTP treatment, and the expressions of both channels even returned to the control level. This observation might be attributed to the loss of many DA neurons.
More importantly, our study demonstrated the iron continuous accumulation in SN was accompanied by the progression of PD, yet isradipine reversed this phenomenon effectively in MPTP-treated mice. In MPP + -induced PD cell model, we also observed MPP + treatment was able to accelerate iron transport in MES23.5 cells. Activation No significant changes at the 1 and 4 weeks, while a significant up-regulation at the 2 and 3 weeks were observed in the MPTP treatment group compared with control. These results could be partly restored by isradipine (*P < 0.05, **P < 0.01, ***P < 0.001, compared with control; # P < 0.05, ### P < 0.001, compared with MPTP treatment group; n = 5). α1 subunits in the SN markedly increased compared with the controls after treated with MPTP for 2 and 3 weeks. The peak of Cav1.2 α1 subunit mRNA appeared after MPTP treated for 2 weeks, while the peak of Cav1.3 α1 subunit mRNA appeared after MPTP treated for 3 weeks. This effect was partly restored by co-treatment with isradipine (**P < 0.01, ***P < 0.001, compared with control; ## P < 0.01, ### P < 0.001, compared with MPTP treatment group; n = 5). www.impactjournals.com/oncotarget of LTCCs by Bayk8644 further accelerated this process. The isradipine that successfully inhibited iron influx in the cells, may indicate that iron might be transported across cell membrane via the LTCCs. All these results directed the fact that LTCCs participate in iron accumulation in the SN. It is known that iron plays a key role in the development of PD [29]. Prevention of iron in the SN provides an effective method to slow down or terminate the progression of PD [25]. However, the cause of the selectivity of the iron deposit in the SN remain unknown. LTCCs were reported to serve as a major pathway of iron entry into cardiomyocytes [22,23]. Iron may enter into neurons via LTCCs as well [30]. These findings suggest that LTCCs may provide an alternative route for iron import into DA and its metaboites contents in the MPTP treatment group decreased compared with that of control. The dopamine content was partly restored by isradipine compared with that of MPTP treatment group. The DOPAC and HVA contents were also partly restored by isradipine, however, no significance were observed (*P < 0.05, **P < 0.01, ***P < 0.001, compared with control; # P < 0.05, compared with MPTP treatment group; n = 5). www.impactjournals.com/oncotarget neuronal cells. Compared with previous studies, our study first demonstrated the persistent accumulation of iron in the SN progresses with PD progression, rather than suddenly appears at the final stage of PD. Isradipine apparently inhibits the increase of iron level in the SN and inhibits iron influx into cells. These results indicate that LTCCs at least partly contribute to the iron accumulation in the SN. However, we still do not know, whether Cav1.2 or Cav1.3 calcium channels mediated iron deposit. To date, we cannot find a DHPs which selectively blocks Cav1.2 or Cav1.3 calcium channels. Most of the DHPs, including nimodipine and nifedipine, are more effective blockers for
Figure 6: Isradipine inhibited the increase in the numbers of iron staining cells in the SN induced by MPTP. Original
figures showed the iron staining cells in different treatment groups (A) Summarized data showed the numbers of iron staining cells in different treatment groups (B) (*P,<0.05, ***P < 0.001, compared with control; # P < 0.05, ### P < 0.001, compared with MPTP treatment group; n = 5). www.impactjournals.com/oncotarget Cav1.2 than for Cav1.3 calcium channels. However, low potency DHPs such as nimodipine could fail to antagonize Cav1.3 against MPTP on dopaminergic terminals, while Cav1.3 is still involved in terminal degeneration [15,31]. Isradipine, one of the DHPs, from earlier studies on these drugs, has a roughly 40 fold higher affinity for Cav1.3 calcium channel than other DHPs, and has neuroprotective effect on both MPTP and 6-OHDA-induced PD models [11,32]. We cannot rule out the fact that Cav1.3 calcium channels mediate iron deposit, which could be responsible for PD pathogenesis.
In conclusion, our study indicates that the L-type calcium channels are associated with the development and progression of DA neuron degeneration via accelerating calcium and iron accumulation. Isradipine, a calcium channel blocker, may slow down DA neuron degeneration by the blockage of Cav1.2 and/or Cav1.3 calcium channels.
Materials and animal preparation
All of the reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise indicated. All procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Ethics Committee of Qingdao University. For the in vivo experiments, 120 male C57BL/6 mice (8-10 weeks old) weighting 22-25g were included in this study. The mice were obtained from Beijing Vitalriver Laboratory Animal Technology Co. Ltd. (Beijing, China). The mice were housed for 1 week with a 12-h light-dark cycled and free access to food and water before experiments. The room temperature was adjusted to 19 ± 2°C, humidity was 60 ± 5%.
MPTP and isradipine were dissolved respectively in 0.9% saline and 2% dimethysulfoxide (DMSO). These solutions were formulated freshly before used. The mice were randomly divided into 3 groups: control group, MPTP treatment group, and isradipine with MPTP co-treatment group. Each group was divided into 4 subgroups (1, 2, 3 and 4 weeks treatment subgroups). Each subgroup has 10 mice. (1) MPTP treatment group: mice were received intraperitoneal injection of MPTP (30 mg/kg) twice per week. (2) Isradipine and MPTP co-treatment group: mice undergoing same procedures mentioned above plus isradipine with a dosage of 3 mg/kg was subcutaneous injected to mice once per day [15]. (3) Control group: the same procedures were followed except MPTP and isradipine were both replaced by 0.9% saline. In each subgroup, the mice were received rotarod behavior test after treated for 1, 2, 3 or 4 weeks respectively and then were decapitated on the next day. The brains were isolated for different assays. In each subgroup, 5 of the brains were used for TH immunofluorescent and iron staining, the others were used for real time PCR, western blots, and HPLC detection.
Rotarod behavioral test
The rotarod behavior test was used to evaluate the ability of balance and motor coordination of mice. The diameter of the rod is 5 cm. First, the mice were placed on this rod to adapted for 2 min, then the rotarod uniform accelerated from 4-40 rpm in 5 min. During this period, Fluorescence quenching in the MPP + treatment group was blocked by isradipine (*P < 0.05,**P < 0.01, compared with control; # P < 0.05, compared with MPP + treatment; ^P < 0.05, ^^P < 0.01, ^^^P < 0.001, compared with MPP + treatment; n = 6). if any mice drop down, the system would automatically stop and record the time that mice stayed on the rotation bar. This experiment was repeated for 3 times. The interval between each two tests was not less than 1 h.
RNA isolation and analysis of Cav1.2 and Cav1.3 α1 subunit mRNA
To measure mRNA expression of Cav1.2 and Cav1.3 α1 subunits in the SN, mice were decapitated and the brains were isolated for further experiments. Left side of SN was isolated and put into RNase free centrifuge tubes (1.5 ml), stored at −80°C before used. Each sample was homogenized in 500 μl TRIzol reagent to purification total RNA then quantified its concentration. The total mRNA was reverse transcribed to cDNA used the firststrand cDNA synthesis kit (Thermo, Waltham, USA). The mRNA expressions were analyzed by Eppendorf system (Eppendorf, Hamburg, GER) using SYBR Green PCR Master Mix (Qiagen, GER) with 2-step PCR program (95°C for 5 s, 60°C for 10 s, 40 cycles). Each sample was performed in triplicate and values were averaged. Cycle threshold (Ct) values for target genes were normalized to the housekeeping gene β-actin. The 2 −ΔΔCt method was used to calculate the amount of target gene. PCRs were performed by using the following primers: Reverse 5′AGGCAACGATGATGATGTAGATG3′ β-actin: Forward 5′ TGCTGTCCCTGTATGCC TCT3′ Reverse 5′TTGATGTCACGCACGATTTC3′
Immunofluorescent labeling for tyrosine hydroxylase (TH) positive neurons
After anesthetized by 8% chloral hydrate (1.6 mg/kg, i.p.), the mice were perfused with 0.9% saline for 15 min then followed by 4% paraformaldehyde (PFA) in 0.1 mol/L phosphate buffered saline (PBS, pH7.4) for 20 min. The brains were isolated and post-fixed overnight in 4% PFA and followed by cryoprotection in 30% sucrose at 4 °C for at least 72 h. After the brains sank to the bottom of the centrifuge tube, they were taken out for the next steps. 20 μm serial coronal slices containing SNpc were collected in a −20°C freezing cryostat (Leica, GER). We collected every 4th serial section as 4 sets of sections and alternate set of sections were stained for TH or iron. Each set of sections contained about 15 such sections. For TH immunohistochemical detection, all the sections were kept free-floating in 0.01 mol/L PBS (pH7.4). The brain sections were blocked with 10% normal goat serum containing 0.3% Triton-X 100 for 1 h at 37°C. Rabbit anti-mouse TH primary antibody (1:2000) was used to incubate the sections overnight at 4°C temperature (AB152; Milipore, Billerica, Massachusetts, USA). The sections were treated with Alexa Fluor ® 488 donkey anti-rabbit IgG (A21206; Molecular Probs, Eugene, Oregon, USA) at room temperature for 2 h. After wash, sections were pasted on glass slides. The results were analyzed by counting the numbers of TH positive neurons on a Zeiss microscope (Carl Zeiss AG, Germany). All the micrographs were quantified using Image J software, which is developed by NIH.
HPLC-ECD to detected the contents of dopamine (DA) and its metabolites (DOPAC and HVA)
Both sides of striatum were isolated and transferred into liquid nitrogen for storage. Samples were prepared by using previously described techniques in our laboratory [33]. Briefly, the striatums were homogenized in 0.1 ml A solution (0.4 mol/L perchloric acid) and followed with centrifuge at 12000 rpm for 20 min at 4°C, then 40 μl solution B (20 mmol/L citromalic acid-potassium, 300 mmol/L dipotassium phosphate, and 2 mmol/L EDTA-2Na) were added to the supernatant. The HPLC (Waters Corp., Milford, MA, USA) was used to determine the contents of DA, DOPAC and HVA. Separation was achieved by a PE C18 reverse-phase column.
Perls' iron staining
Perls' iron staining was carried out according to previous report in our laboratory [34]. Freshly prepared solution (2% HCl and 2% potassium ferrocyanide) was used to incubated the sections for 30 min. Negative control was prepared without adding the freshly prepared solution. After washing 3 times with 0.01 mol/L PBS, sections were immersed in 99% methanol containing 1% hydrogen peroxide for 20 min to quench endogenous peroxidase activity. After washed 3 times with 0.01 mol/L PBS, the slices were incubated in a solution of diaminobenzidine (DAB). All incubations and washes were performed in polypropylene troughs that had been washed in 10% HCl (< 0.02 ppm Fe) overnight and rinsed in Milli-Q water. Staining was analyzed by counting the number of positive cells at 400 × magnification on an Olympus microscope. We used the average number of positive cells in four nonoverlapped fields per slide to conduct iron density. All counts were carried out blindly by a person unaware of the groups of the animals.
Cell culture
The MES23.5 cells were a generous gift from Dr. WD Le (Baylor College of Medicine, TX, USA). It is a dopaminergic cell line hybridized from murine neuroblastoma-glioma N18TG2 cells with rat mesencephalic neurons, which exhibits several properties that are similar to the primary neurons originated in the SN [35]. Cells were cultured in DMEM/F12 growth medium supplemented with 10% FBS, 2% Sato's, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37 °C and 5% CO 2 95% air environment. For experiments, cells were seeded at a density of 2×10 4 /ml in the 24-well plastic plates with a glass coverslips.
Calcein loading of cells and iron influx assay
MES23.5 cells were divided into four groups: Control group, MPP + group, MPP + with isradipine treatment group, and MPP + with Bayk8644 treatment group. Ferrous iron influx into MES23.5 cells was determined by the quenching of calcein fluorescence as described before [36,37]. Control group: MES23.5 cells were cultured in serum-free DMEM/F12 medium for 24 h. MPP + group: MES23.5 Cells were cultured in serumfree DMEM/F12 medium with 200 μmol/L MPP + for 24 h. The cells were incubated with calcein-AM at a final concentration of 1 mmol/L in Hepes-buffered saline (HBS; 10 mmol/L Hepes, 150 mmol/L NaCl, pH 6.8) for 30 min at 37°C. Excess calcein on cell surface was washed out 3 times with HBS. The coverslips were mounted in a perfused chamber. 488 nm excitation and 525 nm emission wavelengths were used to record calcein fluorescence. Fluorescence intensity was measured every 3 min for 30 min while perfusing with 1 mmol/L ferrous iron (ferrous sulfate in ascorbic acid solution, 1:44 molar ratio, pH 6.0) in control and MPP + groups. In the MPP + with isradipine and MPP + with Bayk8644 groups, MPP + treated cells undergoing same procedures except that 0.02 mmol/L isradipine or 0.01 mmol/L Bayk8644 were included in the perfusing fluid. The mean fluorescence intensity of 25-30 single cell in four separate fields was monitored at 200 × magnification and processed with Fluoview 5.0 Software [38]. Data represent the means of three independent experiments.
Statistical analysis
The results were represented as means ± SEM. The data were analyzed by two-way ANOVA followed by Bonferroni post hoc comparison of the means by using GraphPad 5.0 software (Graphpad Software, USA). P values that were less than 0.05 were considered to be significant. | v3-fos-license |
2020-08-06T09:04:06.268Z | 2020-07-30T00:00:00.000 | 221712494 | {
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} | pes2o/s2orc | Antitumor Activity In Vitro Provided by N-Alkyl-Nitroimidazole Compounds
Grupo de Investigaciones Biomédicas, Facultad de Ciencias de la Salud, Corporación Universitaria Remington, Calle 51 No 51-27 Medellín, Colombia. Laboratorio de Diseño y Formulación de Productos Químicos y Derivados, Departamento de Ciencias Farmacéuticas, Facultad de Ciencias Naturales, Universidad ICESI, Calle 18 No. 122 -135, Cali, 760035, Colombia. Grupo de Investigación en Química y Biotecnología (QUIBIO), Facultad de Ciencias Básicas, Universidad Santiago de Cali, calle 5 No. 62-00, Cali 760035, Colombia.
INTRODUCTION
Cancer is considered a public health problem worldwide and in Colombia. According to the International Agency for Research on Cancer, it is estimated that there were 18.1 million new cancer cases and 9.6 million cancer deaths in 2018 [1,2].
The most commonly diagnosed cancer is lung cancer in men and breast cancer in women [3]. Due to the high number ofcases that occur, the large number of adverse effects and the low effectiveness that is achieved in many of the treatments, it is of great importance for many organizations to investigate new alternatives for their treatment. The current treatment modalities are surgical resection, radiation therapy, hormone therapy, anti-hormone therapy, combined therapy as chemotherapy and radiation therapy, where the most commonly used chemotherapeutic agents are cytotoxic agents for cancer cells [4]. Some of these agents correspond to the imidazole compounds, which are developed towards their application in medicinal chemistry due to their pharmacological properties, such as anticancer activity with high efficacy and low toxicity [5,6]. In this context, the imidazole ring containing two nitrogen atoms and desirable πconjugated backbone can interfere with DNA synthesis, altering enzymes involved in DNA replication and the expression of genes associated with cancer cell growth such as tumor suppressor and cell cycle genes [7]. There are enough studies where imidazole ring is combined with heteroatomcontaining groups in a single compound in order to generate compounds with improved anticancer activity [7]. Previous studies have reported that N-alkyl imidazoles display potent anticancer activity via inhibition heme oxygenase (HO-1 and HO-2), an enzyme related to certain types of cancers [8,9].
On the other hand, the length of the alkyl chain on the imidazole ring is relevant for antimicrobial activity. Khabnadideh et al., 2003 [10] showed that antibacterial activity of 1-alkylimidazoles against Gram-negative and Gram-positive bacteria increases, as the number of carbons in the alkyl chain rises up to nine and then decreases. However, the knowledge about the contributions of the number of carbons in the acyl chain on anticancer activity is reduced. N-alkyl-nitroimidazoles with different carbon numbers in the alkyl chain were synthesized and theoretically studied in previous research [11]. Herein, we describe the effect of the alkyl chain length substituted in the N-imidazole ring on the anticancer activity against cell lines A549 (Human lung carcinoma) and MDA-MB-231 (Human breast adenocarcinoma).
MATERIALS AND METHODS
The N-alkyl-nitroimidazoles used in this study were previously synthesized, characterized, cryopreserved (-20 ° C) and supplied by the laboratory of design and formulation of the Icesi University [11], which were used as received. The human cancer cell lines A549 (Human lung carcinoma) and MDA-MB-231 (Human breast adenocarcinoma) were obtained from the American Type Culture Collection (ATCC). The cells were cultured in culture flasks with DMEM (Gibco, Life Technologies Corporation), supplemented with 10% FBS and penicillin (100 U/mL) and streptomycin (100 µg/mL) and were kept in a humidified incubator containing 5% CO 2 at 37°C.
Cell Growth Inhibition Assay
Cell proliferation and viability were assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, through MTT conversion into colored formazan product by metabolically active cells [12]. Cells (1,5 x 10 3 in 100 μL in 96-well flat-bottomed microliter plates) were incubated in DMEM culture medium containing 10% heatinactivated FBS and were permitted to adhere for 12 h. After the culture medium was removed, 100 µL of different concentrations of nitroimidazoles (1, 5, 10, 25, 50 and 100 µM), prepared in DMEM, were added to each well. Following 48 h of incubation at 37ºC in a humidified atmosphere of air/CO 2 (19/1), the MTT assay was performed. Measurements were done in triplicate. Absorbance was measured using an IMARK TM reader with a reference filter at 630 nm and a reading filter at 570 nm. The fraction of viable cells was calculated relative to control cells based on the following equation: (A1/A2) 100%, where A1 and A2 represent the absorbance of the sample and control groups, respectively. Each determination was performed in triplicate.
RESULTS
The in vitro antitumor activity for compounds was determined by the measurement of their cytostatic and cytotoxic properties in human tumor cell lines by MTT assays. The human cell lines used were MDA-MB-231 (human breast adenocarcinoma) and A549 (human lung carcinoma). As shown in Table 1, both breast cancer cells and lung cancer cells are sensitive to the compounds used; however, lung cancer cells were more sensitive to N-methyl-nitroimidazole and Nethyl-nitroimidazole. Furthermore, the length of the alkyl chain influenced on the antitumoral activity against only A549 cell lines. As can be seen, all the compounds showed less activity against normal cells, as are Vero cells. Doxorubicin used as a positive control rendered IC 50 values in the order of 10 −7 -10 −8 M (data not shown).
In order to determine which of N-alkyl-nitroimidazole compounds displayed a higher cytotoxic activity, cell viability was evaluated, through the incorporation MTT assay A concentration-response curve was observed between concentration and viability. A decrease in the viability of both cancer cell lines regarding the increase in the N-alkylnitroimidazole concentration was evaluated and is depicted in Fig. (1). The results showed that the four N-alkylnitroimidazole compounds described greater activity against breast cancer than against lung cancer, where the N-methylnitroimidazole and N-ethyl-nitroimidazole compounds corresponded to those with the highest cytotoxic activity. (Fig. 1). Fig. (1). Cell viability analysis. The viability of the cell lines exposed to different concentrations of the compounds was evaluated by MTT assay. In the figure, it was observed (A) breast cancer and (B) lung cancer. Data are means ± SD or representative of three independent experiments.
DISCUSSION
Anticancer activity measured as the LC50 in cancer cell lines decreased as the length of the substituted N-alkyl chain increased, solely for the A549 cell line. Regarding the MDA-MB231 cell line, the anticancer activity did not vary considerably when the length of the alkyl chain increased, suggesting that the number of carbons in the substituted Nalkyl did not influence on the activity against this type of cell.
There are several studies where the antibacterial and antiparasitic activity of nitroimidazoles is described [13][14][15][16][17][18]. Al-Masoudi et al., reported that nitroimidazoles synthesized by a simple method exhibited some cytostatic activity against leukemia and melanoma cell lines [19]. In a previous study, nitroimidazole derivative of polypyridyl ruthenium complex was employed in order to evaluate the anticancer activity against breast cancer (4T1) and A549 cell lines and they reported that the tested cell lines were strongly inhibited with IC50 ranging from 1. 5 to 18.8 µM [20], a range covering the LC 50 values exhibited by N-methyl and N-ethylnitroimidazoles. In a study conducted by Kumar et al., 1H-1,2,3-Triazole Tethered Nitroimidazole−Isatin Conjugates showed IC50s of 54.25 and 26.12 μM against MCF-7 and MDA-MB-231 cell lines, respectively, when a butyl alkyl chain length as a spacer and a halogen-substituent on the isatin ring was placed [6]. LC50 values reported in our study are below those reported by Kumar et al., reported that the activity tends to increase with the increase in chain length in contrast with our results where activity decreased and was maintained with increasing chain length, against A549 and MDA-MB-231 cell lines, respectively. Furthermore, unlike our results, another study has reported that anticancer activity increases with the increase in the alkyl chain at imidazolium-based ionic liquids [21], suggesting an interesting and poorly reported finding that could be explored in subsequent studies.
Here, the results also suggest a high selectivity of N-alkylnitroimidazoles toward cancer cells. Potential targets can be provided from imidazole derivatives to understand such selectivity. Heme oxygenase, an enzyme involved in the heme group catabolism, has been associated with the proliferation of A549 cancer cell line [22] and has been identified as a selective target for functional groups such as azolyl moiety, phenyl groups, and alkyl linkages [23]. Another selective target of imidazole derivatives towards tumor cells is the cRAF-kinase protein, which is up-regulated in melanomas and alteration of this signaling protein would play a major role in melanoma progression [24,25]. DNA is also known as another anticancer action target for imidazole derivatives. Chen et al. [26] described that triaryl-substituted imidazole was a telomeric Gquadruplex ligand which alters telomere maintenance, a crucial event for the unlimited proliferative potential of cancer cells.
CONCLUSION
In conclusion, compounds with increased antitumor activity and low cytotoxic effect in normal cells, may become therapeutic candidates for the treatment of cancer. Here, Nalkyl nitroimidazoles showed a reduced LC50 in lung cancer cells A549 and breast cancer MDA-MB231, while in Vero kidney cells, the LC50 was almost double for the case of Nethyl-nitroimidazole. Interestingly, only in lung tumor cells, the length of the N-alkyl chain of nitroimidazole is inversely proportional to the anticancer activity. Therefore, the pronounced selectivity of these compounds to tumor cells is a property that should be explored in future trials to contribute to the design of new drugs that can potentially be used in clinical practice.
ETHICS APPROVAL AND CONSENT TO PARTI-CIPATE
Not applicable.
HUMAN AND ANIMAL RIGHTS
Not applicable.
CONSENT FOR PUBLICATION
Not applicable. | v3-fos-license |
2016-05-04T20:20:58.661Z | 2013-12-12T00:00:00.000 | 14437327 | {
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} | pes2o/s2orc | Alginate Encapsulation of Begonia Microshoots for Short-Term Storage and Distribution
Synthetic seeds were formed from shoot tips of two in vitro grown Begonia cultivars using 3% sodium alginate in Murashige and Skoog medium (MS) salt solution as the gel matrix and 100 mM calcium chloride for complexation. Synthetic seed formation was achieved by releasing the sodium alginate/explant combination into 100 mM calcium chloride (CaCl2 ·H2O) solution for 30 or 45 min. Both control and encapsulated shoots were transferred into sterile Petri dishes and stored at 4°C or 22°C for 0, 2, 4, 6, or 8 weeks. Conversion of synthetic seeds into plantlets for both storage environments was assessed in MS medium or peat-based substrate. No significant difference was found between the 30 and 45 min CaCl2 ·H2O treatments or the two cultivars. Encapsulation of explants improved survival rate over time irrespective of the medium type or storage environment. Survival rates of 88, 53, 28, and 11% for encapsulated microshoots versus 73, 13, 0, and 0% for control explants were achieved in microshoots stored for 2, 4, 6, and 8 weeks, respectively. The best results were obtained when synthetic seeds were stored at 4°C and germinated on MS medium. Regenerated plantlets were successfully established in potting soil.
Introduction
Recently, the use of alginate encapsulation of in vitro cultured shoot tips as an alternative to somatic embryos to develop synthetic seeds has increased [1,2]. This increase in the use of microshoots in synthetic seed development is due to the fact that encapsulation of vegetative propagules offers an efficient and cost-effective system for clonal propagation of plant species [3,4]. Moreover, most plant species are more readily amenable to in vitro shoot culture than to regeneration via somatic embryogenesis, which is, for the most part, highly species or genotype dependent. Synthetic seed technology can be used for several purposes. For example, it can be used in conjunction with micropropagation for in vitro conservation, germplasm storage, and reduction of the need for transferring and subculturing during off-season periods [5]. Cold storage of encapsulated or synthetic seeds has the potential to reduce the cost of maintaining germplasm cultures as well as to reduce the possibility of genetic instability that could result from frequent subculturing [5,6]. The technology is particularly useful for the propagation of rare hybrids, elite genotypes, and genetically engineered plants whose seeds are either too expensive or not readily available [7].
Begonias are among the most popular ornamental plants in the world thanks to their large, showy, and long-lasting multicolor flowers, ranging from white to pink, red, and yellow. They are used as garden plants and potted plants, in hanging baskets, and as greenhouse flowers [8]. Begonias have also been used as potherbs or leaf vegetables in many parts of the world, and the roots and tubers of some species have been reported to possess antimicrobial activities and are used to treat various ailments [9][10][11]. Begonias are divided into three categories based on rootstocks: tuberous, fibrous, and rhizomatous. Tuberous and fibrous begonias are grown mostly for their flowers, whereas rhizomatous begonias are cultivated for their large, attractive foliage. As one the largest angiosperm genera containing about 2000 species [8] and with new species continuing to be discovered in various parts of the world [12][13][14][15][16], Begonia is a major component of the floriculture industry with a wholesale value of over $4 billion in 2011 statistics reported for 15 US states [17]. The main purpose of the current study was to establish an encapsulation method for Begonia microshoots as explants for short-term storage and germplasm exchange. Two Begonia semperflorens cultivars, Sweetheart Mix and BabyWing White, were used.
Encapsulation Procedure and Duration of Exposure to
Calcium Chloride. The steps involved in the scheme used for encapsulation, germination, and maturation of Begonia microshoots are shown in Figure 1. Synthetic seeds were formed using 3% sodium alginate in MS salt solution as the gel matrix and 100 mM calcium chloride (CaCl 2 ⋅H 2 O) for complexation. Both the sodium alginate and calcium chloride solutions were sterilized by autoclaving at 121 ∘ C and 1.1 kg/cm 2 for 15 minutes. Explants were submerged in sodium alginate solution for ∼2 minutes. Explants, along with enough sodium alginate solution to encapsulate each one, were individually suctioned into a sterile, disposable pipette that had been modified by cutting the tip so that its diameter was ∼7 mm.
During synthetic seed formation, the sodium alginate/explant combination was released into CaCl 2 ⋅H 2 O and maintained for either 30 or 45 minutes to examine how duration in CaCl 2 ⋅H 2 O might affect germination percentage. After the allotted time, the calcium chloride was drained from the newly formed synthetic seeds (encapsulated explants) by pouring the mixture into a sterile strainer. The synthetic seeds were rinsed at least three times with sterilized distilled water to remove any remaining CaCl 2 ⋅H 2 O.
Storage Temperature and Duration.
Two storage environments, 4 ∘ C refrigeration and room temperature (∼22 ∘ C), and five storage durations (0, 2, 4, 6, and 8 weeks) were evaluated. Synthetic seeds as well as nonencapsulated explants (control) were placed in empty, sterile Petri dishes and stored under 4 ∘ C refrigeration or at room temperature (∼22 ∘ C) for selected durations (0, 2, 4, 6, or 8 weeks). After the allotted storage period, the synthetic seeds and the control explants were placed in either MS media or peat-based substrate as described below. Germination percentages were monitored for all groups beginning with week 0 and extending through week 8.
In Vitro and Ex Vitro Germination.
Two types of growth media, an in vitro germination medium and an ex vitro nonsterile peat-based substrate (PBS; Jiffy-7 Pellets, Shippegan, NB, Canada), were used. The culture medium consisted of Murashige and Skoog (MS) [18] medium supplemented with 2% sucrose, 0.75 g/L MgCl 2 and adjusted to pH 5.8. All media were sterilized by autoclaving at 121 ∘ C for 15 minutes after addition of 2 g/L Gelrite. Synthetic seeds were planted in both The Scientific World Journal 3 MS medium and nonsterile PBS as parallel studies. The synthetic seeds that were plated on MS media were maintained at 22 ∘ C, 50% humidity, and 16-hour days under fluorescent lights with a photon flux averaging 154 moL m −2 s −1 . Those in PBS were placed under grow lights and maintained at 23-25 ∘ C, 47-50% relative humidity, and 16-hour days under fluorescent lights with a photon flux of 71 mol m −2 s −1 . A set of nonencapsulated explants served as a control and was maintained on the media described above and under the same environmental conditions. Germination, also referred to as conversion or regrowth by various authors [19][20][21][22], was defined as the point at which plant growth was observed outside of the synthetic seed matrix.
Experimental Design and Statistical
Analysis. The experiment was conducted in a five-factorial randomized complete block design, with two media (MS medium and Jiffy 7 peat pellets), two cultivars (BabyWing White and Sweetheart Mix), three CaCl 2 ⋅H 2 O exposure durations (0, 30, and 45 min), two storage temperatures (4 and 22 ∘ C), and five storage durations (0, 2, 4, 6, and 8 weeks). There were five replications for each treatment, with three microshoots per replication. The experiment was repeated twice. Data were collected as the count of shoots that "germinated" (developed successfully) in each experimental unit. Data were analyzed using generalized linear mixed models with the GLIMMIX procedure of SAS (version 9.3; SAS Institute Inc., Cary, NC) and the Poisson distribution. Models were evaluated beginning with a full model containing main factors and all interactions. Models were reduced by sequentially eliminating nonsignificant ( ≤ 0.05) interaction terms. The Schaffer-Simulated method was used for multiple mean comparisons.
Initial analyses determined that two factors, medium and storage duration, were highly influential in significant interaction terms; therefore, subsequent analyses were conducted individually by combination of medium and storage duration. Further analyses using cultivar, CaCl 2 ⋅H 2 O exposure duration, and storage temperature determined that there were no significant interactions; therefore, final models contained only the three main effects. Means are presented as percentages.
Results and Discussion
Germination percentage of encapsulated as well as nonencapsulated (control) Begonia microshoots stored in two environments, namely, at 4 ∘ C and room temperature (∼22 ∘ C) for various durations (0, 2, 4, 6, and 8 weeks) was evaluated. In both environments, Begonia shoot tips were dipped into 3% sodium alginate in MS salt solution as the gel matrix developed into nicely formed beads (hydrogels) and subsequently mature plants, irrespective of time of exposure (30 or 45 min) to the complexing agent, 100 mM CaCl 2 ⋅H 2 O, or the growing medium, MS or PBS medium (Figure 1).
Effect of Cultivar on Germination.
There was no significant difference ( = 0.05) in germination percentages between synthetic seeds of the two cultivars, BabyWing White and Sweetheart Mix, grown in MS medium in vitro ( Figure 2(a)). The results obtained under ex vitro conditions mirrored those achieved with MS medium, except that germination percentage for BabyWing White synthetic seeds in peatbased substrate was significantly higher ( = 0.05) than that obtained for Sweetheart Mix at week 2, with 66 and 45% germination percentage for BabyWing White and Sweetheart Mix, respectively (Figure 2(b)). In fact, germination percentage for BabyWing White was consistently higher, albeit not significantly, than that for Sweetheart Mix even after 8 weeks. For BabyWing White, germination percentages of 39% and 29% were obtained after 4 weeks when encapsulated shoots were grown in MS medium and peat-based substrate, respectively (Figures 2(a) and 2(b)). Similarly, for Sweetheart Mix, germination percentages of 36% and 21% were obtained after 4 weeks in MS medium and peat-based substrate, respectively. However, germination capacity for both cultivars decreased substantially with extended storage duration. Similar results have been reported by other workers [7]. In short, although low germination (3.3% for BabyWing White and 13.3% for Sweetheart Mix) could be achieved for synthetic seeds of both genotypes in an in vitro environment even at 8 weeks of storage duration (Figures 2(a) and 2(b)), similarly, encapsulation of explants improved germination under ex vitro conditions when Jiffy-7 pellets were used as nonsterile substrate (Figures 2(a) and 2(b)).
Effect of Exposure Duration of CaCl 2 ⋅H 2 O and Encapsulation Matrix on Germination.
The germination percentage was not significantly ( = 0.05) affected by the duration of exposure to calcium chloride whether the encapsulated seeds were maintained for 30 or 45 min in 100 mM CaCl 2 ⋅H 2 O and whether MS medium or PBS was used (Figures 2(c) and 2(d)). Daud et al. [23] found that 30 min was the optimal exposure time for best germination percentage and that both shorter (10 min) and longer (90 min) exposure durations resulted in reduced germination of African violet (Saintpaulia ionantha Wendl.). On the other hand, Castillo et al. [24] reported that a relatively short (10 min) duration of exposure to CaCl 2 ⋅H 2 O and only 2.5% alginate provided uniform encapsulation of embryos and the highest germination percentage (77.5%) of Carica papaya L. Successful production of synthetic seeds depends on several factors, including the concentration and type of gel needed for encapsulation of microshoots or somatic embryos, the duration of exposure of encapsulated seeds to CaCl 2 ⋅H 2 O, and plant species [23,[25][26][27][28]. Several gel types are used for encapsulation, but the most commonly used matrix is sodium alginate because of low cost, gelling properties, and nontoxic nature [29]. The integrity or hardness of the hydrogels depends for the most part on the number of Na + ions (in sodium alginate solution) exchanged with Ca ++ ions (in CaCl 2 ⋅H 2 O solution) resulting in the formation of insoluble calcium alginate [23]. In the current study, nicely formed beads were easily obtained with 100 mM CaCl 2 ⋅H 2 O and 3% sodium alginate. Our results were in agreement with those of Daud et al. [23] who reported that an encapsulation matrix of 3% alginate with 100 mM CaCl 2 ⋅H 2 O was ideal for the formation of Saintpaulia ionantha Wendl. microshoot beads as higher concentration of sodium alginate (4-5%) Storage duration (weeks) Figure 2: Percentages of encapsulated microshoots (synthetic seeds) and nonencapsulated microshoots of two Begonia cultivars "germinating" (developing successfully) after storage and subsequent transfer to substrate (MS medium (in vitro) or peat pellets (ex vitro)). Encapsulated microshoots were exposed to CaCl 2 ⋅H 2 O after encapsulation, whereas nonencapsulated microshoots served as a control for this factor. Medium and storage duration were highly influential in significant interaction terms; therefore, analyses of cultivar ((a) and (b)), CaCl 2 ⋅H 2 O exposure duration ((c) and (d)), and storage temperature ((e) and (f)) were conducted for each combination of medium and storage duration. Cultivar, CaCl 2 ⋅H 2 O exposure duration, and storage temperature factors showed no significant interactions; therefore, means shown (dots) in each subfigure are averages over the other two factors. Means for a specific storage duration (vertical pairs or trios) within each figure are significantly different ( = 0.05) when labeled with "a" and "b, " or not significantly different when labeled with "ns. " The Schaffer-Simulated method was used for multiple mean comparisons.
The Scientific World Journal 5 resulted in beads that were too hard, causing a germination delay, while lower concentration of sodium alginate (1-2%) produced beads that readily burst because they were too fragile and difficult to handle. Indeed, a 3% sodium alginate appears to be the optimum concentration for a great number of species as low concentrations (1-2%) result in beads too soft to handle and higher concentrations (≥4%) in beads too hard, preventing the emergence of shoots and roots [29][30][31][32][33]. On the other hand, germination percentage was significantly affected by encapsulation as marked improvement was achieved in germination percentage for encapsulated microshoots compared with nonencapsulated explants in both in vitro and ex vitro environments (Figures 2(c) and 2(d)). The germination percentage of encapsulated microshoots, whether exposed to CaCl 2 ⋅H 2 O for 30 or 45 min, was consistently higher than that of nonencapsulated microshoots up to 6 and 4 weeks in MS medium and PBS, respectively. These improved germination percentages using synthetic Begonia seeds are in agreement with other findings from other workers for several other species including Camellia spp., Zingiber officinale, and Ruta graveolens [20,21,34].
Effect of Storage Temperature and Substrate on Germination.
Storage temperature significantly ( = 0.05) affected germination frequency in both in vitro and ex vitro environments as germination percentage was significantly higher for encapsulated microshoots stored at 4 ∘ C (up to 6 weeks) than for those grown at 22 ∘ C (up to 4 weeks). Germination percentage of encapsulated microshoots in both growing environments declined over time, but this decrease was more pronounced when explants were stored at room temperature than under refrigeration (Figures 2(e) and 2(f)). For example, in MS medium, germination percentage was still relatively high at 32% after 6 weeks, whereas only 8% germination was obtained at room temperature at the same time point (Figure 2(e)). Similarly, in PBS, germination percentages after 4 weeks were 47% and 3% for 4 and 22 ∘ C, respectively ( Figure 2(f)). In other words, germination was almost 16fold higher when encapsulated explants were stored at low temperature. It is noteworthy that, even at room temperature and under nonsterile conditions, 41% of synthetic seeds still germinated after 2 weeks (Figure 2(f)).
Overall, significantly better germination percentages were obtained over time when the encapsulated seeds were grown in MS medium in vitro than in peat pellets ex vitro ( Figure 3). Furthermore, the improved germination percentage of encapsulated microshoots over control explants, even after a considerable amount of storage time, can be attributed to the inclusion of MS salts in encapsulation matrix, which serves as an artificial endosperm to the encapsulated microshoots for conversion to plantlets [35,36]. Hung and Trueman [37] reported that direct transfer of synthetic seeds of African mahogany (Khaya senegalensis) to nonsterile substrates was ineffective unless the seeds were allowed to preconvert (i.e., to form roots in vitro with the aid of growth regulators) prior to transfer as most of the seeds were contaminated with fungi within a week. However, in the current study, such an extra step was not required as synthetic Begonia seeds readily b * * * * * * * * * * * * * * * * * * * * * Figure 3: Percentages of encapsulated microshoots (synthetic seeds) and nonencapsulated microshoots of two Begonia cultivars "germinating" (developing successfully) after storage and subsequent transfer to MS medium (in vitro) or peat pellets (ex vitro). Medium and storage duration were highly influential in significant interaction terms among five treatment factors; therefore, analyses were run at each combination of these two factor levels. Means shown (dots) are averages among the three additional treatment factors (cultivar, CaCl 2 ⋅H 2 O exposure duration, and storage temperature), which showed no significant interaction effects when analyzed by medium and storage duration. Means for the two media at a specific storage duration (vertical pairs) are significantly different ( = 0.05) when labeled with "a" and "b" or not significantly different when labeled with "ns. " Means for storage durations connected by a line segment are significantly different at = 0.05 ( * ), 0.01 ( * * ), or 0.001 ( * * * ). The Schaffer-Simulated method was used for multiple mean comparisons. germinated, formed roots, and were easily acclimated when transferred to nonsterile peat-based media (Figure 1(g)).
Conclusions
Production of synthetic seeds of both Begonia cultivars, BabyWing White and Sweetheart Mix, using the protocol developed in this study was relatively easy. Encapsulated seeds kept at low temperature (4 ∘ C) could be stored for a longer period of time and had a higher germination percentage than those stored at room temperature (∼22 ∘ C). Also, better germination percentages were obtained over time when the encapsulated seeds were grown in MS medium than in PBS, irrespective of the storage environment (4 ∘ C or 22 ∘ C). However, storage at room temperature has the advantage of avoiding costs associated with refrigeration equipment. Seeds of Sweetheart Mix are actually a mixture of seeds from different genotypes, so the fact that synthetic seeds of this cultivar could be easily formed and germinated using our protocol suggests that this procedure could be applied for an efficient production of synthetic seeds from microshoots of other Begonia species and cultivars.
Disclosure
Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. | v3-fos-license |
2018-09-23T21:23:24.108Z | 2018-09-01T00:00:00.000 | 52293561 | {
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} | pes2o/s2orc | Optimization of Aminoimidazole Derivatives as Src Family Kinase Inhibitors
Protein kinases have emerged as crucial targets for cancer therapy over the last decades. Since 2001, 40 and 39 kinase inhibitors have been approved by FDA and EMA, respectively, and the majority are antineoplastic drugs. Morevoer, many candidates are currently in clinical trials. We previously reported a small library of 4-aminoimidazole and 2-aminothiazole derivatives active as Src family kinase (SFK) inhibitors. Starting from these results, we decided to perform an optimization study applying a mix and match strategy to identify a more potent generation of 4-aminoimidazoles. Firstly, a computational study has been performed, then compounds showing the best predicted docking scores were synthesized and screened in a cell-free assay for their SFK inhibitory activity. All the new chemical entities showed IC50s in the nanomolar range, with 2–130 fold increased activities compared to the previously reported inhibitors. Finally, the most active compounds have been tested on three cancer cell lines characterized by Src hyperactivation. Compounds 4k and 4l showed an interesting antiproliferative activity on SH-SY5Y neuroblastoma (NB) cell line. In this assay, the compounds resulted more potent than dasatinib, a tyrosine kinase inhibitor approved for the treatment of leukemias and in clinical trials for NB.
Introduction
Protein kinases are a large class of enzymes (to date 518 members have been identified) which are involved in different phases of the cell life [1]. These proteins are overexpressed and/or hyperactivated in several diseases, including cancer, neurodegenerative disorders and inflammation [2]. For this reason, protein kinases have become a thoroughly studied target in medicinal chemistry and, to date, 40 and 39 kinase inhibitors have been approved by the FDA and EMA, respectively, and the majority are antineoplastic drugs [3].
TKs are further divided in two main families: receptor tyrosine kinases and non-receptor tyrosine kinases. Into the latter, Src family kinases (SFKs) are the biggest subfamily of enzymes. This class includes nine highly homologous members, i.e., Src, Fyn, Yes, Blk, Yrk, Fgr, Hck, Lck, and Lyn. All SFKs present a common structure characterized by a N-terminal Src homology domain (SH4), a 'unique' region-different among SFK members, two Src homology domains (SH2 and SH3), a catalytic domain (SH1), and a short C-terminal tail. The catalytic domain constitutes the core of the enzyme since it possesses the kinase activity. SH1 presents a bilobal structure, with a small Nterminal lobe and a large C-terminal lobe, linked by a flexible chain, named hinge region [5]. At the interlobe cleft there are the ATP-and substrate-binding sites. Under basal conditions, SFKs present a close and inactive conformation that prevents ATP and substrate binding. After phosphorylation of a specific tyrosine residue (Tyr419 in human Src) in the C-terminal lobe by upstream kinases, a structural rearrangement occurs and results in a flip to an open and active conformation. At this point, the enzyme is able to bind ATP and the opportune peptide substrate, that will be phosphorylated [6,7]. SFKs are involved in the regulation of different signal transduction pathways such as growth, proliferation, differentiation, migration, metabolism, and apoptosis and, as reported for kinases in general, their overexpression and/or hyperactivation have been shown in many types of tumors [8,9]. Interestingly, despite the high homology among SFK members, different pathological implications have been detected inside the family. For instance, Fyn plays a key role in brain pathologies, such as Alzheimer's disease [10], whereas Hck, Fgr, and Lyn are the main SFK members involved in inflammation [11].
To date, three molecules active as SFK inhibitors have been approved for clinical use, i.e., dasatinib, bosutinib, and ponatinib ( Figure 1) [3,12,13]. Anyway, these compounds are not selective SFK inhibitors, but also inhibit other kinases. Dasatinib (Sprycel ® , Bristol-Myers Squibb, approved in 2006), and bosutinib (Bosulif ® , Wyeth, approved in 2012), are two dual Src/Bcr-Abl (another nonreceptor TK) inhibitors which bind the enzymes in their active conformation. Dasatinib potently inhibits all nine members of SFKs [14] and, in detail, a KINOMEscan analysis published by Davis et al. in 2011 [15] showed that it possesses Kd values of 0.21, 0.30, 0.53, and 0.79 nM on c-Src, Yes, Lyn, and Fyn, respectively. The compound also inhibits other TKs, including Kit, PDGFR, ephrin A receptor kinase, and the Tec kinase Btk [16]. Dasatinib has been approved for the treatment of chronic myeloid leukemia (CML) and acute lymphoblastic leukemia Philadelphia chromosome-positive (ALL Ph+), and is currently in clinical trials for NB and other solid tumors [17]. NB is an extracranial solid tumor of childhood often characterized by poor prognosis and for which an effective treatment still lacks. Bosutinib has been approved for the treatment of CML Ph+ with resistance or intolerance to prior therapy, and in December 2017, the FDA granted accelerated approval as first line therapy [18]. Ponatinib (Iclusig ® , Ariad Pharmaceuticals, approved in 2012) is a so-called "pan-kinase" inhibitor, since it is active against many kinases, including SFKs, Bcr-Abl, VEGFR, PDGFR, and Ret. Ponatinib binds Src and Bcr-Abl in their inactive conformation. It has been approved for CML or ALL Ph+, in particular for resistant T315I-positive CML or T315I-positive, Ph+ ALL [19]. Although the majority of protein kinase inhibitors occupy the ATP binding site, few compounds are able to bind pockets that are far from the catalytic cleft and act as allosteric inhibitors [20]. The complexity in regulating kinases' activity offers many potential routes for pursuing their inhibition Although the majority of protein kinase inhibitors occupy the ATP binding site, few compounds are able to bind pockets that are far from the catalytic cleft and act as allosteric inhibitors [20]. The complexity in regulating kinases' activity offers many potential routes for pursuing their inhibition and, despite the initial concerns about the possibility to identify potent and selective kinases inhibitors, significant advances have been made over the past two decades [21][22][23].
As widely reported in the literature, kinase inhibitors usually have heteroaromatic scaffolds capable of interacting with the hinge region [8]. Therefore, the synthesis of new hinge interacting moieties is crucial to obtain new molecules that may be selective for one or a few kinases and endowed with a high inhibitory potency. Zhang et al. described in detail the binding site of different kinase inhibitors and showed many heterocyclic ring systems that occupy the purine binding site [24]. The strength of the binding between these heterocyclic moieties and the target kinase is due to electrostatic interactions; in particular, some hinge binders were designed to establish one to three hydrogen bonds to the hinge region.
From an extensive literature study, the 4-aminoimidazole and the 2-aminothiazole rings emerged as interesting starting entities for the development of new ATP pocket binders. The 4-aminoimidazole moiety has been first explored by AstraZeneca [25], who highlighted the ability of this scaffold to bind the Janus kinase hinge region. The authors compared their clinical candidate AZD1480 (a Jak2 inhibitor with an IC 50 of 58 nM, Figure 2), containing the 3-aminopyrazole moiety, with the related 4-aminoimidazole derivative (IC 50 value of 120 nM towards Jak2), and found that this bioisosteric substitution could be an effective replacement for the 3-aminopyrazole ring. The subsequent modulation of this first hit led to the discovery of the potent and orally bioavailable Jak2 inhibitor 1 (IC 50 < 3 nM, Figure 2) [25].
Molecules 2018, 23, x FOR PEER REVIEW 3 of 17 and, despite the initial concerns about the possibility to identify potent and selective kinases inhibitors, significant advances have been made over the past two decades [21][22][23].
As widely reported in the literature, kinase inhibitors usually have heteroaromatic scaffolds capable of interacting with the hinge region [8]. Therefore, the synthesis of new hinge interacting moieties is crucial to obtain new molecules that may be selective for one or a few kinases and endowed with a high inhibitory potency. Zhang et al. described in detail the binding site of different kinase inhibitors and showed many heterocyclic ring systems that occupy the purine binding site [24]. The strength of the binding between these heterocyclic moieties and the target kinase is due to electrostatic interactions; in particular, some hinge binders were designed to establish one to three hydrogen bonds to the hinge region.
From an extensive literature study, the 4-aminoimidazole and the 2-aminothiazole rings emerged as interesting starting entities for the development of new ATP pocket binders. The 4aminoimidazole moiety has been first explored by AstraZeneca [25], who highlighted the ability of this scaffold to bind the Janus kinase hinge region. The authors compared their clinical candidate AZD1480 (a Jak2 inhibitor with an IC50 of 58 nM, Figure 2), containing the 3-aminopyrazole moiety, with the related 4-aminoimidazole derivative (IC50 value of 120 nM towards Jak2), and found that this bioisosteric substitution could be an effective replacement for the 3-aminopyrazole ring. The subsequent modulation of this first hit led to the discovery of the potent and orally bioavailable Jak2 inhibitor 1 (IC50 < 3 nM, Figure 2) [25]. On the other hand, the 2-aminothiazole represents the hinge binder moiety of dasatinib, the potent SFK inhibitor already reported.
In a previous work, we described the synthesis and the biological evaluation of a set of 4aminoimidazole and 2-aminothiazole derivatives as SFK inhibitors [26]. The 4-aminoimidazole ring was demonstrated to be an effective hinge binder for c-Src kinase, suggesting that this moiety, properly functionalized, is a good replacement for the aminothiazole ring [26]. Indeed, the most active compound 2 ( Figure 3) showed IC50 values of 220, 689, 1300, and 167 nM for the isolated enzymes Src, Fyn, Lyn, and Yes, respectively (Table 1), and resulted active on SH-SY5Y neuroblastoma (NB) cell line with an IC50 of 25 μM. On the other hand, 2 showed a weak activity on K562 CML cell line, possessing an IC50 > 25 μM. As a continuation of our work, herein we present an optimization study aimed at obtaining a new generation of (1H-imidazol-4-yl)-pyrimidin-4-ylamines endowed with a higher affinity towards SFKs and a stronger activity on cells compared to compounds previously reported by us. On the other hand, the 2-aminothiazole represents the hinge binder moiety of dasatinib, the potent SFK inhibitor already reported.
In a previous work, we described the synthesis and the biological evaluation of a set of 4-aminoimidazole and 2-aminothiazole derivatives as SFK inhibitors [26]. The 4-aminoimidazole ring was demonstrated to be an effective hinge binder for c-Src kinase, suggesting that this moiety, properly functionalized, is a good replacement for the aminothiazole ring [26]. Indeed, the most active compound 2 ( Figure 3) showed IC 50 values of 220, 689, 1300, and 167 nM for the isolated enzymes Src, Fyn, Lyn, and Yes, respectively (Table 1), and resulted active on SH-SY5Y neuroblastoma (NB) cell line with an IC 50 of 25 µM. On the other hand, 2 showed a weak activity on K562 CML cell line, possessing an IC 50 > 25 µM. As a continuation of our work, herein we present an optimization study aimed at obtaining a new generation of (1H-imidazol-4-yl)-pyrimidin-4-yl-amines endowed with a higher affinity towards SFKs and a stronger activity on cells compared to compounds previously reported by us.
Docking Studies
Starting from our hit 2 [26] and 3 (a potent Src inhibitor) [27], a mix and match strategy combined with the use of computational tools has been applied for the identification of new chemical entities (NCEs) acting as ATP pocket binders ( Figure 3). As a first step towards a better understanding of the molecular determinants for the inhibitory activity of this class of compounds against Src, a molecular docking simulation has been performed on NCEs and the upcoming results were compared with the best aminoimidazole hit 2 reported in our previous work [26]. In particular, more than one hundred NCEs were designed and docked into the ATP binding site of Src by using the 3G5D X-ray structure [28]. The mix and match strategy has been applied in order to (i) introduce different alkyl groups as linkers to investigate the steric hindrance allowed around the hinge region; (ii) introduce hydrophobic and hydrophilic moieties interacting with the hydrophobic region I (HRI) to improve the primary activity by achieving electrostatic interactions that would be missing with an unsubstituted phenyl group; (iii) replace the solvent exposed substituent with different types of heterocycles.
Docking Studies
Starting from our hit 2 [26] and 3 (a potent Src inhibitor) [27], a mix and match strategy combined with the use of computational tools has been applied for the identification of new chemical entities (NCEs) acting as ATP pocket binders ( Figure 3). As a first step towards a better understanding of the molecular determinants for the inhibitory activity of this class of compounds against Src, a molecular docking simulation has been performed on NCEs and the upcoming results were compared with the best aminoimidazole hit 2 reported in our previous work [26]. In particular, more than one hundred NCEs were designed and docked into the ATP binding site of Src by using the 3G5D X-ray structure [28]. The mix and match strategy has been applied in order to (i) introduce different alkyl groups as linkers to investigate the steric hindrance allowed around the hinge region; (ii) introduce hydrophobic and hydrophilic moieties interacting with the hydrophobic region I (HRI) to improve the primary activity by achieving electrostatic interactions that would be missing with an unsubstituted phenyl group; (iii) replace the solvent exposed substituent with different types of heterocycles. Docking studies were performed by means of Glide [29] software and the reliability of the applied protocol was first assessed by reproducing the experimental binding mode of two known inhibitors of Src: dasatinib (PDB code: 3G5D) [28] and CGP77675 (PDB code: 1YOL [30], Figure 2). Compounds were drawn, minimized and finally docked into the ATP-binding site of Src (3G5D) [28]. Docking studies were performed by means of Glide [29] software and the reliability of the applied protocol was first assessed by reproducing the experimental binding mode of two known inhibitors of Src: dasatinib (PDB code: 3G5D) [28] and CGP77675 (PDB code: 1YOL [30], Figure 2). Compounds were drawn, minimized and finally docked into the ATP-binding site of Src (3G5D) [28].
As a result, the program was able to reproduce the experimental poses of the two compounds with a RMSD of 0.55 Å suggesting that the docking procedure could be reliable to predict the binding mode of our NCEs ( Figure 4).
Docking Studies
Starting from our hit 2 [26] and 3 (a potent Src inhibitor) [27], a mix and match strategy combined with the use of computational tools has been applied for the identification of new chemical entities (NCEs) acting as ATP pocket binders ( Figure 3). As a first step towards a better understanding of the molecular determinants for the inhibitory activity of this class of compounds against Src, a molecular docking simulation has been performed on NCEs and the upcoming results were compared with the best aminoimidazole hit 2 reported in our previous work [26]. In particular, more than one hundred NCEs were designed and docked into the ATP binding site of Src by using the 3G5D X-ray structure [28]. The mix and match strategy has been applied in order to (i) introduce different alkyl groups as linkers to investigate the steric hindrance allowed around the hinge region; (ii) introduce hydrophobic and hydrophilic moieties interacting with the hydrophobic region I (HRI) to improve the primary activity by achieving electrostatic interactions that would be missing with an unsubstituted phenyl group; (iii) replace the solvent exposed substituent with different types of heterocycles. Docking studies were performed by means of Glide [29] software and the reliability of the applied protocol was first assessed by reproducing the experimental binding mode of two known inhibitors of Src: dasatinib (PDB code: 3G5D) [28] and CGP77675 (PDB code: 1YOL [30], Figure 2). Compounds were drawn, minimized and finally docked into the ATP-binding site of Src (3G5D) [28]. Compounds 4a-g (Table 1), showing the best predicted docking scores, are characterized by a series of polar moieties in the solvent exposed region, and hydroxyl or methoxyl groups on the phenyl ring of the N-1-(2-phenylethyl)-1H-imidazol-4-yl side chain. These substitution patterns have been selected with the aim of improving the water solubility and getting further insights into this class of inhibitors. All compounds showed a similar interaction pattern characterized by two hydrogen bonds involving the imidazole nucleus and the hinge region: one between the N3 and the NH backbone of Met341, and one between the 4-NH and the CO backbone of Met341. The phenyl ring was located into the HR1, forming Van der Waals interactions with hydrophobic amino acids of this region. Moreover, the new N-[1-(2-phenylethyl)-1H-imidazol-4-yl]pyrimidinamines interacted with two different residues in HR1 and in solvent exposed area. The best compounds in terms of docking scores show a meta or ortho hydroxyl group on the phenyl ring and an amide, ester, or carbamate group in N4 position of the piperazine chain. In detail, the pose example of compound 4j (GB = −11.33 kcal/mol) has been reported in Figure 5: the meta hydroxyl group acts as both H-bond donor and acceptor in the interactions with Glu310 and Asp464 respectively. The ortho substituted derivative 4g (GB = −11.23 kcal/mol) establishes a hydrogen bond interaction with Asp464 belonging to the DFG motif.
with a RMSD of 0.55 Å suggesting that the docking procedure could be reliable to predict the binding mode of our NCEs ( Figure 4).
Compounds 4a-g (Table 1), showing the best predicted docking scores, are characterized by a series of polar moieties in the solvent exposed region, and hydroxyl or methoxyl groups on the phenyl ring of the N-1-(2-phenylethyl)-1H-imidazol-4-yl side chain. These substitution patterns have been selected with the aim of improving the water solubility and getting further insights into this class of inhibitors. All compounds showed a similar interaction pattern characterized by two hydrogen bonds involving the imidazole nucleus and the hinge region: one between the N3 and the NH backbone of Met341, and one between the 4-NH and the CO backbone of Met341. The phenyl ring was located into the HR1, forming Van der Waals interactions with hydrophobic amino acids of this region. Moreover, the new N-[1-(2-phenylethyl)-1H-imidazol-4-yl]pyrimidinamines interacted with two different residues in HR1 and in solvent exposed area. The best compounds in terms of docking scores show a meta or ortho hydroxyl group on the phenyl ring and an amide, ester, or carbamate group in N4 position of the piperazine chain. In detail, the pose example of compound 4j (GB = −11.33 kcal/mol) has been reported in Figure 5: the meta hydroxyl group acts as both H-bond donor and acceptor in the interactions with Glu310 and Asp464 respectively. The ortho substituted derivative 4g (GB = −11.23 kcal/mol) establishes a hydrogen bond interaction with Asp464 belonging to the DFG motif. phenyl ring of the N-1-(2-phenylethyl)-1H-imidazol-4-yl side chain. These substitution patterns have been selected with the aim of improving the water solubility and getting further insights into this class of inhibitors. All compounds showed a similar interaction pattern characterized by two hydrogen bonds involving the imidazole nucleus and the hinge region: one between the N3 and the NH backbone of Met341, and one between the 4-NH and the CO backbone of Met341. The phenyl ring was located into the HR1, forming Van der Waals interactions with hydrophobic amino acids of this region. Moreover,
the new N-[1-(2-phenylethyl)-1H-imidazol-4-yl]pyrimidinamines interacted
with two different residues in HR1 and in solvent exposed area. The best compounds in terms of docking scores show a meta or ortho hydroxyl group on the phenyl ring and an amide, ester, or carbamate group in N4 position of the piperazine chain. In detail, the pose example of compound 4j (GB = −11.33 kcal/mol) has been reported in Figure 5: the meta hydroxyl group acts as both H-bond donor and acceptor in the interactions with Glu310 and Asp464 respectively. The ortho substituted derivative 4g (GB = −11.23 kcal/mol) establishes a hydrogen bond interaction with Asp464 belonging to the DFG motif. -3OH -COOtert-butyl −10.99 50 ± 3 14 ± 1.5 26 ± 3 7 ± 0.35 a The compound was tested in two independent experiments, and IC 50 values are the mean ± SD. b Dasatinb was used as reference; the IC 50 values of dasatinib were less than that of enzyme concentrations, which were 4, 9, 0.9, and 3 nm for Src, Fyn, Lyn, and Yes, respectively.
As shown in Table 1, compounds 4g and 4j, having the highest values of docking score, are predicted to be the most active compounds on the selected kinase, while compounds 4b and 4d (−7.266 and −8.336 kcal/mol respectively) resulted as the least active ones.
Chemistry
The best predicted derivatives, in respect to our previous reported hit 2, were selected to be synthesized and tested (Table 1). 4b and 4d were also prepared as negative controls in enzymatic assays. Compounds 4a-d, bearing a hydrophobic group -OMe exposed to HR1, were first synthesized (Scheme 1) [26]. The commercially available 4-nitro-1H-imidazole 5 was regioselectively functionalized using opportune alkylating agents to give intermediates 6a,b in high purity and yield [26,31]. Subsequent palladium mediated hydrogenation of 6a,b afforded the corresponding amino derivatives as free bases, which were immediately converted to hydrochloride salts, since the compounds are unstable as free bases, to yield derivatives 7a,b. Compounds 7a,b were regioselectively coupled with the commercially available 4,6-dichloro-2-methylpyrimidine at 50 • C in the presence of N,N-diisopropylethylamine (DIPEA) to afford intermediates 8a,b.
Dasatinb was used as reference; the IC50 values of dasatinib were less than that of enzyme concentrations, which were 4, 9, 0.9, and 3 nm for Src, Fyn, Lyn, and Yes, respectively.
As shown in Table 1, compounds 4g and 4j, having the highest values of docking score, are predicted to be the most active compounds on the selected kinase, while compounds 4b and 4d (−7.266 and −8.336 kcal/mol respectively) resulted as the least active ones.
Chemistry
The best predicted derivatives, in respect to our previous reported hit 2, were selected to be synthesized and tested (Table 1). 4b and 4d were also prepared as negative controls in enzymatic assays. Compounds 4a-d, bearing a hydrophobic group -OMe exposed to HR1, were first synthesized (Scheme 1) [26]. The commercially available 4-nitro-1H-imidazole 5 was regioselectively functionalized using opportune alkylating agents to give intermediates 6a,b in high purity and yield [26,31]. Subsequent palladium mediated hydrogenation of 6a,b afforded the corresponding amino derivatives as free bases, which were immediately converted to hydrochloride salts, since the compounds are unstable as free bases, to yield derivatives 7a,b. Compounds 7a,b were regioselectively coupled with the commercially available 4,6-dichloro-2-methylpyrimidine at 50 °C in the presence of N,N-diisopropylethylamine (DIPEA) to afford intermediates 8a,b. The intermediates 8a,b were reacted with the appropriate piperazines at 110 • C under microwave irradiation in the presence of DIPEA to afford the final compounds 4a,c and the intermediates 9a-c, used for the synthesis of phenol derivatives 4f,i,l described in the Scheme 2. Furthermore, 9a,b were reacted with methyl bromoacetate to obtain intermediates 10a,b, that were treated with ammonia 7N in MeOH to yield the final compounds 4b,d, according to the procedure used by Novartis (Scheme 1) [27].
Furthermore, 9a,b were reacted with methyl bromoacetate to obtain intermediates 10a,b, that were treated with ammonia 7N in MeOH to yield the final compounds 4b,d, according to the procedure used by Novartis (Scheme 1) [27].
Enzymatic Assays
Compounds 4a-l have been tested against the isolated Src enzyme and showed IC50 values in the nanomolar range. Derivatives 4e-l were found more active on Src than the previous reported hit 2, showing IC50 values from 93 nM to 40 nM. In particular, compounds 4g, 4j and 4k, bearing a hydroxyl group in the ortho or meta positions of the phenyl ring and an amide or methylester substituent as side chain, resulted to have the highest inhibitory activity (IC50 values of 40 nM). On the other hand, the methoxy derivatives 4a-d are less potent on Src (IC50 values 225-1533 nM) compared with the phenolic derivatives, confirming the importance of the hydroxyl group, as predicted by modeling studies. In addition, all new compounds were tested for their activity against other members of SFKs. As expected, the most promising compounds were also potent inhibitors of Yes, Lyn, and Fyn with IC50 values in the range 3-73 nM. These results confirmed the hypothesis that the 4-aminoimidazole template, properly decorated, is an effective hinge binder for SFKs and has a good/high in vitro potency on these enzymes.
Cellular Assays
Starting from these promising results in enzymatic assays, we decided to test NCEs 4 on K562 CML and SH-SY5Y NB cell lines, to evaluate if they are endowed with an increased antiproliferative activity compared with the hit compound 2. A hyperactivation of SFKs has been detected in both K562 and SH-SY5Y cell lines [9,32,33]. Cells were treated with increasing concentrations of compounds and cell proliferation was measured by counting viable cells after 72 h of incubation. Dasatinib and 2 were used as reference compounds. In Figure 6 we show the activity of 4k and 4l that demonstrated, in comparison with the other NCEs (see Supplementary Materials, Figure S1), the best antiproliferative activity on SH-SY5Y cells. In detail, 4k and 4l possess IC50 values of 8.6 and 7.8 μM, respectively, and show a more than 2-fold increased activity compared to the hit compound 2. Importantly, in NB cells, 4k and 4l exerted an antiproliferative effect similar or higher than dasatinib. The activity of these compounds could be due not only to Src inhibition, but also to their effect on Fyn and Lyn, both involved in NB development [34]. Furthermore, both the compounds showed a similar activity on K562 cells, with an antiproliferative effect comparable with the one observed on NB cells. In fact, compounds 4k and 4l show IC50 values of 11.7 and 18.9 μM, respectively, and are more active than 2, but less active than dasatinib ( Figure 6).
Enzymatic Assays
Compounds 4a-l have been tested against the isolated Src enzyme and showed IC 50 values in the nanomolar range. Derivatives 4e-l were found more active on Src than the previous reported hit 2, showing IC 50 values from 93 nM to 40 nM. In particular, compounds 4g, 4j and 4k, bearing a hydroxyl group in the ortho or meta positions of the phenyl ring and an amide or methylester substituent as side chain, resulted to have the highest inhibitory activity (IC 50 values of 40 nM). On the other hand, the methoxy derivatives 4a-d are less potent on Src (IC 50 values 225-1533 nM) compared with the phenolic derivatives, confirming the importance of the hydroxyl group, as predicted by modeling studies. In addition, all new compounds were tested for their activity against other members of SFKs. As expected, the most promising compounds were also potent inhibitors of Yes, Lyn, and Fyn with IC 50 values in the range 3-73 nM. These results confirmed the hypothesis that the 4-aminoimidazole template, properly decorated, is an effective hinge binder for SFKs and has a good/high in vitro potency on these enzymes.
Cellular Assays
Starting from these promising results in enzymatic assays, we decided to test NCEs 4 on K562 CML and SH-SY5Y NB cell lines, to evaluate if they are endowed with an increased antiproliferative activity compared with the hit compound 2. A hyperactivation of SFKs has been detected in both K562 and SH-SY5Y cell lines [9,32,33]. Cells were treated with increasing concentrations of compounds and cell proliferation was measured by counting viable cells after 72 h of incubation. Dasatinib and 2 were used as reference compounds. In Figure 6 we show the activity of 4k and 4l that demonstrated, in comparison with the other NCEs (see Supplementary Materials, Figure S1), the best antiproliferative activity on SH-SY5Y cells. In detail, 4k and 4l possess IC 50 values of 8.6 and 7.8 µM, respectively, and show a more than 2-fold increased activity compared to the hit compound 2. Importantly, in NB cells, 4k and 4l exerted an antiproliferative effect similar or higher than dasatinib. The activity of these compounds could be due not only to Src inhibition, but also to their effect on Fyn and Lyn, both involved in NB development [34]. Furthermore, both the compounds showed a similar activity on K562 cells, with an antiproliferative effect comparable with the one observed on NB cells. In fact, compounds 4k and 4l show IC 50 values of 11.7 and 18.9 µM, respectively, and are more active than 2, but less active than dasatinib ( Figure 6).
On the basis of the exciting activity of 4k and 4l on NB cell lines in comparison with dasatinib, we decided to test NCEs also on U87 glioblastoma multiforme (GBM), another tumor characterized by Src hyperactivation (Figure 6) [35]. Compounds 4k and 4l showed IC 50 s of 12.6 and 13.3, respectively, but, unfortunately, resulted less active than dasatinib. A possible explanation could be the multidrug resistance mechanisms that GBM cells usually carry: an example is the overexpression of membrane channels (ABCB1) that are able to pump different kind of drugs out of the cells [36]. In Supplementary Materials, Figure S1 On the basis of the exciting activity of 4k and 4l on NB cell lines in comparison with dasatinib, we decided to test NCEs also on U87 glioblastoma multiforme (GBM), another tumor characterized by Src hyperactivation (Figure 6) [35]. Compounds 4k and 4l showed IC50s of 12.6 and 13.3, respectively, but, unfortunately, resulted less active than dasatinib. A possible explanation could be the multidrug resistance mechanisms that GBM cells usually carry: an example is the overexpression of membrane channels (ABCB1) that are able to pump different kind of drugs out of the cells [36]. In Supplementary Materials, Figure S1, the activity of other NCEs on U87 GBM cell line are reported.
In conclusion, a small library of aminoimidazole derivatives was synthesized and screened in a cell-free assay for their SFK inhibitory activity. Enzymatic assays showed an increase in potency against isolated Src (from a micromolar range of our previously reported compounds [13] to nanomolar of the new molecules), with an exceptional increase in potency also against other SFK members. Furthermore, the most active inhibitors have been tested on three different cancer cell lines, i.e., NB, GBM, and CML cell lines. Interestingly, compounds 4k and 4l showed good antiproliferative activity in the SH-SY5Y NB cell line. In this assay the compounds resulted more potent than dasatinib, a TKI inhibitor which is currently in clinical trials for NB [17].
Further studies on this class of compounds will be focused on the improvement of the ADME properties, with the aim of obtaining more potent compounds in cell assays.
Protein Preparation
Crystal structures of c-Src in complex with dasatinib and CGP77675 (PDB IDs:3G5D [28] and 1YOL [30]), were retrieved from the RCSB Protein Data Bank. After removal of bound ligands, the proteins were prepared by using the Protein Preparation Wizard [37] workflow (Schrodinger Suite). In particular, all water molecules were deleted, hydrogen atoms were added, and partial charges assigned. In addition, the ionization and tautomeric states of His, Asp, Glu, Arg, and Lys were adjusted to match pH 7.4. Next, optimization of the hydrogen bonding network was obtained by reorienting hydroxyl and thiol groups, amide groups of Asn and Gln, and the His imidazole ring. Finally, the systems were refined by running a restrained minimization (OPLS3 force field) which was stopped when the RMSD of heavy atoms reached 0.30 Å , the default limit. In conclusion, a small library of aminoimidazole derivatives was synthesized and screened in a cell-free assay for their SFK inhibitory activity. Enzymatic assays showed an increase in potency against isolated Src (from a micromolar range of our previously reported compounds [13] to nanomolar of the new molecules), with an exceptional increase in potency also against other SFK members. Furthermore, the most active inhibitors have been tested on three different cancer cell lines, i.e., NB, GBM, and CML cell lines. Interestingly, compounds 4k and 4l showed good antiproliferative activity in the SH-SY5Y NB cell line. In this assay the compounds resulted more potent than dasatinib, a TKI inhibitor which is currently in clinical trials for NB [17].
Ligands Preparation
Further studies on this class of compounds will be focused on the improvement of the ADME properties, with the aim of obtaining more potent compounds in cell assays.
Protein Preparation
Crystal structures of c-Src in complex with dasatinib and CGP77675 (PDB IDs:3G5D [28] and 1YOL [30]), were retrieved from the RCSB Protein Data Bank. After removal of bound ligands, the proteins were prepared by using the Protein Preparation Wizard [37] workflow (Schrodinger Suite). In particular, all water molecules were deleted, hydrogen atoms were added, and partial charges assigned. In addition, the ionization and tautomeric states of His, Asp, Glu, Arg, and Lys were adjusted to match pH 7.4. Next, optimization of the hydrogen bonding network was obtained by reorienting hydroxyl and thiol groups, amide groups of Asn and Gln, and the His imidazole ring. Finally, the systems were refined by running a restrained minimization (OPLS3 force field) which was stopped when the RMSD of heavy atoms reached 0.30 Å, the default limit.
Molecular Docking
Docking simulations were performed using the Glide program (Schrödinger, LLC, New York, NY, USA) [38,39] within the ATP binding site of Src (3G5D). A grid box of default size was centered on the X-ray ligand. No constraints were included during grid generation while rotation of the hydroxyl groups was allowed only for those residues closer to the ligand. After grid preparation, NCEs were flexibly docked and scored using the Glide standard-precision (SP) mode, treating the proteins as rigid. Docking experiments were performed using 0.80 factor to scale vdW radii of the nonpolar ligand atoms with a partial atomic charge of <0.15. Flash silica gel chromatography was performed on Biotage automatic flash chromatography systems (Uppsala, Sweden) (Isolera or SP1) using Biotage SNAP HP silica cartridges or Biotage SNAP KP-NH cartridges. Reverse phase chromatography was performed on a Biotage automatic flash chromatography system (Isolera) using Redisep Gold C-18Aq cartridges. Purification was also done by SPE with SCX cartridges. Reactions were monitored by thin-layer chromatography on 0.25 mm E. Merck silica gel plates (60F-254), visualized with UV light. Melting points (Mp) were determined with a Büchi B-540 apparatus.
Chemistry
Microwave irradiation experiments were conducted on a Biotage Initiator microwave reactor.
To obtain compounds 4a,c and 9c the column was eluted with 2CV of water + 0.1% AcOH and then the eluent was gradually changed to acetonitrile + 0.1% AcOH over 13CV. The fractions were combined and evaporated. The orange-brown residue was dissolved in DCM and treated with a saturated solution of sodium bicarbonate to avoid the acetic acid salt formation. Finally, the organic phase was dried, filtered and evaporated under vacuum affording the desired compounds.
To obtain compounds 9a,b, the column was eluted with 3CV of water + 0.1% AcOH and then the eluent was gradually changed to acetonitrile + 0.1% AcOH over 13CV. The fractions containing the desired product were combined and evaporated to obtain a brown residue. It was dissolved in MeOH and loaded onto a SPE-SCX (5 g) eluted first with methanol and then with ammonia in methanol (2N). Basic fractions were collected and evaporated affording the desired compounds. and Yes1, all human recombinant full-length proteins) according to a procedure already reported. Final results have been expressed as percent inhibition, and IC 50 values were calculated by non-linear curve fitting using GraphPad Prism software (version 6 for Windows). The inter-experimental variability of IC 50 values resulted within accepted limits of ±0.5 log units.
Cellular Assays
In vitro experiments were carried out using human neuroblastoma cell line SH-SY5Y, human glioblastoma cell line U-87, and human erythroleukemia cell line K-562. Cell lines were obtained from American Tissue Culture Collection (ATCC, SH-SY5Y CRL-2266; U-87 HTB-14; K-562 CCL-243). K-562 cells were cultured in RPMI medium with 10% FCS. SH-SY5Y and U-87 cells were cultured in DMEM medium with 10% FCS. In order to determine antiproliferative effect of NCE compounds cells were seeded at density of 5 × 10 4 cells/mL (K-562) or 10 × 10 4 cells/cm 2 (SH-SY5Y, U-87) and treated with increasing concentrations of NCE compounds. Control cells were treated with the vehicle of the experimental point containing the highest percentage of DMSO. Cell cultures were maintained at 37 • C in 5% v/v CO 2 for 72 h. Cell number and vitality were evaluated on cell suspension using the automatic cell counter NucleoCounter ® (Chemometec, Denmark). Results from the NucleoCounter represented either total or non-viable cell concentration, depending on the sample preparation indicated by the manufacturer. Each experiment was performed at least three times and results were expressed as mean and standard deviation.
Conflicts of Interest:
The authors declare no conflict of interest. | v3-fos-license |
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} | pes2o/s2orc | Metabolic-Hydroxy and Carboxy Functionalization of Alkyl Moieties in Drug Molecules: Prediction of Structure Influence and Pharmacologic Activity.
Alkyl moieties—open chain or cyclic, linear, or branched—are common in drug molecules. The hydrophobicity of alkyl moieties in drug molecules is modified by metabolic hydroxy functionalization via free-radical intermediates to give primary, secondary, or tertiary alcohols depending on the class of the substrate carbon. The hydroxymethyl groups resulting from the functionalization of methyl groups are mostly oxidized further to carboxyl groups to give carboxy metabolites. As observed from the surveyed cases in this review, hydroxy functionalization leads to loss, attenuation, or retention of pharmacologic activity with respect to the parent drug. On the other hand, carboxy functionalization leads to a loss of activity with the exception of only a few cases in which activity is retained. The exceptions are those groups in which the carboxy functionalization occurs at a position distant from a well-defined primary pharmacophore. Some hydroxy metabolites, which are equiactive with their parent drugs, have been developed into ester prodrugs while carboxy metabolites, which are equiactive to their parent drugs, have been developed into drugs as per se. In this review, we present and discuss the above state of affairs for a variety of drug classes, using selected drug members to show the effect on pharmacologic activity as well as dependence of the metabolic change on drug molecular structure. The review provides a basis for informed predictions of (i) structural features required for metabolic hydroxy and carboxy functionalization of alkyl moieties in existing or planned small drug molecules, and (ii) pharmacologic activity of the metabolites resulting from hydroxy and/or carboxy functionalization of alkyl moieties.
Introduction
Nonpolar alkyl moieties are frequently incorporated into drug molecules to serve pharmacodynamic and/or pharmacokinetic purposes. Being lipophilic, alkyl moieties are metabolized in phase I via hydroxy functionalization to alcohols-a process which is, in some cases, followed by carboxy functionalization. Usually, the carboxyl and sterically unhindered hydroxyl groups in the resulting metabolites are conjugated in phase II by glucuronic acid. The chemical forms and metabolic products of the alkyl moieties surveyed in this review are summarized in Table 1. primary alcohols are further oxidized to carboxylic acids Unhindered methylene groups in alicycles and aliphatic heterocycles (usually at the farthest position from the monosubstituent)
Secondary alcohols
Benzylic methyl groups Primary alcohols, followed by oxidation to carboxyl group Methyl groups bonded to alicycles or heterocycles Primary alcohols, followed by oxidation to carboxyl groups Methylene groups alpha to both carbonyl and imino groups Secondary alcohols Carbons α to a heteroatom in a heterocycle Secondary alcohol; usually followed by carbonyl compound (aldehyde or ketone) elimination
Allylic carbons Primary alcohols in open-chain alkyls and secondary alcohols in alicycles
Generally, alkyls are found in drug molecules in their capacity as functional groups or as frameworks for (or carriers of) hydrophilic or other hydrophobic functional groups. Usually, internal linear alkyls assume the role of frameworks, while ω methyls of terminal linear or branched alkyls assume primary or auxiliary pharmacophoric roles by interacting with biological targets through van der Waals binding. In addition, internal linear alkyls may be used as spacers between functional groups for different purposes-mainly to extend the chain for one of the functional groups to reach a binding site. Further, branching of alkyl chains results in compactness: this feature will cause less disruption of the hydrogen-bonding network of water. Consequently, the lipophilicity of the drug containing the branched alkyl group will decrease, and if the drug's mechanism of action is related to its lipophilicity, then a significant alteration in the biologic effect will ensue [1].
Cycloalkyl groups encountered in drug molecules mostly extend from cyclopropyl to cyclohexyl. In drug design, cycloalkyl groups are substituted for open-chain alkyl groups to (i) better fill a hydrophobic pocket in a receptor, and (ii) introduce rigidity and limit the number of conformations a drug molecule may adopt. Both effects contribute to attaining more drug affinity and selectivity of the drug containing such groups [2][3][4]. In monosubstituted cyclohexyl groups-the most common in drug molecules-metabolic hydroxylation is stereoselective, favoring the trans isomer for its higher stability with respect to the cis isomer ( Figure 1) [5].
Mechanism of Metabolic Oxidation of Alkyl Moieties
Metabolic hydroxylation of alkyl groups is catalyzed by a family of monooxygenase enzymes, known as the "cytochrome P450" family, that contain heme redox centers. The heme group is Molecules 2020, 25, 1937 3 of 29 characterized by an iron atom coordinated to the nitrogen atoms of four linked pyrrole rings. The mechanism of metabolic hydroxylation involves free-radical formation at the substrate carbon in the alkyl moiety, as illustrated in Figure 2 [6][7][8][9][10]. In drug molecules containing more than one class of carbon atoms, the priority of metabolic hydroxylation is dictated by the stability of the intermediate free radicals; however, anomalies may occur due to prevailing electronic or steric effects in the molecule. Due to electronic effects, the stability of alkyl free radicals follows this sequence: benzyl, allyl > 3 • > 2 • > 1 • > methyl [11]. The different classes of alkyl carbons are shown in Figure 1.
Mechanism of Metabolic Oxidation of Alkyl Moieties
Metabolic hydroxylation of alkyl groups is catalyzed by a family of monooxygenase enzymes, known as the "cytochrome P450" family, that contain heme redox centers. The heme group is characterized by an iron atom coordinated to the nitrogen atoms of four linked pyrrole rings. The mechanism of metabolic hydroxylation involves free-radical formation at the substrate carbon in the alkyl moiety, as illustrated in Figure 2 [6][7][8][9][10]. In drug molecules containing more than one class of carbon atoms, the priority of metabolic hydroxylation is dictated by the stability of the intermediate free radicals; however, anomalies may occur due to prevailing electronic or steric effects in the molecule. Due to electronic effects, the stability of alkyl free radicals follows this sequence: benzyl, allyl > 3° > 2° > 1° > methyl [11]. The different classes of alkyl carbons are shown in Figure 1. Figure 2. Free-radical metabolic alkyl hydroxylation (adapted from [3,4]).
Data on Selected Groups of Drugs Containing Alkyl Moieties
The selection of drug candidates for metabolic alkyl-moiety hydroxylation and carboxyl functionalization was based on the presence of the groups in Table 1.
NSAIDS
The chemical classification of NSAIDS is given in the first part of this review series [12]. The two NSAIDS considered in this section, ibuprofen and tolmetin are of the arylalkanoic acid class.
Ibuprofen
Ibuprofen ( Figure 3) is an arylpropionic acid NSAID used in the management of arthritis as well as for its analgesic and antipyretic properties. It acts as an NSAID by inhibiting COX and consequently PGE2, which is implicated in the inflammation process [13]. Ibuprofen is a chiral drug existing in two enantiomeric forms: S-(+) and R-(−). The NSAID activity of ibuprofen has been reported to reside in the S-(+)-enantiomer [14][15][16], which is now marketed in a number of countries as dexibuprofen; however, in most countries, the drug is used as the racemate. The possible reason why large pharmaceutical companies tend to market racemic equivalent ibuprofen is that the levo enantiomer is metabolically converted in vivo to the dextro enantiomer [17]. The isobutyl group in ibuprofen contains three classes of carbon: two primary (C3, C3), one tertiary (C2), and one benzylic (C1) ( Figure 3). As depicted in Figure 4, phase I metabolic hydroxylation occurs at the three carbons to varying extents [17][18][19][20][21][22]. 3-Hydroxyibuprofen is further oxidized via the aldehyde intermediate to the carboxylic acid metabolite. The benzylic-carbon oxidation results in the formation of a chiral secondary alcohol (1-hydroxyibuprofen). Both the intrinsic and the metabolically generated carboxyl groups are further metabolized in phase II to glucuronide conjugates. All the metabolites of ibuprofen are devoid of pharmacological activity [20,23]. Free-radical metabolic alkyl hydroxylation (adapted from [3,4]).
Data on Selected Groups of Drugs Containing Alkyl Moieties
The selection of drug candidates for metabolic alkyl-moiety hydroxylation and carboxyl functionalization was based on the presence of the groups in Table 1.
NSAIDS
The chemical classification of NSAIDS is given in the first part of this review series [12]. The two NSAIDS considered in this section, ibuprofen and tolmetin are of the arylalkanoic acid class.
Ibuprofen
Ibuprofen ( Figure 3) is an arylpropionic acid NSAID used in the management of arthritis as well as for its analgesic and antipyretic properties. It acts as an NSAID by inhibiting COX and consequently PGE 2 , which is implicated in the inflammation process [13]. Ibuprofen is a chiral drug existing in two enantiomeric forms: S-(+) and R-(−). The NSAID activity of ibuprofen has been reported to reside in the S-(+)-enantiomer [14][15][16], which is now marketed in a number of countries as dexibuprofen; however, in most countries, the drug is used as the racemate. The possible reason why large pharmaceutical companies tend to market racemic equivalent ibuprofen is that the levo enantiomer is metabolically converted in vivo to the dextro enantiomer [17]. The isobutyl group in ibuprofen contains three classes of carbon: two primary (C3, C3), one tertiary (C2), and one benzylic (C1) ( Figure 3). As depicted in Figure 4, phase I metabolic hydroxylation occurs at the three carbons to varying extents [17][18][19][20][21][22]. 3-Hydroxyibuprofen is further oxidized via the aldehyde intermediate to the carboxylic acid metabolite. The benzylic-carbon oxidation results in the formation of a chiral secondary alcohol (1-hydroxyibuprofen). Both the intrinsic and the metabolically generated carboxyl groups are further metabolized in phase II to glucuronide conjugates. All the metabolites of ibuprofen are devoid of pharmacological activity [20,23].
Tolmetin
Tolmetin ( Figure 4) is a pyrrole acetic acid NSAID. It is metabolized by the hydroxylation of the benzylic methyl group to the active hydroxymethyl derivative, which is further oxidized to the inactive 5-p-carboxybenzoyl-1-methylpyrrole-2-acetic acid in rat, monkey, and human [24,25]. Both the intrinsic and metabolically produced carboxyl groups are further metabolized in phase II to the inactive glucuronide conjugates. The retention of COX-inhibiting activity by the hydroxymethyl metabolite may indicate an auxiliary pharmacophoric role of the benzylic methyl group, since the relatively large phenyl group is responsible for the primary pharmacophoric role.
Sulfonylurea Oral Antidiabetics
Sulfonylurea oral antidiabetics have the general structure shown in Figure 5, with the framed moiety representing the pharmacophore. In the first-generation sulfonylureas in Figure 5, R1 is a small lipophilic group such as methyl or chloro, while R2 is a lipophilic alkyl or cycloalkyl group, mostly cyclohexyl. In the second-generation sulfonylureas, the alkyl and cycloalkyl substituents at R2 are mostly maintained while the substituent at R1 is a large p-(β-arylcarboxyamidoethyl) group ( Figure 5). This latter group enhances antidiabetic activity through strong binding affinity to the ATP K + channel [26]. On the other hand, according to
Tolmetin
Tolmetin ( Figure 4) is a pyrrole acetic acid NSAID. It is metabolized by the hydroxylation of the benzylic methyl group to the active hydroxymethyl derivative, which is further oxidized to the inactive 5-p-carboxybenzoyl-1-methylpyrrole-2-acetic acid in rat, monkey, and human [24,25]. Both the intrinsic and metabolically produced carboxyl groups are further metabolized in phase II to the inactive glucuronide conjugates. The retention of COX-inhibiting activity by the hydroxymethyl metabolite may indicate an auxiliary pharmacophoric role of the benzylic methyl group, since the relatively large phenyl group is responsible for the primary pharmacophoric role.
Sulfonylurea Oral Antidiabetics
Sulfonylurea oral antidiabetics have the general structure shown in Figure 5, with the framed moiety representing the pharmacophore.
where R 3 is aryl or heterocycle substituent H H Figure 5. General structure of sulfonylurea antidiabetics depicting the pharmacophore.
In the first-generation sulfonylureas in Figure 5, R1 is a small lipophilic group such as methyl or chloro, while R2 is a lipophilic alkyl or cycloalkyl group, mostly cyclohexyl. In the second-generation sulfonylureas, the alkyl and cycloalkyl substituents at R2 are mostly maintained while the substituent at R1 is a large p-(β-arylcarboxyamidoethyl) group ( Figure 5). This latter group enhances antidiabetic activity through strong binding affinity to the ATP K + channel [26]. On the other hand, according to
Tolmetin
Tolmetin ( Figure 4) is a pyrrole acetic acid NSAID. It is metabolized by the hydroxylation of the benzylic methyl group to the active hydroxymethyl derivative, which is further oxidized to the inactive 5-p-carboxybenzoyl-1-methylpyrrole-2-acetic acid in rat, monkey, and human [24,25]. Both the intrinsic and metabolically produced carboxyl groups are further metabolized in phase II to the inactive glucuronide conjugates. The retention of COX-inhibiting activity by the hydroxymethyl metabolite may indicate an auxiliary pharmacophoric role of the benzylic methyl group, since the relatively large phenyl group is responsible for the primary pharmacophoric role.
Sulfonylurea Oral Antidiabetics
Sulfonylurea oral antidiabetics have the general structure shown in Figure 5, with the framed moiety representing the pharmacophore. Figure 3. Metabolism of ibuprofen.
Tolmetin
Tolmetin ( Figure 4) is a pyrrole acetic acid NSAID. It is metabolized by the hydroxylation of the benzylic methyl group to the active hydroxymethyl derivative, which is further oxidized to the inactive 5-p-carboxybenzoyl-1-methylpyrrole-2-acetic acid in rat, monkey, and human [24,25]. Both the intrinsic and metabolically produced carboxyl groups are further metabolized in phase II to the inactive glucuronide conjugates. The retention of COX-inhibiting activity by the hydroxymethyl metabolite may indicate an auxiliary pharmacophoric role of the benzylic methyl group, since the relatively large phenyl group is responsible for the primary pharmacophoric role.
Sulfonylurea Oral Antidiabetics
Sulfonylurea oral antidiabetics have the general structure shown in Figure 5, with the framed moiety representing the pharmacophore.
where R 3 is aryl or heterocycle substituent H H Figure 5. General structure of sulfonylurea antidiabetics depicting the pharmacophore.
In the first-generation sulfonylureas in Figure 5, R1 is a small lipophilic group such as methyl or chloro, while R2 is a lipophilic alkyl or cycloalkyl group, mostly cyclohexyl. In the second-generation sulfonylureas, the alkyl and cycloalkyl substituents at R2 are mostly maintained while the substituent at R1 is a large p-(β-arylcarboxyamidoethyl) group ( Figure 5). This latter group enhances antidiabetic activity through strong binding affinity to the ATP K + channel [26]. On the other hand, according to In the first-generation sulfonylureas in Figure 5, R 1 is a small lipophilic group such as methyl or chloro, while R 2 is a lipophilic alkyl or cycloalkyl group, mostly cyclohexyl. In the second-generation sulfonylureas, the alkyl and cycloalkyl substituents at R 2 are mostly maintained while the substituent Molecules 2020, 25, 1937 5 of 29 at R 1 is a large p-(β-arylcarboxyamidoethyl) group ( Figure 5). This latter group enhances antidiabetic activity through strong binding affinity to the ATP K + channel [26]. On the other hand, according to Foye (2020) [26], the small lipophilic groups at R 1 in the first-generation sulfonylureas have little influence over activity. Hence, they may have been included to play auxiliary pharmacophoric or auxophoric roles. Nevertheless, only methyl groups at R 1 are within the scope of this review.
Mechanistically, the sulfonylurea antidiabetics act by binding to the specific receptor for sulfonylureas on β-pancreatic cells, blocking the inflow of potassium (K + ) through the ATP-dependent channel. The flow of K + within the β-cell goes to zero; the cell membrane becomes depolarized, thus removing the electric screen, which prevents the diffusion of calcium into the cytosol. The increased flow of calcium into β-cells causes contraction in the filaments of actomyosin responsible for the exocytosis of insulin, which is therefore promptly secreted in large amounts [28].
Acetohexamide
Acetohexamide ( Figure 6) is metabolized by (i) reduction of the carbonyl group to give a hydroxy metabolite that is 2.5 times as active as the parent drug, as well as (ii) stereoselective oxidation of the cyclohexyl ring to trans-4 -hydroxyacetohexamide, which is inactive as an oral antidiabetic [29,30]. Foye (2020) [26], the small lipophilic groups at R1 in the first-generation sulfonylureas have little influence over activity. Hence, they may have been included to play auxiliary pharmacophoric or auxophoric roles. Nevertheless, only methyl groups at R1 are within the scope of this review. The first-generation sulfonylurea oral antidiabetics surveyed in this review include acetohexamide, tolbutamide, chlorpropamide, and tolazamide, while the second-generation members include glyburide (glibenclamide), glimepiride, and glipizide [27].
Mechanistically, the sulfonylurea antidiabetics act by binding to the specific receptor for sulfonylureas on β-pancreatic cells, blocking the inflow of potassium (K + ) through the ATPdependent channel. The flow of K + within the β-cell goes to zero; the cell membrane becomes depolarized, thus removing the electric screen, which prevents the diffusion of calcium into the cytosol. The increased flow of calcium into β-cells causes contraction in the filaments of actomyosin responsible for the exocytosis of insulin, which is therefore promptly secreted in large amounts [28].
Acetohexamide
Acetohexamide ( Figure 6) is metabolized by (i) reduction of the carbonyl group to give a hydroxy metabolite that is 2.5 times as active as the parent drug, as well as (ii) stereoselective oxidation of the cyclohexyl ring to trans-4′-hydroxyacetohexamide, which is inactive as an oral antidiabetic [29,30]. Figure 6. Metabolic pathways of acetohexamide.
Tolbutamide
Tolbutamide ( Figure 7) is primarily metabolized by benzylic methyl oxidation to the primary alcoholic hydroxymethyl group, which is further oxidized to the carboxyl group ( Figure 7) [31,32]. While the hydroxymethyl metabolite is equiactive with tolbutamide, the carboxy metabolite is inactive [31]. A minor route of tolbutamide metabolism occurs via butyl chain oxidation at the ω and ω-1 carbons to give primary and secondary alcohol metabolites, respectively, which have minimal antidiabetic activity (Figure 7). An inference can be made from the ratio of the hydroxy metabolites of tolbutamide: when a benzylic methyl group and an alkyl chain are present in the same drug molecule, the preference of metabolic oxidation is for the benzylic methyl group. Substantiation of the inference is given by the higher stability of the benzylic free radical involved in the oxidation of the benzylic methyl group compared to the alkyl-chain free radicals. The stability of the benzylic free radical is due to resonance stabilization [33], as depicted in Figure 8. The metabolic oxidation of the benzylic methyl group occurs as per the mechanism of alkyl hydroxylation shown in Figure 2.
Tolbutamide
Tolbutamide ( Figure 7) is primarily metabolized by benzylic methyl oxidation to the primary alcoholic hydroxymethyl group, which is further oxidized to the carboxyl group ( Figure 7) [31,32]. While the hydroxymethyl metabolite is equiactive with tolbutamide, the carboxy metabolite is inactive [31]. A minor route of tolbutamide metabolism occurs via butyl chain oxidation at the ω and ω-1 carbons to give primary and secondary alcohol metabolites, respectively, which have minimal antidiabetic activity (Figure 7). An inference can be made from the ratio of the hydroxy metabolites of tolbutamide: when a benzylic methyl group and an alkyl chain are present in the same drug molecule, the preference of metabolic oxidation is for the benzylic methyl group. Substantiation of the inference is given by the higher stability of the benzylic free radical involved in the oxidation of the benzylic methyl group compared to the alkyl-chain free radicals. The stability of the benzylic free radical is due to resonance stabilization [33], as depicted in Figure 8. The metabolic oxidation of the benzylic methyl group occurs as per the mechanism of alkyl hydroxylation shown in Figure 2. Molecules 2020, 25,
Chlorpropamide
Chlorpropamide ( Figure 9) has been developed as a variant of tolbutamide in order to prolong the drug's antidiabetic effect with the consequent enhancement of potency. Chlorpropamide is slowly metabolized by alkyl-chain oxidation at the ω and ω-1 carbons to give primary and secondary alcohols, respectively ( Figure 9) [34]. Both metabolites have minimal antidiabetic activity [20]. It should be noted that in chlorpropamide metabolism, the secondary alcohol (55% of dose) predominates over the primary alcohol (2%) [34] (Figure 9). A possible explanation of this finding resides in the higher stability of the intermediate secondary-propyl free radical compared to the primary-propyl free radical. In the absence of corresponding data on metabolite concentrations, the analogy could be extended to the butyl group in tolbutamide (Section 2.2.2).
Tolazamide
Tolazamide ( Figure 10) contains an azepane ring bonded to the terminal sulfonylurea nitrogen and a methyl group bonded to the aromatic ring. The metabolism of tolazamide is depicted in Figure 12. The azepane ring is oxidized at position 4′ to the 4′ hydroxy group, while the benzylic methyl group is oxidized sequentially to the hydroxymethyl and carboxyl groups [35]. It is noteworthy that the concentrations of the two alcoholic metabolites are almost equal, which leads to the inference that the benzyl and azepanyl free radicals are almost of equal stability. However, as far as activity is concerned, the hydroxymethyl metabolite is equiactive with the parent drug, while the azepanyl alcohol metabolite is only weakly active. Furthermore, the carboxy metabolite resulting from the hydroxymethyl group oxidation is inactive.
Chlorpropamide
Chlorpropamide ( Figure 9) has been developed as a variant of tolbutamide in order to prolong the drug's antidiabetic effect with the consequent enhancement of potency. Chlorpropamide is slowly metabolized by alkyl-chain oxidation at the ω and ω-1 carbons to give primary and secondary alcohols, respectively ( Figure 9) [34]. Both metabolites have minimal antidiabetic activity [20]. It should be noted that in chlorpropamide metabolism, the secondary alcohol (55% of dose) predominates over the primary alcohol (2%) [34] (Figure 9). A possible explanation of this finding resides in the higher stability of the intermediate secondary-propyl free radical compared to the primary-propyl free radical. In the absence of corresponding data on metabolite concentrations, the analogy could be extended to the butyl group in tolbutamide (Section 2.2.2).
Tolazamide
Tolazamide ( Figure 10) contains an azepane ring bonded to the terminal sulfonylurea nitrogen and a methyl group bonded to the aromatic ring. The metabolism of tolazamide is depicted in Figure 12. The azepane ring is oxidized at position 4′ to the 4′ hydroxy group, while the benzylic methyl group is oxidized sequentially to the hydroxymethyl and carboxyl groups [35]. It is noteworthy that the concentrations of the two alcoholic metabolites are almost equal, which leads to the inference that the benzyl and azepanyl free radicals are almost of equal stability. However, as far as activity is concerned, the hydroxymethyl metabolite is equiactive with the parent drug, while the azepanyl alcohol metabolite is only weakly active. Furthermore, the carboxy metabolite resulting from the hydroxymethyl group oxidation is inactive.
Chlorpropamide
Chlorpropamide ( Figure 9) has been developed as a variant of tolbutamide in order to prolong the drug's antidiabetic effect with the consequent enhancement of potency. Chlorpropamide is slowly metabolized by alkyl-chain oxidation at the ω and ω-1 carbons to give primary and secondary alcohols, respectively ( Figure 9) [34]. Both metabolites have minimal antidiabetic activity [20]. It should be noted that in chlorpropamide metabolism, the secondary alcohol (55% of dose) predominates over the primary alcohol (2%) [34] (Figure 9). A possible explanation of this finding resides in the higher stability of the intermediate secondary-propyl free radical compared to the primary-propyl free radical. In the absence of corresponding data on metabolite concentrations, the analogy could be extended to the butyl group in tolbutamide (Section 2.2.2).
Chlorpropamide
Chlorpropamide ( Figure 9) has been developed as a variant of tolbutamide in order to prolong the drug's antidiabetic effect with the consequent enhancement of potency. Chlorpropamide is slowly metabolized by alkyl-chain oxidation at the ω and ω-1 carbons to give primary and secondary alcohols, respectively ( Figure 9) [34]. Both metabolites have minimal antidiabetic activity [20]. It should be noted that in chlorpropamide metabolism, the secondary alcohol (55% of dose) predominates over the primary alcohol (2%) [34] (Figure 9). A possible explanation of this finding resides in the higher stability of the intermediate secondary-propyl free radical compared to the primary-propyl free radical. In the absence of corresponding data on metabolite concentrations, the analogy could be extended to the butyl group in tolbutamide (Section 2.2.2).
Tolazamide
Tolazamide ( Figure 10) contains an azepane ring bonded to the terminal sulfonylurea nitrogen and a methyl group bonded to the aromatic ring. The metabolism of tolazamide is depicted in Figure 12. The azepane ring is oxidized at position 4′ to the 4′ hydroxy group, while the benzylic methyl group is oxidized sequentially to the hydroxymethyl and carboxyl groups [35]. It is noteworthy that the concentrations of the two alcoholic metabolites are almost equal, which leads to the inference that the benzyl and azepanyl free radicals are almost of equal stability. However, as far as activity is concerned, the hydroxymethyl metabolite is equiactive with the parent drug, while the azepanyl alcohol metabolite is only weakly active. Furthermore, the carboxy metabolite resulting from the hydroxymethyl group oxidation is inactive.
Tolazamide
Tolazamide ( Figure 10) contains an azepane ring bonded to the terminal sulfonylurea nitrogen and a methyl group bonded to the aromatic ring. The metabolism of tolazamide is depicted in Figure 12. The azepane ring is oxidized at position 4 to the 4 hydroxy group, while the benzylic methyl group is oxidized sequentially to the hydroxymethyl and carboxyl groups [35]. It is noteworthy that the concentrations of the two alcoholic metabolites are almost equal, which leads to the inference that the benzyl and azepanyl free radicals are almost of equal stability. However, as far as activity is concerned, the hydroxymethyl metabolite is equiactive with the parent drug, while the azepanyl alcohol metabolite is only weakly active. Furthermore, the carboxy metabolite resulting from the hydroxymethyl group oxidation is inactive. (equiactive) Figure 10. Metabolic pathways of tolazamide.
Glibenclamide
Glibenclamide (also known as glyburide) ( Figure 11) is a second-generation sulfonylurea oral antidiabetic. It contains a cyclohexyl group bonded to the terminal nitrogen of the sulfonylurea moiety. The cyclohexyl ring forms the main site of metabolism of the drug; it is stereoselectively hydroxylated to 3-cis and 4-trans isomers ( Figure 11) with the latter isomer being the major metabolite [36,37]. The two metabolites have little hypoglycemic effect compared to the parent drug. However, retention of 4-trans-hydroxyglyburide may prolong the hypoglycemic effect of the agent in those with severe renal impairment [37].
Glimepiride
The cyclohexylmethyl group in glimepiride ( Figure 12) allows the drug to exist in cis and trans isomeric forms; the active antidiabetic form is the trans isomer. The latter is metabolized, as shown in Figure 12, through the sequential oxidation of the cyclohexylmethyl group to the hydroxymethyl and carboxy metabolites [38,39]. The hydroxymethyl metabolite is an active antidiabetic in animal models, while the carboxyl metabolite is inactive [39].
Glibenclamide
Glibenclamide (also known as glyburide) ( Figure 11) is a second-generation sulfonylurea oral antidiabetic. It contains a cyclohexyl group bonded to the terminal nitrogen of the sulfonylurea moiety. The cyclohexyl ring forms the main site of metabolism of the drug; it is stereoselectively hydroxylated to 3-cis and 4-trans isomers ( Figure 11) with the latter isomer being the major metabolite [36,37]. The two metabolites have little hypoglycemic effect compared to the parent drug. However, retention of 4-trans-hydroxyglyburide may prolong the hypoglycemic effect of the agent in those with severe renal impairment [37]. (equiactive) Figure 10. Metabolic pathways of tolazamide.
Glibenclamide
Glibenclamide (also known as glyburide) ( Figure 11) is a second-generation sulfonylurea oral antidiabetic. It contains a cyclohexyl group bonded to the terminal nitrogen of the sulfonylurea moiety. The cyclohexyl ring forms the main site of metabolism of the drug; it is stereoselectively hydroxylated to 3-cis and 4-trans isomers ( Figure 11) with the latter isomer being the major metabolite [36,37]. The two metabolites have little hypoglycemic effect compared to the parent drug. However, retention of 4-trans-hydroxyglyburide may prolong the hypoglycemic effect of the agent in those with severe renal impairment [37].
Glimepiride
The cyclohexylmethyl group in glimepiride ( Figure 12) allows the drug to exist in cis and trans isomeric forms; the active antidiabetic form is the trans isomer. The latter is metabolized, as shown in Figure 12, through the sequential oxidation of the cyclohexylmethyl group to the hydroxymethyl and carboxy metabolites [38,39]. The hydroxymethyl metabolite is an active antidiabetic in animal models, while the carboxyl metabolite is inactive [39].
Glimepiride
The cyclohexylmethyl group in glimepiride ( Figure 12) allows the drug to exist in cis and trans isomeric forms; the active antidiabetic form is the trans isomer. The latter is metabolized, as shown in Figure 12, through the sequential oxidation of the cyclohexylmethyl group to the hydroxymethyl and carboxy metabolites [38,39]. The hydroxymethyl metabolite is an active antidiabetic in animal models, while the carboxyl metabolite is inactive [39].
Glibenclamide
Glibenclamide (also known as glyburide) ( Figure 11) is a second-generation sulfonylurea oral antidiabetic. It contains a cyclohexyl group bonded to the terminal nitrogen of the sulfonylurea moiety. The cyclohexyl ring forms the main site of metabolism of the drug; it is stereoselectively hydroxylated to 3-cis and 4-trans isomers ( Figure 11) with the latter isomer being the major metabolite [36,37]. The two metabolites have little hypoglycemic effect compared to the parent drug. However, retention of 4-trans-hydroxyglyburide may prolong the hypoglycemic effect of the agent in those with severe renal impairment [37].
Glimepiride
The cyclohexylmethyl group in glimepiride ( Figure 12) allows the drug to exist in cis and trans isomeric forms; the active antidiabetic form is the trans isomer. The latter is metabolized, as shown in Figure 12, through the sequential oxidation of the cyclohexylmethyl group to the hydroxymethyl and carboxy metabolites [38,39]. The hydroxymethyl metabolite is an active antidiabetic in animal models, while the carboxyl metabolite is inactive [39]. Glipizide ( Figure 13) is a second-generation sulfonylurea with R 2 in Figure 6 as cyclohexyl substituent. It is metabolized by stereoselective hydroxylation to 4-trans and 4-cis-hydroxyglipizide [40]. No data are available on the activity of the hydroxy metabolites of glipizide.
Glipizide
Glipizide ( Figure 13) is a second-generation sulfonylurea with R2 in Figure 6 as cyclohexyl substituent. It is metabolized by stereoselective hydroxylation to 4-trans and 4-cis-hydroxyglipizide [40]. No data are available on the activity of the hydroxy metabolites of glipizide.
Barbiturates
Barbiturates are CNS depressants used as sedatives and hypnotics, anesthetics, and anti-seizure drugs. Barbiturates' primary mechanism of action is inhibition of the central nervous system (CNS). The CNS depression is brought about by stimulating the inhibitory neurotransmitter system in the brain called the gamma-aminobutyric acid (GABA) system. The GABA channel is a chloride channel that has five subunits at its gate. When barbiturates bind to the GABA channel, they cause the chloride ion channel to open, which allows chloride ions into the cells in the brain. The entry of the chloride ions into the brain leads to increased negative charge and alteration of the voltage across the brain cells. This change in voltage makes the brain cells resistant to nerve impulses, thus depressing them [41].
Most barbiturates contain alkyl groups of varying lengths. Being lipophilic, these alkyl groups are functionalized by metabolic hydroxylation at different positions. The primary alcohols resulting from oxidation of ω carbons are usually further metabolized to carboxylic acids. Amobarbital and pentobarbital ( Figures 14 and 15, respectively) are taken as representative examples of barbiturates that contain alkyl groups. Amobarbital has ethyl and amyl groups bonded to C5 of barbituric acid, while pentobarbital has ethyl and 2-methylbutyl groups bonded to C5 of barbituric acid ( Figures 14 and 15). Amobarbital is metabolized by 3′-hydoxylation to give 3′-hydroxyamobarbital as nearly the sole metabolite ( Figure 14) [42,43]. On the other hand, the 2-methylbutyl group in pentobarbital undergoes ω and ω-1 metabolic oxidation to give primary-and secondary-alcohol metabolites, respectively ( Figure 15) [44]. The primary alcohol metabolite of pentobarbital is further oxidized to the carboxylic acid. The three metabolites of pentobarbital (the two alcoholic metabolites and the carboxyl metabolite) are further conjugated by glucuronic acid in phase II ( Figure 15) [44]. The alcoholic and the carboxylic acid metabolites of pentobarbital, and their glucuronide conjugates, are inactive as sedative-hypnotics [44]. In contrast to the alcoholic metabolites of pentobarbital, 3′hydroxyamobarbital has not been reported to undergo glucuronide conjugation-possibly because, being a tertiary alcohol, it is sterically hindered from such a metabolic pathway. In addition to metabolism, redistribution of barbiturates has been reported to play an important role in their deactivation [45]. Redistribution of the lipophilic barbiturates from the brain to other body compartments, such as adipose tissue, will lead to a reduction of their effective concentration at the receptors in the brain, thus leading to a loss of sedative-hypnotic activity. On the other hand, amobarbital is rapidly metabolized; however, its extended activity has been attributed to the 3′hydroxy metabolite, which is present in diminished concentration but is, nevertheless, longer acting than the parent drug [43].
Barbiturates
Barbiturates are CNS depressants used as sedatives and hypnotics, anesthetics, and anti-seizure drugs. Barbiturates' primary mechanism of action is inhibition of the central nervous system (CNS). The CNS depression is brought about by stimulating the inhibitory neurotransmitter system in the brain called the gamma-aminobutyric acid (GABA) system. The GABA channel is a chloride channel that has five subunits at its gate. When barbiturates bind to the GABA channel, they cause the chloride ion channel to open, which allows chloride ions into the cells in the brain. The entry of the chloride ions into the brain leads to increased negative charge and alteration of the voltage across the brain cells. This change in voltage makes the brain cells resistant to nerve impulses, thus depressing them [41].
Most barbiturates contain alkyl groups of varying lengths. Being lipophilic, these alkyl groups are functionalized by metabolic hydroxylation at different positions. The primary alcohols resulting from oxidation of ω carbons are usually further metabolized to carboxylic acids. Amobarbital and pentobarbital ( Figures 14 and 15, respectively) are taken as representative examples of barbiturates that contain alkyl groups. Amobarbital has ethyl and amyl groups bonded to C5 of barbituric acid, while pentobarbital has ethyl and 2-methylbutyl groups bonded to C5 of barbituric acid ( Figures 14 and 15). Amobarbital is metabolized by 3 -hydoxylation to give 3 -hydroxyamobarbital as nearly the sole metabolite ( Figure 14) [42,43]. On the other hand, the 2-methylbutyl group in pentobarbital undergoes ω and ω-1 metabolic oxidation to give primary-and secondary-alcohol metabolites, respectively ( Figure 15) [44]. The primary alcohol metabolite of pentobarbital is further oxidized to the carboxylic acid. The three metabolites of pentobarbital (the two alcoholic metabolites and the carboxyl metabolite) are further conjugated by glucuronic acid in phase II ( Figure 15) [44]. The alcoholic and the carboxylic acid metabolites of pentobarbital, and their glucuronide conjugates, are inactive as sedative-hypnotics [44]. In contrast to the alcoholic metabolites of pentobarbital, 3 -hydroxyamobarbital has not been reported to undergo glucuronide conjugation-possibly because, being a tertiary alcohol, it is sterically hindered from such a metabolic pathway. In addition to metabolism, redistribution of barbiturates has been reported to play an important role in their deactivation [45]. Redistribution of the lipophilic barbiturates from the brain to other body compartments, such as adipose tissue, will lead to a reduction of their effective concentration at the receptors in the brain, thus leading to a loss of sedative-hypnotic activity. On the other hand, amobarbital is rapidly metabolized; however, its extended activity has been attributed to the 3 -hydroxy metabolite, which is present in diminished concentration but is, nevertheless, longer acting than the parent drug [43].
Valproic Acid
Valproic acid ( Figure 16) is an anticonvulsant drug used in the treatment of epilepsy. Its mechanism of action involves the blockage of voltage-gated sodium channels and increased brain levels of gamma-aminobutyric acid (GABA) [46]. Valproic acid is mainly metabolized by oxidation to alkene and hydroxy products [47,48]. The two major hydroxy metabolites are the 4-and 5-isomers. The primary alcoholic metabolite (i.e., 5-hydroxyvalproic acid) is further oxidized to the carboxylic acid to give 2-n-propylglutaric acid ( Figure 16). The substantial reduction in anticonvulsant activities of the valproic acid hydroxy and carboxy metabolites has been attributed to their increased molecular size and surface, steric effects, and reduced log P, all of which are features that lower the extent of blood-brain barrier crossing [47,48].
Risperidone/Paliperidone
Risperidone ( Figure 17) blocks the formation of serotonin and dopamine, thus decreasing psychotic and aggressive behavior. By targeting serotonin 5HT2A and D2 receptors, risperidone is considered an atypical antipsychotic drug and is used in the treatment of schizophrenia. In addition, it is used off-label in the treatment of ADHD in children. The metabolism of risperidone is stereoselectively catalyzed (i) by CYP2D6 at the aliphatic heterocycle to give the major enantiomer
Valproic Acid
Valproic acid ( Figure 16) is an anticonvulsant drug used in the treatment of epilepsy. Its mechanism of action involves the blockage of voltage-gated sodium channels and increased brain levels of gamma-aminobutyric acid (GABA) [46]. Valproic acid is mainly metabolized by oxidation to alkene and hydroxy products [47,48]. The two major hydroxy metabolites are the 4-and 5-isomers. The primary alcoholic metabolite (i.e., 5-hydroxyvalproic acid) is further oxidized to the carboxylic acid to give 2-n-propylglutaric acid ( Figure 16). The substantial reduction in anticonvulsant activities of the valproic acid hydroxy and carboxy metabolites has been attributed to their increased molecular size and surface, steric effects, and reduced log P, all of which are features that lower the extent of blood-brain barrier crossing [47,48].
Risperidone/Paliperidone
Risperidone ( Figure 17) blocks the formation of serotonin and dopamine, thus decreasing psychotic and aggressive behavior. By targeting serotonin 5HT2A and D2 receptors, risperidone is considered an atypical antipsychotic drug and is used in the treatment of schizophrenia. In addition, it is used off-label in the treatment of ADHD in children. The metabolism of risperidone is stereoselectively catalyzed (i) by CYP2D6 at the aliphatic heterocycle to give the major enantiomer
Valproic Acid
Valproic acid ( Figure 16) is an anticonvulsant drug used in the treatment of epilepsy. Its mechanism of action involves the blockage of voltage-gated sodium channels and increased brain levels of gamma-aminobutyric acid (GABA) [46]. Valproic acid is mainly metabolized by oxidation to alkene and hydroxy products [47,48]. The two major hydroxy metabolites are the 4-and 5-isomers. The primary alcoholic metabolite (i.e., 5-hydroxyvalproic acid) is further oxidized to the carboxylic acid to give 2-n-propylglutaric acid ( Figure 16). The substantial reduction in anticonvulsant activities of the valproic acid hydroxy and carboxy metabolites has been attributed to their increased molecular size and surface, steric effects, and reduced log P, all of which are features that lower the extent of blood-brain barrier crossing [47,48].
Valproic Acid
Valproic acid ( Figure 16) is an anticonvulsant drug used in the treatment of epilepsy. Its mechanism of action involves the blockage of voltage-gated sodium channels and increased brain levels of gamma-aminobutyric acid (GABA) [46]. Valproic acid is mainly metabolized by oxidation to alkene and hydroxy products [47,48]. The two major hydroxy metabolites are the 4-and 5-isomers. The primary alcoholic metabolite (i.e., 5-hydroxyvalproic acid) is further oxidized to the carboxylic acid to give 2-n-propylglutaric acid ( Figure 16). The substantial reduction in anticonvulsant activities of the valproic acid hydroxy and carboxy metabolites has been attributed to their increased molecular size and surface, steric effects, and reduced log P, all of which are features that lower the extent of blood-brain barrier crossing [47,48].
Risperidone/Paliperidone
Risperidone ( Figure 17) blocks the formation of serotonin and dopamine, thus decreasing psychotic and aggressive behavior. By targeting serotonin 5HT2A and D2 receptors, risperidone is considered an atypical antipsychotic drug and is used in the treatment of schizophrenia. In addition, it is used off-label in the treatment of ADHD in children. The metabolism of risperidone is stereoselectively catalyzed (i) by CYP2D6 at the aliphatic heterocycle to give the major enantiomer
Risperidone/Paliperidone
Risperidone ( Figure 17) blocks the formation of serotonin and dopamine, thus decreasing psychotic and aggressive behavior. By targeting serotonin 5HT2A and D2 receptors, risperidone is considered an atypical antipsychotic drug and is used in the treatment of schizophrenia. In addition, it is used off-label in the treatment of ADHD in children. The metabolism of risperidone is stereoselectively catalyzed (i) by CYP2D6 at the aliphatic heterocycle to give the major enantiomer (+)-9-hydroxyrisperidone, and (ii) by CYP3A4 to (−)-9-hydroxyrisperidone ( Figure 17) [49][50][51][52][53]. Both enantiomers are equiactive with risperidone and have been developed into the racemate antipsychotic drug paliperidone [49][50][51][52][53]. Almo and Lopez-Mufioz (2013) [51] have reviewed the clinical use of both risperidone and paliperidone, stressing the pharmacokinetic and pharmacodynamic bases on which the metabolite drug has been developed. Further, the metabolically formed hydroxy group in paliperidone has been esterified with palmitic acid to give paliperidone palmitate. Paliperidone palmitate ( Figure 17) is a depo-long-acting injectable prodrug formulation indicated for a single dose to be given once monthly [53]. The active drug is released in the blood by esterase hydrolysis.
Paliperidone palmitate is an example of a prodrug that has been developed from a metabolite drug; hence, it can be described as a metabolite prodrug. Other metabolite prodrugs will be presented and discussed in due course.
Molecules 2020, 25, x FOR PEER REVIEW 10 of 29 (+)-9-hydroxyrisperidone, and (ii) by CYP3A4 to (−)-9-hydroxyrisperidone ( Figure 17) [49][50][51][52][53]. Both enantiomers are equiactive with risperidone and have been developed into the racemate antipsychotic drug paliperidone [49][50][51][52][53]. Almo and Lopez-Mufioz (2013) [51] have reviewed the clinical use of both risperidone and paliperidone, stressing the pharmacokinetic and pharmacodynamic bases on which the metabolite drug has been developed. Further, the metabolically formed hydroxy group in paliperidone has been esterified with palmitic acid to give paliperidone palmitate. Paliperidone palmitate ( Figure 17) is a depo-long-acting injectable prodrug formulation indicated for a single dose to be given once monthly [53]. The active drug is released in the blood by esterase hydrolysis. Paliperidone palmitate is an example of a prodrug that has been developed from a metabolite drug; hence, it can be described as a metabolite prodrug. Other metabolite prodrugs will be presented and discussed in due course.
Bupropion
Bupropion ( Figure 18) is an atypical antidepressant drug used to treat major depressive disorder (MDD) and seasonal affective disorder; it is also used off-label as a smoking cessation aid. Mechanistically, bupropion enhances both noradrenergic and dopaminergic neurotransmission via reuptake inhibition of the norepinephrine and dopamine transporters. In addition, its mechanism of action may involve the presynaptic release of norepinephrine and dopamine. The major active metabolite of bupropion is hydroxybupropion ( Figure 18) [54,55]. The groups in this metabolite are positioned in such a way as to allow for the occurrence of cyclization, thus preventing further oxidation of the hydroxymethyl group to the carboxyl group and the consequent loss of activity. The cyclic metabolite is an active antidepressant [55].
Bupropion
Bupropion ( Figure 18) is an atypical antidepressant drug used to treat major depressive disorder (MDD) and seasonal affective disorder; it is also used off-label as a smoking cessation aid. Mechanistically, bupropion enhances both noradrenergic and dopaminergic neurotransmission via reuptake inhibition of the norepinephrine and dopamine transporters. In addition, its mechanism of action may involve the presynaptic release of norepinephrine and dopamine. The major active metabolite of bupropion is hydroxybupropion ( Figure 18) [54,55]. The groups in this metabolite are positioned in such a way as to allow for the occurrence of cyclization, thus preventing further oxidation of the hydroxymethyl group to the carboxyl group and the consequent loss of activity. The cyclic metabolite is an active antidepressant [55].
Molecules 2020, 25, x FOR PEER REVIEW 10 of 29 (+)-9-hydroxyrisperidone, and (ii) by CYP3A4 to (−)-9-hydroxyrisperidone ( Figure 17) [49][50][51][52][53]. Both enantiomers are equiactive with risperidone and have been developed into the racemate antipsychotic drug paliperidone [49][50][51][52][53]. Almo and Lopez-Mufioz (2013) [51] have reviewed the clinical use of both risperidone and paliperidone, stressing the pharmacokinetic and pharmacodynamic bases on which the metabolite drug has been developed. Further, the metabolically formed hydroxy group in paliperidone has been esterified with palmitic acid to give paliperidone palmitate. Paliperidone palmitate ( Figure 17) is a depo-long-acting injectable prodrug formulation indicated for a single dose to be given once monthly [53]. The active drug is released in the blood by esterase hydrolysis. Paliperidone palmitate is an example of a prodrug that has been developed from a metabolite drug; hence, it can be described as a metabolite prodrug. Other metabolite prodrugs will be presented and discussed in due course.
Bupropion
Bupropion ( Figure 18) is an atypical antidepressant drug used to treat major depressive disorder (MDD) and seasonal affective disorder; it is also used off-label as a smoking cessation aid. Mechanistically, bupropion enhances both noradrenergic and dopaminergic neurotransmission via reuptake inhibition of the norepinephrine and dopamine transporters. In addition, its mechanism of action may involve the presynaptic release of norepinephrine and dopamine. The major active metabolite of bupropion is hydroxybupropion ( Figure 18) [54,55]. The groups in this metabolite are positioned in such a way as to allow for the occurrence of cyclization, thus preventing further oxidation of the hydroxymethyl group to the carboxyl group and the consequent loss of activity. The cyclic metabolite is an active antidepressant [55].
∆ 9 -Tetrahydrocannabinol
∆ 9 -Tetrahydrocannabinol (∆ 9 -THC, Figure 19) is the psychoactive hallucinogenic constituent in Cannabis sativa (hashish and marijuana). It contains three allylic carbons at positions 11, 8, and 10a ( Figure 19). The allylic positions at C11and C8 are metabolically hydroxylated, with the former hydroxylation resulting in the major equiactive hydroxymethyl metabolite; due to steric hindrance, position 10a is not hydroxylated. The C11 hydroxymethyl metabolite is further metabolically oxidized to the inactive 11-carboxy-∆ 9 -THC metabolite ( Figure 19) [56,57]. The resonance stabilization of the allyl free radical in ∆ 9 -THC that accounts for the formation of the major allylic hydroxy metabolite is depicted in Figure 20.
Molecules 2020, 25, x FOR PEER REVIEW 11 of 29 Δ 9 -Tetrahydrocannabinol (Δ 9 -THC, Figure 19) is the psychoactive hallucinogenic constituent in Cannabis sativa (hashish and marijuana). It contains three allylic carbons at positions 11, 8, and 10a ( Figure 19). The allylic positions at C11and C8 are metabolically hydroxylated, with the former hydroxylation resulting in the major equiactive hydroxymethyl metabolite; due to steric hindrance, position 10a is not hydroxylated. The C11 hydroxymethyl metabolite is further metabolically oxidized to the inactive 11-carboxy-Δ 9 -THC metabolite ( Figure 19) [56,57]. The resonance stabilization of the allyl free radical in Δ 9 -THC that accounts for the formation of the major allylic hydroxy metabolite is depicted in Figure 20.
Tolterodine/Fesoterodine
Tolterodine ( Figure 21) is an antimuscarinic drug used in the treatment of overactive bladder (OAB). As shown in Figure 21, tolterodine is metabolized (i) through mono-deisopropylation to give an inactive metabolite, and (ii) through benzylic-methyl group oxidation to give 5-hydroxymethyl tolterodine (5-HMT), which is equiactive with the parent drug [58][59][60][61]. Despite being equiactive to its parent drug, 5-HMT did not qualify for the status of metabolite drug because of its low log P value of 0.73 and the associated poor bioavailability [58]. However, the problem was resolved by esterifying the aromatic hydroxy (phenolic) group with isobutanoic acid to produce the prodrug fesoterodine, which has a log D7.4 value of 5.7 [58] and hence enjoys a substantial improvement in bioavailability.
Fesoterodine is the second example of parent-drug equiactive metabolites to have been developed into metabolite prodrug. The first example from this class of prodrugs is paliperidone palmitate, presented and discussed in Section 2.4.2. Further discussion of metabolite drugs and prodrugs will be given in Section 3. Δ 9 -Tetrahydrocannabinol (Δ 9 -THC, Figure 19) is the psychoactive hallucinogenic constituent in Cannabis sativa (hashish and marijuana). It contains three allylic carbons at positions 11, 8, and 10a ( Figure 19). The allylic positions at C11and C8 are metabolically hydroxylated, with the former hydroxylation resulting in the major equiactive hydroxymethyl metabolite; due to steric hindrance, position 10a is not hydroxylated. The C11 hydroxymethyl metabolite is further metabolically oxidized to the inactive 11-carboxy-Δ 9 -THC metabolite ( Figure 19) [56,57]. The resonance stabilization of the allyl free radical in Δ 9 -THC that accounts for the formation of the major allylic hydroxy metabolite is depicted in Figure 20.
Tolterodine/Fesoterodine
Tolterodine ( Figure 21) is an antimuscarinic drug used in the treatment of overactive bladder (OAB). As shown in Figure 21, tolterodine is metabolized (i) through mono-deisopropylation to give an inactive metabolite, and (ii) through benzylic-methyl group oxidation to give 5-hydroxymethyl tolterodine (5-HMT), which is equiactive with the parent drug [58][59][60][61]. Despite being equiactive to its parent drug, 5-HMT did not qualify for the status of metabolite drug because of its low log P value of 0.73 and the associated poor bioavailability [58]. However, the problem was resolved by esterifying the aromatic hydroxy (phenolic) group with isobutanoic acid to produce the prodrug fesoterodine, which has a log D7.4 value of 5.7 [58] and hence enjoys a substantial improvement in bioavailability.
Fesoterodine is the second example of parent-drug equiactive metabolites to have been developed into metabolite prodrug. The first example from this class of prodrugs is paliperidone palmitate, presented and discussed in Section 2.4.2. Further discussion of metabolite drugs and prodrugs will be given in Section 3.
Tolterodine/Fesoterodine
Tolterodine ( Figure 21) is an antimuscarinic drug used in the treatment of overactive bladder (OAB). As shown in Figure 21, tolterodine is metabolized (i) through mono-deisopropylation to give an inactive metabolite, and (ii) through benzylic-methyl group oxidation to give 5-hydroxymethyl tolterodine (5-HMT), which is equiactive with the parent drug [58][59][60][61]. Despite being equiactive to its parent drug, 5-HMT did not qualify for the status of metabolite drug because of its low log P value of 0.73 and the associated poor bioavailability [58]. However, the problem was resolved by esterifying the aromatic hydroxy (phenolic) group with isobutanoic acid to produce the prodrug fesoterodine, which has a log D 7.4 value of 5.7 [58] and hence enjoys a substantial improvement in bioavailability.
Fesoterodine is the second example of parent-drug equiactive metabolites to have been developed into metabolite prodrug. The first example from this class of prodrugs is paliperidone palmitate, presented and discussed in Section 2.4.2. Further discussion of metabolite drugs and prodrugs will be given in Section 3. 2.4.6. Terfenadine/Fexofenadine Terfenadine ( Figure 22) is a second generation H1-antihistamine free of the sedative side effect associated with the first-generation H1-antihistamines. Terfenadine is almost completely metabolized by benzylic-methyl-group oxidation to an equiactive carboxy metabolite, as shown in Figure 22 [62], and it is thus considered a prodrug. However, despite this advantage, terfenadine was withdrawn from clinical use because of its cardiotoxic effect [63]. In the interim, its carboxy metabolite, being free of cardiotoxicity, was developed into a drug of its own right under the name of fexofenadine. As shown in Figure 24, fexofenadine is amphoteric and thus is capable of existing as a zwitterion at physiologic pH [64]. The existence of fexofenadine as zwitterion at physiologic pH may be explained by the carboxylic group's interaction with the basic pyridinyl nitrogen via folded conformers [65]. Generally, zwitterions do not cross the blood-brain barrier and hence do not cause sedation [65].
Ebastine/Carebastine
Ebastine ( Figure 23) is a second-generation non-sedating H1-antihistamine. Its structure is similar to that of terfenadine. As in the latter drug, in ebastine the benzylic methyl group is metabolically oxidized to the carboxyl group after an intermediate step in which hydroxymethyl metabolite is formed as shown in Figure 23. The resulting metabolite, given the name of carebastine, is more active than the parent drug and accounts for nearly all the H1-antihistaminic activity [66]. Despite its high log P value of 6.9 [67], ebastine does not cross the blood-brain barrier, and accordingly it does not cause sedation. On the other hand, carebastine, the active metabolite of ebastine, exists as zwitterion at physiologic pH ( Figure 24) and accordingly does not cross the bloodbrain barrier. Further, like terfenadine (Section 2.4.6), ebastine is cardiotoxic [68]. It is worth mentioning that, despite carebastine lack of cardiotoxicity relative to its parent drug, it has not been developed into a fully-fledged drug in analogy with fexofenadine (Section 2.4.6). Almirall-
Terfenadine/Fexofenadine
Terfenadine ( Figure 22) is a second generation H1-antihistamine free of the sedative side effect associated with the first-generation H1-antihistamines. Terfenadine is almost completely metabolized by benzylic-methyl-group oxidation to an equiactive carboxy metabolite, as shown in Figure 22 [62], and it is thus considered a prodrug. However, despite this advantage, terfenadine was withdrawn from clinical use because of its cardiotoxic effect [63]. In the interim, its carboxy metabolite, being free of cardiotoxicity, was developed into a drug of its own right under the name of fexofenadine. As shown in Figure 24, fexofenadine is amphoteric and thus is capable of existing as a zwitterion at physiologic pH [64]. The existence of fexofenadine as zwitterion at physiologic pH may be explained by the carboxylic group's interaction with the basic pyridinyl nitrogen via folded conformers [65]. Generally, zwitterions do not cross the blood-brain barrier and hence do not cause sedation [65].
Terfenadine/Fexofenadine
Terfenadine ( Figure 22) is a second generation H1-antihistamine free of the sedative side effect associated with the first-generation H1-antihistamines. Terfenadine is almost completely metabolized by benzylic-methyl-group oxidation to an equiactive carboxy metabolite, as shown in Figure 22 [62], and it is thus considered a prodrug. However, despite this advantage, terfenadine was withdrawn from clinical use because of its cardiotoxic effect [63]. In the interim, its carboxy metabolite, being free of cardiotoxicity, was developed into a drug of its own right under the name of fexofenadine. As shown in Figure 24, fexofenadine is amphoteric and thus is capable of existing as a zwitterion at physiologic pH [64]. The existence of fexofenadine as zwitterion at physiologic pH may be explained by the carboxylic group's interaction with the basic pyridinyl nitrogen via folded conformers [65]. Generally, zwitterions do not cross the blood-brain barrier and hence do not cause sedation [65].
Ebastine/Carebastine
Ebastine ( Figure 23) is a second-generation non-sedating H1-antihistamine. Its structure is similar to that of terfenadine. As in the latter drug, in ebastine the benzylic methyl group is metabolically oxidized to the carboxyl group after an intermediate step in which hydroxymethyl metabolite is formed as shown in Figure 23. The resulting metabolite, given the name of carebastine, is more active than the parent drug and accounts for nearly all the H1-antihistaminic activity [66]. Despite its high log P value of 6.9 [67], ebastine does not cross the blood-brain barrier, and accordingly it does not cause sedation. On the other hand, carebastine, the active metabolite of ebastine, exists as zwitterion at physiologic pH ( Figure 24) and accordingly does not cross the bloodbrain barrier. Further, like terfenadine (Section 2.4.6), ebastine is cardiotoxic [68]. It is worth mentioning that, despite carebastine lack of cardiotoxicity relative to its parent drug, it has not been developed into a fully-fledged drug in analogy with fexofenadine (Section 2.4.6). Almirall-
Ebastine/Carebastine
Ebastine ( Figure 23) is a second-generation non-sedating H1-antihistamine. Its structure is similar to that of terfenadine. As in the latter drug, in ebastine the benzylic methyl group is metabolically oxidized to the carboxyl group after an intermediate step in which hydroxymethyl metabolite is formed as shown in Figure 23. The resulting metabolite, given the name of carebastine, is more active than the parent drug and accounts for nearly all the H1-antihistaminic activity [66]. Despite its high log P value of 6.9 [67], ebastine does not cross the blood-brain barrier, and accordingly it does not cause sedation. On the other hand, carebastine, the active metabolite of ebastine, exists as zwitterion at physiologic pH ( Figure 24) and accordingly does not cross the blood-brain barrier. Further, like terfenadine (Section 2.4.6), ebastine is cardiotoxic [68]. It is worth mentioning that, despite carebastine lack of cardiotoxicity relative to its parent drug, it has not been developed into a fully-fledged drug in analogy with fexofenadine (Section 2.4.6). Almirall-Prodesfarma, a Spanish pharmaceutical company, reached stage III in the development of carebastine for the treatment of allergic conjunctivitis and allergic rhinitis, but the company subsequently discontinued the endeavor [69]. Prodesfarma, a Spanish pharmaceutical company, reached stage III in the development of carebastine for the treatment of allergic conjunctivitis and allergic rhinitis, but the company subsequently discontinued the endeavor [69].
Metabolic Conversion of Intrinsic Hydroxymethyl Groups in Parent Drugs to Active Carboxy Metabolites
As has been shown in examples in Sections 2.1 through 2.4, carboxy-metabolites can result from the oxidation of ω-methyl groups in alkyl chains or methyl groups attached to cycloalkyl or aromatic rings. In all the cases cited, the carboxy metabolites were found to be pharmacologically inactive, that is, they did not give the same pharmacological effect as the corresponding parent drugs. However, this observation should not be generalized. Three prominent examples in which intrinsic hydroxymethyl groups are metabolically oxidized to the carboxyl groups with retention of activity are hydroxyzine to cetirizine, salicin to salicylic acid, and losartan to losartan carboxylic acid.
Hydroxyzine/Cetirizine
Hydroxyzine ( Figure 24) is a first-generation H1-antihistamine. H1-antihistamines are generally lipophilic in nature, a property that causes them to cross the blood-brain barrier to cause sedation as a main side effect [70]. Hydroxyzine is primarily metabolized by oxidation of the primary alcoholic group to give the equiactive carboxyl metabolite ( Figure 24) [71]. Being appreciably more hydrophilic than hydroxyzine and capable of existing as a zwitterion at the physiologic pH of 7.4 ( Figure 24), the metabolite does not cross the blood-brain barrier and therefore does not cause sedation [72]. As a result of this pharmacokinetic advantage, the carboxy metabolite of hydroxyzine has been developed into a second-generation H1-antihistamine of its own right under the name of cetirizine [62,63]. The existence of cetirizine as zwitterion may be explained by analogy to fexofenadine in Section 2.4.6. Hydroxyzine and cetirizine are used concurrently in clinical settings; night urticaria may be a suitable indication for the sedative hydroxyzine, while in allergic reactions demanding alertness, cetirizine is indicated [72].
Metabolic Conversion of Intrinsic Hydroxymethyl Groups in Parent Drugs to Active Carboxy Metabolites
As has been shown in examples in Sections 2.1 through 2.4, carboxy-metabolites can result from the oxidation of ω-methyl groups in alkyl chains or methyl groups attached to cycloalkyl or aromatic rings. In all the cases cited, the carboxy metabolites were found to be pharmacologically inactive, that is, they did not give the same pharmacological effect as the corresponding parent drugs. However, this observation should not be generalized. Three prominent examples in which intrinsic hydroxymethyl groups are metabolically oxidized to the carboxyl groups with retention of activity are hydroxyzine to cetirizine, salicin to salicylic acid, and losartan to losartan carboxylic acid.
Hydroxyzine/Cetirizine
Hydroxyzine ( Figure 24) is a first-generation H1-antihistamine. H1-antihistamines are generally lipophilic in nature, a property that causes them to cross the blood-brain barrier to cause sedation as a main side effect [70]. Hydroxyzine is primarily metabolized by oxidation of the primary alcoholic group to give the equiactive carboxyl metabolite (Figure 24) [71]. Being appreciably more hydrophilic than hydroxyzine and capable of existing as a zwitterion at the physiologic pH of 7.4 (Figure 24), the metabolite does not cross the blood-brain barrier and therefore does not cause sedation [72]. As a result of this pharmacokinetic advantage, the carboxy metabolite of hydroxyzine has been developed into a second-generation H1-antihistamine of its own right under the name of cetirizine [62,63]. The existence of cetirizine as zwitterion may be explained by analogy to fexofenadine in Section 2.4.6. Hydroxyzine and cetirizine are used concurrently in clinical settings; night urticaria may be a suitable indication for the sedative hydroxyzine, while in allergic reactions demanding alertness, cetirizine is indicated [72].
Metabolic Conversion of Intrinsic Hydroxymethyl Groups in Parent Drugs to Active Carboxy Metabolites
As has been shown in examples in Sections 2.1-2.4, carboxy-metabolites can result from the oxidation of ω-methyl groups in alkyl chains or methyl groups attached to cycloalkyl or aromatic rings. In all the cases cited, the carboxy metabolites were found to be pharmacologically inactive, that is, they did not give the same pharmacological effect as the corresponding parent drugs. However, this observation should not be generalized. Three prominent examples in which intrinsic hydroxymethyl groups are metabolically oxidized to the carboxyl groups with retention of activity are hydroxyzine to cetirizine, salicin to salicylic acid, and losartan to losartan carboxylic acid.
Hydroxyzine/Cetirizine
Hydroxyzine ( Figure 24) is a first-generation H1-antihistamine. H1-antihistamines are generally lipophilic in nature, a property that causes them to cross the blood-brain barrier to cause sedation as a main side effect [70]. Hydroxyzine is primarily metabolized by oxidation of the primary alcoholic group to give the equiactive carboxyl metabolite (Figure 24) [71]. Being appreciably more hydrophilic than hydroxyzine and capable of existing as a zwitterion at the physiologic pH of 7.4 (Figure 24), the metabolite does not cross the blood-brain barrier and therefore does not cause sedation [72]. As a result of this pharmacokinetic advantage, the carboxy metabolite of hydroxyzine has been developed into a second-generation H1-antihistamine of its own right under the name of cetirizine [62,63]. The existence of cetirizine as zwitterion may be explained by analogy to fexofenadine in Section 2.4.6. Hydroxyzine and cetirizine are used concurrently in clinical settings; night urticaria may be a suitable indication for the sedative hydroxyzine, while in allergic reactions demanding alertness, cetirizine is indicated [72].
Salicin/Salicylic Acid/Aspirin
Salicin ( Figure 25) is a natural product found in the bark of the willow tree. The major turning point for salicylate medicines came in 1763, when a letter from the English chaplain Edward Stone was read at a meeting of the Royal Society. Stone's letter described the dramatic power of the willow bark extract to cure intermittent fever, pain, and fatigue [73]. As shown in Figure 25, the metabolism of salicin to salicylic acid involves acetalic ether bridge hydrolysis (reminiscent of aromatic-alkoxy dealkylation) to a phenolic group as well as primary alcohol oxidation to the carboxyl group. The latter metabolic pathway is the subject of this section.
Salicin/Salicylic Acid/Aspirin
Salicin ( Figure 25) is a natural product found in the bark of the willow tree. The major turning point for salicylate medicines came in 1763, when a letter from the English chaplain Edward Stone was read at a meeting of the Royal Society. Stone's letter described the dramatic power of the willow bark extract to cure intermittent fever, pain, and fatigue [73]. As shown in Figure 25, the metabolism of salicin to salicylic acid involves acetalic ether bridge hydrolysis (reminiscent of aromatic-alkoxy dealkylation) to a phenolic group as well as primary alcohol oxidation to the carboxyl group. The latter metabolic pathway is the subject of this section. Research into willow bark extract culminated in 1899, when the German drug company, Bayer, prepared aspirin by acetylating the phenolic hydroxy group in salicylic acid, which was believed to cause gastric irritation and bleeding [74]. However, subsequent research has proven that the acetyl group in aspirin is crucial to aspirin's mode of action as a COX inhibitor in the treatment of inflammation. Through transacetylation, aspirin acetylates the alcoholic hydroxy group of the serine moiety in COX, thus inhibiting it from catalyzing prostaglandin biosynthesis [75].
In addition to being one of the most widely used anti-inflammatory, analgesic, and antipyretic drug, aspirin is now renowned for its use as a thrombolytic agent to prevent blood clotting in patients prone to stroke [76][77][78]. Furthermore, its preventive role in colorectal cancer has almost been established [79,80], and it is now being actively researched for other cancers [79,80].
Three factors played significant roles in the design and development of aspirin: (i) nature, by providing salicin from the willow bark; (ii) metabolism, by converting salicin to salicylic acid; and (iii) medicinal chemistry, by blocking the phenolic hydroxy group of salicylic acid by acetylation. Therefore, from a developmental perspective, aspirin can be described as a natural-productmetabolite-synthetic drug, while salicin can be considered a natural prodrug.
Losartan/Losartan Carboxylic Acid
Losartan ( Figure 26) is a selective, competitive angiotensin II receptor type (AT1) antagonist used as antihypertensive. Through the route shown in Figure 26, the 5-hydroxymethyl group in losartan is metabolized by cytochrome P450 to the 5-carboxylic acid group through intermediate aldehyde formation [81][82][83]. This metabolic route accounts for 14% of losartan dose; the remainder of the drug is excreted unchanged [82,83]. The carboxy metabolite of losartan has 10-40 times the activity of the parent drug [82,83]. Since losartan is only partially converted into an active form, it is not considered a typical prodrug. Research into willow bark extract culminated in 1899, when the German drug company, Bayer, prepared aspirin by acetylating the phenolic hydroxy group in salicylic acid, which was believed to cause gastric irritation and bleeding [74]. However, subsequent research has proven that the acetyl group in aspirin is crucial to aspirin's mode of action as a COX inhibitor in the treatment of inflammation. Through transacetylation, aspirin acetylates the alcoholic hydroxy group of the serine moiety in COX, thus inhibiting it from catalyzing prostaglandin biosynthesis [75].
In addition to being one of the most widely used anti-inflammatory, analgesic, and antipyretic drug, aspirin is now renowned for its use as a thrombolytic agent to prevent blood clotting in patients prone to stroke [76][77][78]. Furthermore, its preventive role in colorectal cancer has almost been established [79,80], and it is now being actively researched for other cancers [79,80].
Three factors played significant roles in the design and development of aspirin: (i) nature, by providing salicin from the willow bark; (ii) metabolism, by converting salicin to salicylic acid; and (iii) medicinal chemistry, by blocking the phenolic hydroxy group of salicylic acid by acetylation. Therefore, from a developmental perspective, aspirin can be described as a natural-product-metabolite-synthetic drug, while salicin can be considered a natural prodrug.
Losartan/Losartan Carboxylic Acid
Losartan ( Figure 26) is a selective, competitive angiotensin II receptor type (AT1) antagonist used as antihypertensive. Through the route shown in Figure 26, the 5-hydroxymethyl group in losartan is metabolized by cytochrome P450 to the 5-carboxylic acid group through intermediate aldehyde formation [81][82][83]. This metabolic route accounts for 14% of losartan dose; the remainder of the drug is excreted unchanged [82,83]. The carboxy metabolite of losartan has 10-40 times the activity of the parent drug [82,83]. Since losartan is only partially converted into an active form, it is not considered a typical prodrug. According to Foye [81], the hydroxymethyl group in losartan can be replaced by other groups including carboxy, keto, or benzimidazole, to give active ARB drugs. Such groups interact with the AT1 receptor via either ionic, ion-dipole, or dipole-dipole bindings. The considerable increase in activity of the carboxy metabolite of losartan compared to the parent drug may be explained by the metabolite's increased affinity to the receptor caused by the stronger ion-ion or ion-dipole binding due to the ionized carboxylate group at physiologic pH compared to the hydrogen bond binding of the hydroxyl group in the parent drug.
Metabolic Oxidation of Methylene Groups Alpha (α) to Carbonyl and Imino Groups
Generally, methylene groups alpha to carbonyl as well as imino groups undergo metabolic oxidation via mixed function oxidases [84,85]. Examples of drugs in which such groups are found are diazepam and alprazolam within the benzodiazepine class, whose members are used as tranquilizers, hypnotics, or anticonvulsants. The mechanism of metabolic oxidation involves, as a first step, the formation of a resonance-stabilized free radical, as depicted in Figure 27. A hydroxyl group will then be transferred to the free radical in accordance with the mechanism of metabolic alkyl oxidation shown in Figure 2.
Diazepam
As shown in Figure 28, diazepam is mainly metabolized by hydroxylation at the carbon atom α to the carbonyl and imino groups at position 3, as well as by N-dealkylation [86][87][88]. Both metabolic routes give equiactive products with respect to diazepam, though with modified pharmacokinetic properties that affect the drugs' duration of action. Both hydroxylation at position 3 and Ndealkylation result in increased metabolite polarity and hence enhanced metabolite elimination. In According to Foye [81], the hydroxymethyl group in losartan can be replaced by other groups including carboxy, keto, or benzimidazole, to give active ARB drugs. Such groups interact with the AT 1 receptor via either ionic, ion-dipole, or dipole-dipole bindings. The considerable increase in activity of the carboxy metabolite of losartan compared to the parent drug may be explained by the metabolite's increased affinity to the receptor caused by the stronger ion-ion or ion-dipole binding due to the ionized carboxylate group at physiologic pH compared to the hydrogen bond binding of the hydroxyl group in the parent drug.
Metabolic Oxidation of Methylene Groups Alpha (α) to Carbonyl and Imino Groups
Generally, methylene groups alpha to carbonyl as well as imino groups undergo metabolic oxidation via mixed function oxidases [84,85]. Examples of drugs in which such groups are found are diazepam and alprazolam within the benzodiazepine class, whose members are used as tranquilizers, hypnotics, or anticonvulsants. The mechanism of metabolic oxidation involves, as a first step, the formation of a resonance-stabilized free radical, as depicted in Figure 27. A hydroxyl group will then be transferred to the free radical in accordance with the mechanism of metabolic alkyl oxidation shown in Figure 2. According to Foye [81], the hydroxymethyl group in losartan can be replaced by other groups including carboxy, keto, or benzimidazole, to give active ARB drugs. Such groups interact with the AT1 receptor via either ionic, ion-dipole, or dipole-dipole bindings. The considerable increase in activity of the carboxy metabolite of losartan compared to the parent drug may be explained by the metabolite's increased affinity to the receptor caused by the stronger ion-ion or ion-dipole binding due to the ionized carboxylate group at physiologic pH compared to the hydrogen bond binding of the hydroxyl group in the parent drug.
Metabolic Oxidation of Methylene Groups Alpha (α) to Carbonyl and Imino Groups
Generally, methylene groups alpha to carbonyl as well as imino groups undergo metabolic oxidation via mixed function oxidases [84,85]. Examples of drugs in which such groups are found are diazepam and alprazolam within the benzodiazepine class, whose members are used as tranquilizers, hypnotics, or anticonvulsants. The mechanism of metabolic oxidation involves, as a first step, the formation of a resonance-stabilized free radical, as depicted in Figure 27. A hydroxyl group will then be transferred to the free radical in accordance with the mechanism of metabolic alkyl oxidation shown in Figure 2.
Diazepam
As shown in Figure 28, diazepam is mainly metabolized by hydroxylation at the carbon atom α to the carbonyl and imino groups at position 3, as well as by N-dealkylation [86][87][88]. Both metabolic routes give equiactive products with respect to diazepam, though with modified pharmacokinetic properties that affect the drugs' duration of action. Both hydroxylation at position 3 and Ndealkylation result in increased metabolite polarity and hence enhanced metabolite elimination. In
Diazepam
As shown in Figure 28, diazepam is mainly metabolized by hydroxylation at the carbon atom α to the carbonyl and imino groups at position 3, as well as by N-dealkylation [86][87][88]. Both metabolic routes give equiactive products with respect to diazepam, though with modified pharmacokinetic properties that affect the drugs' duration of action. Both hydroxylation at position 3 and N-dealkylation result in increased metabolite polarity and hence enhanced metabolite elimination. In addition, glucuronide conjugation taking place at the metabolically generated hydroxy group results in fast elimination and deactivation of the metabolites. The metabolic hydroxylation of diazepam at position 3 results in the generation of chiral centers in both temazepam and oxazepam ( Figure 28). However, despite the presence of several reports in the literature describing the separation of the enantiomers of the drugs [89][90][91][92], studies investigating the activity of their separated enantiomers are lacking.
Alprazolam
The triazolobenzodiazepine alprazolam ( Figure 29) is metabolized (i) by hepatic microsomal oxidation at C4, which is alpha to two imino moieties, to give 4-hydroxyalprazolam, and (ii) at the methyl group at position 1 to give α-hydroxyalprazolam ( Figure 29). Both metabolites have decreased benzodiazepine receptor affinity compared to the parent drug [93].
Oxazaphosphorines
Metabolic alkyl-moiety hydroxylation in prodrug activation is best exemplified by the three oxazaphosphorine alkylating anticancer prodrugs, cyclophosphamide, ifosfamide, and profosfamide [94,95]. The metabolic and chemical processes that lead to the activation of the three drugs in vivo are respectively illustrated in Figures 30-32. The first step in the activation process is the metabolic hydroxylation of the 4-methylene group of the common structural feature, the cyclophosphamide. Generally, carbons α to heteroatoms in heterocycles are activated by metabolic oxidation [96]. Next, the secondary alcohol so produced will tautomerize to the aldehydic group to give the aldo tautomer. This is followed by spontaneous non-enzymic elimination of the aldehydic neutral fragment, acrolein, to give the active alkylating agent, the nitrogen mustard. Acrolein causes hemorrhagic The metabolic hydroxylation of diazepam at position 3 results in the generation of chiral centers in both temazepam and oxazepam ( Figure 28). However, despite the presence of several reports in the literature describing the separation of the enantiomers of the drugs [89][90][91][92], studies investigating the activity of their separated enantiomers are lacking.
Alprazolam
The triazolobenzodiazepine alprazolam ( Figure 29) is metabolized (i) by hepatic microsomal oxidation at C4, which is alpha to two imino moieties, to give 4-hydroxyalprazolam, and (ii) at the methyl group at position 1 to give α-hydroxyalprazolam ( Figure 29). Both metabolites have decreased benzodiazepine receptor affinity compared to the parent drug [93]. The metabolic hydroxylation of diazepam at position 3 results in the generation of chiral centers in both temazepam and oxazepam ( Figure 28). However, despite the presence of several reports in the literature describing the separation of the enantiomers of the drugs [89][90][91][92], studies investigating the activity of their separated enantiomers are lacking.
Alprazolam
The triazolobenzodiazepine alprazolam ( Figure 29) is metabolized (i) by hepatic microsomal oxidation at C4, which is alpha to two imino moieties, to give 4-hydroxyalprazolam, and (ii) at the methyl group at position 1 to give α-hydroxyalprazolam ( Figure 29). Both metabolites have decreased benzodiazepine receptor affinity compared to the parent drug [93].
Oxazaphosphorines
Metabolic alkyl-moiety hydroxylation in prodrug activation is best exemplified by the three oxazaphosphorine alkylating anticancer prodrugs, cyclophosphamide, ifosfamide, and profosfamide [94,95]. The metabolic and chemical processes that lead to the activation of the three drugs in vivo are respectively illustrated in Figures 30-32. The first step in the activation process is the metabolic hydroxylation of the 4-methylene group of the common structural feature, the cyclophosphamide. Generally, carbons α to heteroatoms in heterocycles are activated by metabolic oxidation [96]. Next, the secondary alcohol so produced will tautomerize to the aldehydic group to give the aldo tautomer. This is followed by spontaneous non-enzymic elimination of the aldehydic neutral fragment, acrolein, to give the active alkylating agent, the nitrogen mustard. Acrolein causes hemorrhagic
Oxazaphosphorines
Metabolic alkyl-moiety hydroxylation in prodrug activation is best exemplified by the three oxazaphosphorine alkylating anticancer prodrugs, cyclophosphamide, ifosfamide, and profosfamide [94,95]. The metabolic and chemical processes that lead to the activation of the three drugs in vivo are respectively illustrated in Figures 30-32. The first step in the activation process is the metabolic hydroxylation of the 4-methylene group of the common structural feature, the cyclophosphamide. Generally, carbons α to heteroatoms in heterocycles are activated by metabolic oxidation [96]. Next, the secondary alcohol so produced will tautomerize to the aldehydic group to give the aldo tautomer. This is followed by spontaneous non-enzymic elimination of the aldehydic neutral fragment, acrolein, to give the active alkylating agent, the nitrogen mustard. Acrolein causes hemorrhagic cystitis, an adverse effect that can be offset by the concurrent administration of mesna. The mechanism of action of mesna involves the formation of a highly water-soluble conjugate of acrolein that is excreted in the urine [97] (Figure 33).
Molecules 2020, 25, x FOR PEER REVIEW 17 of 29 cystitis, an adverse effect that can be offset by the concurrent administration of mesna. The mechanism of action of mesna involves the formation of a highly water-soluble conjugate of acrolein that is excreted in the urine [97] (Figure 33).
Aryl-Dialkyl-Triazines
Another class of anticancer prodrugs that are activated by alkyl-group metabolic hydroxylation is the aryl-dialkyl-triazines [98,99]. The antitumor 1-aryl-3,3-dimethyltriazines have the general structure shown in Figure 34. The prototype of this class of drugs is 5-(3,3-Dimethyl-1- cystitis, an adverse effect that can be offset by the concurrent administration of mesna. The mechanism of action of mesna involves the formation of a highly water-soluble conjugate of acrolein that is excreted in the urine [97] (Figure 33). Another class of anticancer prodrugs that are activated by alkyl-group metabolic hydroxylation is the aryl-dialkyl-triazines [98,99]. The antitumor 1-aryl-3,3-dimethyltriazines have the general structure shown in Figure 34. The prototype of this class of drugs is 5-(3,3-Dimethyl-1- Another class of anticancer prodrugs that are activated by alkyl-group metabolic hydroxylation is the aryl-dialkyl-triazines [98,99]. The antitumor 1-aryl-3,3-dimethyltriazines have the general structure shown in Figure 34. The prototype of this class of drugs is 5-(3,3-Dimethyl-1- Another class of anticancer prodrugs that are activated by alkyl-group metabolic hydroxylation is the aryl-dialkyl-triazines [98,99]. The antitumor 1-aryl-3,3-dimethyltriazines have the general structure shown in Figure 34. The prototype of this class of drugs is 5-(3,3-Dimethyl-1-
Further Interpretations
When studying the effect of drug molecules' metabolic hydroxy and carboxy functionalization of alkyl groups on metabolite pharmacologic activity, several factors should first be considered. These factors include: (a) the extent of formation of the hydroxy and carboxy metabolites (b) the hydrophobicity of the parent drug and hydrophilicity of the metabolites (c) the drug's mechanism of action (d) the favored site of metabolic hydroxylation in drug molecules containing more than one alkyl group (e) the molecular size increase and steric effect resulting from the replacement of the small hydrogen atom in the alkyl group in the drug molecule by the larger and bulkier hydroxyl or carboxyl group in the metabolite molecule (f) the creation of new metabolite-receptor binding mechanisms, e.g., hydrogen bonding, ionpairing, and ion-dipole, in contrast to van der Waals binding of the alkyl groups Generally, the extent of the metabolic oxidation of carbon atoms in drug molecules' alkyl chains depends in part on the class of the carbon atom, which in turn dictates the stability of the resulting free radicals: benzylic, allylic > tertiary > secondary > primary > methyl. On the other hand, the hydrophilicity of an alcoholic hydroxyl group is determined by the strength of the intermolecular hydrogen bonds it forms. Due to steric effects, the strength of hydrogen bonding in the different classes of alcohols follows the sequence primary > secondary > tertiary [100]. The order of the hydrophilicity of primary, secondary, and tertiary alcohols follows the same sequence.
From the aliphatic hydroxy and carboxy metabolites of the cases surveyed in this review, we observe three effects on the pharmacologic activity of the metabolites relevant to their parent drugs: loss, attenuation, or retention.
NSAIDS
Loss of pharmacologic activity has been observed for ibuprofen upon metabolic hydroxylation of the isobutyl group at C1, C2, and C3 ( Figure 3). In analogy with the O-demethylation of the
Further Interpretations
When studying the effect of drug molecules' metabolic hydroxy and carboxy functionalization of alkyl groups on metabolite pharmacologic activity, several factors should first be considered. These factors include: (a) the extent of formation of the hydroxy and carboxy metabolites (b) the hydrophobicity of the parent drug and hydrophilicity of the metabolites (c) the drug's mechanism of action (d) the favored site of metabolic hydroxylation in drug molecules containing more than one alkyl group (e) the molecular size increase and steric effect resulting from the replacement of the small hydrogen atom in the alkyl group in the drug molecule by the larger and bulkier hydroxyl or carboxyl group in the metabolite molecule (f) the creation of new metabolite-receptor binding mechanisms, e.g., hydrogen bonding, ion-pairing, and ion-dipole, in contrast to van der Waals binding of the alkyl groups Generally, the extent of the metabolic oxidation of carbon atoms in drug molecules' alkyl chains depends in part on the class of the carbon atom, which in turn dictates the stability of the resulting free radicals: benzylic, allylic > tertiary > secondary > primary > methyl. On the other hand, the hydrophilicity of an alcoholic hydroxyl group is determined by the strength of the intermolecular hydrogen bonds it forms. Due to steric effects, the strength of hydrogen bonding in the different classes of alcohols follows the sequence primary > secondary > tertiary [100]. The order of the hydrophilicity of primary, secondary, and tertiary alcohols follows the same sequence.
From the aliphatic hydroxy and carboxy metabolites of the cases surveyed in this review, we observe three effects on the pharmacologic activity of the metabolites relevant to their parent drugs: loss, attenuation, or retention.
NSAIDS
Loss of pharmacologic activity has been observed for ibuprofen upon metabolic hydroxylation of the isobutyl group at C1, C2, and C3 ( Figure 3). In analogy with the O-demethylation of the methoxy-group-containing NSAIDS discussed in the first part of this review series [12], both pharmacodynamic and pharmacokinetic effects may account for the ibuprofen isobutyl-hydroxy metabolites' loss of pharmacologic activity. The SAR of ibuprofen dictates that the branched isobutyl moiety is essential for optimum COX-inhibitory effect; in this context, n-butyl substitution has led to significant loss of activity [101]. This finding should imply that each of the methyl groups in the isobutyl moiety occupies a small hydrophobic pocket in COX, enabling a pharmacophoric effect that is essential for optimum activity. The hydroxy and carboxy groups in metabolite III and metabolite IV, respectively (Figure 3), are detrimental to the hydrophobic binding of the isobutyl group, and accordingly, they precipitate a loss of COX-inhibiting activity [102]. Further, by replacing a hydrogen atom in the isobutyl group in ibuprofen, the bulkier hydroxyl or carboxyl groups in the metabolites will impart molecular-size increase and steric effects-factors that are detrimental to optimum binding between isobutyl group and COX [102]. In addition, being hydrophilic, the hydroxyl or carboxyl group will increase the water solubility of the metabolites, hence leading to their elimination and termination of their action. Furthermore, glucuronide conjugation of the hydroxyl and carboxyl groups will considerably enhance the prospects of metabolite elimination and activity termination through substantially increasing aqueous solubility.
Sulfonylurea Oral Antidiabetics
For the sake of discussing the effect of metabolic oxidation of alkyl and aliphatic cyclic groups in the sulfonylurea antidiabetics, we dissect the general structure of these agents, as depicted in Figure 5. To reiterate, in the first-generation sulfonylureas (Section 2.2), R 1 (Figure 7) is a small lipophilic group, such as methyl or chloro, while R 2 is an alkyl or aliphatic cyclic group. According to Foye (2020) [26], the R 1 groups do little to increase the binding efficiency of the pharmacophore to the ATP-sensitive K + channel. As such, R 1 groups may be playing weak auxiliary pharmacophoric and/or auxophoric roles. On the other hand, the R 2 groups in both first-and second-generation sulfonylurea antidiabetics have the auxophoric role of optimizing the pKa of the sulfonylurea group to~5. At this pKa value, a sulfonylurea anion is formed that is essential for interaction with the pancreatic β-cell subtypes (SUR1, SURA1, and SUR2A) through ion-ion and ion-dipole bindings [103]. Generally, metabolic change at auxiliary pharmacophores or auxophores is associated with retention of pharmacologic activity [12]. However, a discrepancy is observed for some members of the first-generation sulfonylurea antidiabetics in this respect. For instance, while the aliphatic-ring hydroxy metabolite of tolazamide ( Figure 12) is active with a prolonged duration of action, the counterpart metabolite of acetohexamide ( Figure 8) is inactive.
The lipophilic methyl group at R 1 in the general structure of sulfonylureas ( Figure 5) is metabolized by oxidation, via hydroxymethyl formation, to the carboxyl group with loss of activity in both tolbutamide and tolazamide (Figures 7 and 9, respectively). Generally, the loss of activity caused by metabolically formed carboxyl groups can be explained by two effects. Firstly, the carboxyl group is almost fully ionized at the physiologic pH of 7.4. Secondly, the carboxyl group is, in most cases, glucuronide conjugated in phase II. These two effects will result in a substantial increase of water solubility and elimination of the metabolite with the consequent loss of activity due to reduced effective concentration of the metabolite at the receptor. It is noteworthy that the metabolic functionalization of the lipophilic benzylic methyl group to the carboxyl group in tolbutamide (Figure 7), with the consequent enhanced elimination and loss of activity, has led to the development of chlorpropamide ( Figure 9). By employing bioisosterism, medicinal chemists replaced the benzylic methyl group in tolbutamide with a chloro group, which is not prone to metabolism, to obtain chlorpropamide. Due to this manipulation of metabolic stability, chlorpropamide can be used at a lower dose and frequency than tolbutamide [104].
Molecules 2020, 25, 1937 20 of 29 In the second-generation sulfonylurea antidiabetics, the small lipophilic groups of the first generation at R 1 ( Figure 5) have been replaced by the larger p-(β-arylcarboxyamidoethyl) group, such as in glimepiride (Figure 14), in order to attain strong binding affinity to the ATP-sensitive K + channel [26]. Metabolism of this group is not within the scope of this review.
Barbiturates
The metabolic oxidative hydroxylation of alkyl chains in barbiturates has resulted in variable levels of activity subject to the class of the resulting alcohol. In pentobarbital (Figure 17), the primary and secondary alcohols, respectively resulting from ω and ω-1 oxidation, are sufficiently hydrophilic to jeopardize the hydrophobicity requirement for blood-brain barrier crossing [105]. In addition to hydrophilicity, the factors of increased steric effect, molecular size, and surface area may come into play to hinder the hydroxy metabolite from fitting in the receptor, thus leading to either attenuation or loss of activity as governed by the extent of each factor. On the other hand, in amobarbital ( Figure 16), metabolic oxidation occurs mainly at the ω-1 tertiary carbon, resulting in a tertiary alcohol, 3 -hydroxyamobarbital. Despite being less active, 3 -hydroxyamobarbital has been reported to be responsible for the sedative-hypnotic activity of amobarbital [43]. With reduced hydrogen bonding ability, and the consequent diminishment of hydrophilicity, due to steric effects in tertiary alcohols, 3 -hydroxyamobarbital is expected to cross the blood-brain barrier in sufficient concentration to produce sedative-hypnotic effects.
The loss of activity of the carboxy metabolites of barbiturates may be explained similarly to the NSAIDS-carboxy metabolites (Section 3.1).
Accounting for the Activity of the H1-Antihistamines' Carboxy Metabolites: Hydroxyzine, Terfenadine, and Ebastine
Methyl groups that are bonded to aromatic or cycloalkyl rings, or terminal in alkyl chains (i.e., ω methyls) in drug molecules are usually oxidized to inactive carboxy metabolites through the formation of mostly active hydroxymethyl intermediates. The loss of pharmacologic activity indicates that the methyl groups in such cases play pharmacophoric roles, at least of an auxiliary nature. However, when the methyl or hydroxymethyl group is distant from a predetermined pharmacophore, the situation is different: metabolic oxidation of either group to the carboxyl group does not cause loss of activity of the resulting metabolite. This has been the case with the three H1-antihistamines hydroxyzine, terfenadine, and ebastine, which are respectively metabolized to equiactive cetirizine ( Figure 24), fexofenadine ( Figure 22), and carebastine ( Figure 23).
Hydroxyzine is a first generation H1-antihistamine. With a log P value of 3.5 [106], it is hydrophobic enough to cross the blood-brain barrier, interact with cholinergic, serotonergic, and adrenergic receptors and cause sedation [107]. On the other hand, cetirizine, the carboxy-metabolite drug of hydroxyzine, has a log P value of 1.5 [108] and exists as zwitterion at physiologic pH of 7.4. Due to these properties, cetirizine does not cross the blood-brain barrier and does not accordingly cause sedation. As shown in Figure 24, in both hydroxyzine and cetirizine, the metabolically exchanged groups are distant from the pharmacophore, and accordingly, the two drugs are therapeutically equiactive as H1-antihistamines. The ethoxyethanol group in hydroxyzine and the ethoxyacetic acid group in cetirizine each play an auxophoric role. A similar situation can be observed for the terfenadine/fexofenadine H1-antihistamine pair ( Figure 23). However, here, the carboxyl group in fexofenadine plays a pharmacodynamic rather that a pharmacokinetic role. Terfenadine causes heart arrhythmias by blocking the hERG channel K + current [109]. On the other hand, the ionized carboxylate group (COO − ) in fexofenadine reduces this blockage by over three orders of magnitude, thus rendering this drug almost free of the cardiotoxic effect [110].
The inference that can be made from the two H1-antihistamine pairs presented above is that when metabolic changes occur at groups distant from the primary pharmacophores (i.e., at auxophoric groups), the original pharmacologic activity will not be affected. Further, beneficial pharmacokinetic and/or pharmacodynamic modifications may result in the metabolites warranting their development into fully-fledged drugs. An extended definition of pharmacophores is given in Section 3.6.7.
Aspirin Is an NSAID of Its Own Disposition
In salicin (Figure 27), the acetalic group is metabolically converted to a hydroxyl group in a reaction reminiscent of O-dealkylation, while the hydroxymethyl group is oxidized to the carboxyl group to give salicylic acid. The phenolic hydroxyl group in salicylic acid was suspected to be the cause of stomach irritation and bleeding, and it was hence esterified by acetic anhydride to give aspirin. However, later, it was proven [111][112][113] that the gastrointestinal adverse effects of aspirin were associated with the inhibition of COX1 and accordingly the inhibition of PGE1 formation, i.e., synthesis of the prostaglandin involved in the protection of gastric mucosa against acid attack. Sometime then elapsed before the mechanism of the anti-inflammatory activity of aspirin was understood to be caused by acetylation of the serine moiety in COX [114]. That being the case, the benzene ring and the carboxyl group in aspirin are likely playing auxiliary pharmacophoric roles by properly anchoring the aspirin molecule in the COX-active cavity, thus facilitating the transfer of the acetyl group to the serine moiety.
Subtexts Arising from Hydroxy and Carboxy Metabolic Functionalization of Alkyl Moieties in Drug Molecules
The aim of this section is to provide focused information on some general and specific issues that have been extracted from the individual cases of alkyl-moiety metabolic hydroxy and carboxy functionalization. The information presented and discussed includes definitions, significance, implications, and/or applications of selected topics, which include: 3.6.1. Metabolism of methyl groups in drug molecules 3.6.2. Metabolic hydroxylation of alicycles and aliphatic heterocycles in drug molecules 3.6.3. Inferences from hydroxymethyl group in drug metabolites regarding origin and significance 3.6.4. Development of metabolite drugs and prodrugs from metabolites equiactive with parent drugs 3.6.5. Pharmacologic activity of carboxy metabolites 3.6.6. Significance of the carboxy metabolite of ∆ 9 -tetrahydrocannabinol 3.6.7. Primary and auxiliary pharmacophoric properties 3.6.1.
Metabolism of Methyl Groups in Drug Molecules
Methyl groups assume their importance in drug molecules due to their small size, higher steric effect with respect to the hydrogen atom, hydrophobicity, isosterism with a number of groups, and historical inclusion in drug molecules. They are found in drug molecules at ω-carbons in both straightand branched-chain alkyls, as substituents in aromatic (benzene) rings and alicycles, and as substituents in secondary and tertiary amino moieties. In branched-chain alkyls, methyl groups are found as isopropyl, isobutyl, or tert-butyl moieties. In all of these forms, the methyl group is metabolically oxidized by CYP450 enzymes to the hydroxymethyl group. The sequential oxidation of the latter group to the carboxylic acid follows in most cases via primary alcohol formation. When there is more than one equivalent methyl group in a drug molecule, only one group will be metabolically oxidized.
Metabolic Hydroxylation of Alicycles and Aliphatic Heterocycles in Drug Molecules
Six-membered alicycle (cyclohexyl) and heterocycle (piperidinyl) groups are often encountered in drug molecules of various pharmacologic classes. For the most part, rings are stereoselectively metabolized by hydroxylation at the positions 3 and 4, which are less sterically hindered, compared to other positions (in the ring), to form cis and trans isomers. Pharmacologic action may also be a function of stereoselective metabolism. For instance, the oral antidiabetic acetohexamide is mainly metabolized to trans-4 -hydroxyacetohexamide, which is inactive. The cyclohexyl ring in glibenclamide is metabolically oxidized to 3-cis and 4-trans-hydroxy metabolites with a substantial attenuation of antidiabetic activity. A similar effect has been observed for tolazamide, in which the azepane ring is metabolically hydroxylated at position 4 ( Figure 11) with a substantial loss of activity.
An interesting case of metabolic hydroxylation of alicycles and aliphatic heterocycles is given by the psychotropic drug phencyclidine, which contains both cyclohexyl and piperidinyl groups. Phencyclidine is mainly metabolized by hydroxylation at position 4 of the cyclohexyl ring to the active cisand trans-4-phenyl-4-(1-piperidinyl)cyclohexanol ( Figure 35) [115]. In addition, the piperidinyl ring in phencyclidine is metabolically hydroxylated to a minor extent at position 4 to give 4-phenyl-4-(1-coclohexyl)piperidinyl alcohol [116]. The pharmacologic activity of the piperidinyl-hydroxy metabolite of phencyclidine has not been reported. A tentative inference can be made heeding the phencyclidine metabolic hydroxylation example: when an alicycle and aliphatic heterocycle are parts of the same molecule, metabolic hydroxylation favors the alicycle over the heterocycle. active cis-and trans-4-phenyl-4-(1-piperidinyl)cyclohexanol ( Figure 35) [115]. In addition, the piperidinyl ring in phencyclidine is metabolically hydroxylated to a minor extent at position 4 to give 4-phenyl-4-(1-coclohexyl)piperidinyl alcohol [116]. The pharmacologic activity of the piperidinylhydroxy metabolite of phencyclidine has not been reported. A tentative inference can be made heeding the phencyclidine metabolic hydroxylation example: when an alicycle and aliphatic heterocycle are parts of the same molecule, metabolic hydroxylation favors the alicycle over the heterocycle.
Inferences from Hydroxymethyl Metabolites
Hydroxymethyl groups (-CH2OH), either intrinsic to drug molecules or metabolically formed, play pharmacophoric and/or auxophoric roles. Here, we highlight the significance of the hydroxymethyl group as derived from the relevant cases in Section 2.
Hydroxymethyl groups may be intrinsic to drug molecules, or they may result from the metabolic oxidation of methyl groups bonded to aromatic or alicyclic rings or terminal methyl groups in alkyl chains-i.e., ω carbons. Intrinsic or metabolically formed hydroxymethyl groups are almost invariably metabolically oxidized to carboxyl groups.
The hydroxymethyl metabolites are almost invariably equiactive with the parent drugs, whereas the carboxy metabolites (resulting from the sequential oxidation of the hydroxymethyl metabolites) are mostly inactive with only a few exceptions being equiactive with the parent drugs. These exceptional cases are those in which the hydroxymethyl groups are distant from the primary pharmacophore.
The fact that hydroxymethyl metabolites invariably retain the pharmacologic activity due to the parent drug probably reflects the auxiliary pharmacophoric status of the methyl group from which they have resulted.
Of the hydroxymethyl metabolites that are equiactive with their parent drugs, only that of tolterodine has been developed into a prodrug, which carries the name of fesoterodine ( Figure 20).
Development of Metabolite Drugs and Prodrugs from Parent-Drug Equiactive Metabolites
The metabolite drugs presented in Section 1 include the H1-antihistamines cetirizine ( Figure 24) and fexofenadine (Figure 22), the carboxy metabolites of hydroxyzine and terfenadine, respectively. Both drugs are more hydrophilic than their respective parent drugs and are capable of existing as zwitterions at physiologic pH. Due to these properties, the two drugs do not cross the blood-brain barrier, and accordingly, they do not cause sedation. In addition, the carboxyl group in fexofenadine seems to offer an ionic binding site that is responsible for the removal of cardiotoxic adverse effects from the parent drug terfenadine. The advantages of both cetirizine and fexofenadine that warranted their development into metabolite drugs may be described as intrinsic. However, cases are known in which these advantages are artificially produced, such as in metabolite prodrugs. In such cases, the
Inferences from Hydroxymethyl Metabolites
Hydroxymethyl groups (-CH 2 OH), either intrinsic to drug molecules or metabolically formed, play pharmacophoric and/or auxophoric roles. Here, we highlight the significance of the hydroxymethyl group as derived from the relevant cases in Section 2.
Hydroxymethyl groups may be intrinsic to drug molecules, or they may result from the metabolic oxidation of methyl groups bonded to aromatic or alicyclic rings or terminal methyl groups in alkyl chains-i.e., ω carbons. Intrinsic or metabolically formed hydroxymethyl groups are almost invariably metabolically oxidized to carboxyl groups.
The hydroxymethyl metabolites are almost invariably equiactive with the parent drugs, whereas the carboxy metabolites (resulting from the sequential oxidation of the hydroxymethyl metabolites) are mostly inactive with only a few exceptions being equiactive with the parent drugs. These exceptional cases are those in which the hydroxymethyl groups are distant from the primary pharmacophore.
The fact that hydroxymethyl metabolites invariably retain the pharmacologic activity due to the parent drug probably reflects the auxiliary pharmacophoric status of the methyl group from which they have resulted.
Of the hydroxymethyl metabolites that are equiactive with their parent drugs, only that of tolterodine has been developed into a prodrug, which carries the name of fesoterodine ( Figure 20).
Development of Metabolite Drugs and Prodrugs from Parent-Drug Equiactive Metabolites
The metabolite drugs presented in Section 1 include the H1-antihistamines cetirizine ( Figure 24) and fexofenadine (Figure 22), the carboxy metabolites of hydroxyzine and terfenadine, respectively. Both drugs are more hydrophilic than their respective parent drugs and are capable of existing as zwitterions at physiologic pH. Due to these properties, the two drugs do not cross the blood-brain barrier, and accordingly, they do not cause sedation. In addition, the carboxyl group in fexofenadine seems to offer an ionic binding site that is responsible for the removal of cardiotoxic adverse effects from the parent drug terfenadine. The advantages of both cetirizine and fexofenadine that warranted their development into metabolite drugs may be described as intrinsic. However, cases are known in which these advantages are artificially produced, such as in metabolite prodrugs. In such cases, the metabolite is equiactive with the parent drug, but due to high hydrophilicity, it suffers the disadvantages of low bioavailability and short duration of action. Chemists have responded to this situation by producing ester prodrugs. The two metabolite prodrugs presented in Section 2 are the antipsychotic paliperidone palmitate (Section 2.4.2, Figure 17) and the antimuscarinic fesoterodine (Section 2.4.5, Figure 21). The development of ester prodrugs of hydroxy metabolites equiactive with their parent drugs to attain improved pharmacokinetic properties may be extended to other cases if sufficiently warranted.
Pharmacologic Activity of Carboxy Metabolites
In most of the surveyed drugs in Section 2, where terminal methyls in alkyl chains (ω-carbons), benzylic methyls, and methyls directly bonded to alicycles are metabolically oxidized to the carboxy group, a loss of pharmacologic activity due to the parent drug has been observed. However, two cases, terfenadine and ebastine, are unique in that the oxidation of the benzylic methyl group has resulted in retention of pharmacologic activity. In these two particular cases, the metabolic oxidation of the methyl group to the carboxyl group has taken place at positions distant from the primary pharmacophores: terfenadine and ebastine (Figures 20 and 21, respectively). Of all the metabolically generated polar functional groups in drug molecules, the carboxyl group stands alone in that it is almost completely ionized at the physiologic pH of 7.4. If such metabolic change occurs at a pharmacophoric site, the new state of ion-pairing interactions replacing the van der Waals interaction of the methyl group will tend to change the pharmacodynamics of the parent drug, leading to a loss of activity. On the other hand, the ionized carboxyl group introduces a pharmacokinetic dimension-which substantially enhances the polarity, water solubility, and elimination of the metabolite as per se or as the glucuronide conjugate, thus causing a significant reduction of the metabolite's effective concentration at the receptor. It is noteworthy that when a carboxyl group is involved in zwitterion formation with an aliphatic amino group, such as in fexofenadine, carebastine, and cetirizine, it (the carboxy group) will not be subject to glucuronide conjugation. In fact, glucuronide conjugation has not been reported as a metabolic route for any of the three aforementioned drugs.
3.6.6. Significance of the Carboxy Metabolite of ∆ 9 -Tetrahydrocannabinol The use of Cannabis products, hashish and marijuana, is illegal in many countries and is punishable by law. The detection of cannabis-product use is based on urinalysis of the constituent metabolites. For this purpose, presumptive immunoassays have been developed based on the carboxy metabolite of ∆ 9 -tetrahydrocannabinol, i.e., carboxy-∆ 9 -tetrahydrocannabinol ( Figure 21). Confirmation tests of the presence of this metabolite are carried out by chromatography-mass spectrometry methods such as GC-MS after trimethylsilyl derivatization or LC-MS [117,118].
Primary and Auxiliary Pharmacophores
In the first part of this review series [12], we classified the pharmacophore as primary and auxiliary (secondary or logistic) based on the "message/address" concept suggested by Dr. Portoghese [119]. The case of fexofenadine development as a metabolite drug of terfenadine ( Figure 24) due to the cardiotoxicity of the latter has prompted us to extend the definition of an auxiliary pharmacophore. An auxiliary pharmacophore is a group that may play one of two roles: (a) properly anchoring the primary pharmacophore in the active site of the receptor or enzyme, or (b) interacting with a site other than the primary site (i.e., an auxiliary site) to produce or override an adverse effect or to account for an off-label use of the drug. Hence, the cardiotoxic effect of terfenadine may be explained by the interaction of the three benzylic methyl groups with an auxiliary receptor via van der Waals binding. On the other hand, in fexofenadine, one of the benzylic methyl groups (in terfenadine) has been metabolically oxidized to a carboxyl group, which interacts with the auxiliary site via ionic binding. Due to its higher strength, ionic binding strongly predominates over van der Waals binding and thus dictates, in part, the nature of the pharmacologic activity of drugs in which it occurs.
Conclusions
The occurrence and extent of alkyl moiety hydroxy functionalization in drug molecules is predictable based on the feasibility of intermediate free radicals' formation and stability. On the other hand, the pharmacologic activities of the alkyl moieties-hydroxy and carboxy metabolites may be predicted based on analogy with the reviewed cases. All hydroxymethyl metabolites are pharmacologically equiactive with their parent drugs while most carboxy metabolites are inactive. The development of metabolite ester prodrugs has an extendable potential when the equiactive hydroxy metabolite is characterized by poor bioavailability and/or short duration of action. The pharmacologic activities of the hydroxy and carboxy metabolites resulting respectively from alkyl or hydroxymethyl moiety functionalization are explicable on pharmacodynamic and/or pharmacokinetic grounds. In some cases, metabolic hydroxy and carboxy functionalization of alkyl or hydroxymethyl moieties has enabled distinctions to be made between primary pharmacophores, auxiliary pharmacophores, and auxophores. | v3-fos-license |
2020-07-02T10:31:58.852Z | 2020-06-26T00:00:00.000 | 220647712 | {
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} | pes2o/s2orc | Crystal structure and Hirshfeld surface analysis of 2-{[(4-iodophenyl)imino]methyl}-4-nitrophenol
In the title Schiff base compound, C13H9IN2O3, the hydroxy group forms a intramolecular hydrogen bond to the imine N atom and generates an S(6) ring motif. The 4-iodobenzene ring is inclined to the phenol ring by 39.1 (2)°. The configuration about the C=N bonds is E. The crystal structure features C—H⋯O hydrogen-bonding interactions.
Chemical context
Over the past 25 years, extensive research has been directed towards the synthesis and use of Schiff base compounds in organic and inorganic chemistry as they have important medicinal and pharmaceutical applications. These compounds exhibit biological activities, including antibacterial, antifungal, anticancer and herbicidal properties (Desai et al., 2001;Singh & Dash, 1988;Karia & Parsania, 1999). They may also show useful photochromic properties, leading to applications in various fields such as the measurement and control of radiation intensities in imaging systems and optical computers, electronics, optoelectronics and photonics (Iwan et al., 2007). Schiff bases derived from 2-hydroxy-5-nitrobenzaldehyde are widely used either as materials or as intermediates in explosives, dyestuffs, pesticides and organic synthesis (Yan et al., 2006). Intramolecular hydrogen-atom transfer (tautomerism) from the o-hydroxy group to the imine-N atom is of prime importance with respect to the solvato-, thermo-and photochromic properties of o-hydroxy Schiff bases (Filarowski, 2005;Hadjoudis & Mavridis 2004). Such proton-exchanging materials can be utilized for the design of various molecular electronic devices (Alarcó n et al., 1999). The present work is a part of an ongoing structural study of Schiff bases and their utilization in the synthesis of quinoxaline derivatives , fluorescence sensors (Faizi et al., 2016;Mukherjee et al., 2018;Kumar et al., 2017; and non-linear optical properties (Faizi et al., 2020). We report herein the synthesis (from 2-hydroxy-5-nitrobenzaldehyde and 4-iodoaniline) and crystal structure of the title compound (I), along with the findings of a Hirshfeld surface analysis.
Figure 2
A partial packing plot showing the C-HÁ Á ÁO hydrogen-bonded (thick dashed lines) helical chains about the crystallographic 2 1 screw axis parallel to c.
Figure 3
A partial packing plot showing close contacts (dashed lines) between iodine and the phenolic oxygen of glide-related (x + 1 significant intermolecular interactions present in the crystal. The Hirshfeld surface analysis confirms the role of the C-HÁ Á ÁO interactions in the packing arrangement.
Hirshfeld surface analysis
In order to visualize the intermolecular interactions in the crystal packing of (I), a Hirshfeld surface (HS) analysis (Hirshfeld, 1977;Spackman & Jayatilaka, 2009) was carried out using Crystal Explorer 17.5 (Turner et al., 2017). In the HS plotted over d norm (Fig. 4), white surfaces indicate contacts with distances equal to the sum of van der Waals radii, and the red and blue colours indicate distances shorter (i.e., in close contact) or longer than the van der Waals radii sum, respectively (Venkatesan et al., 2016). The two-dimensional finger print plots are depicted in Fig. 5 Ojala et al., 1999), the OH group is absent and the NO 2 group is replaced by a cyano group. In (E)-5-(diethylamino)-2-[(4-iodophenylimino)methyl]phenol (VEFPED; Kaştaş et al., 2012), the NO 2 is replaced by an N,N diethyl group. In N-(3,5-di-tert-butylsalicylidene)-4-iodobenzene; (MILFET; Spangenberg et al., 2007), the NO 2 group is absent but a pair of t Bu groups occupy the 3,5 positions of the salicylidene group. In 2-{[(4-iodophenyl)imino]methyl}-6-methoxyphenol (SEDBIP; Carletta, et al., 2017), the NO 2 group is absent and a methoxy group is ortho to the hydroxyl. Lastly, in N-(2-cyanobenzylidene)-4-iodoaniline (XOXKIF; Ojala et al., 1999) the NO 2 is absent and the OH is replaced by cyano. All these compounds have an E configuration about the C N bond and form the S(6) ring motif.
Synthesis and crystallization
The title compound was synthesized by condensation of 2-hydroxy-5-nitrobenzaldehyde (11.0 mg, 0.066 mmol) and 4-iodoaniline (14.4 mg, 0.066 mmol) in ethanol (15 ml). After the mixture had refluxed for about 15 h, the orange product was washed with ether and dried at room temperature (yield 60%, m.p. 484-486 K). Crystals suitable for X-ray analysis were obtained by slow evaporation of an ethanol solution.
Refinement
Crystal data, data collection and structure refinement details are summarized in Two-dimensional fingerprint plots of the crystal with the relative contributions of the atom pairs to the Hirshfeld surface along with d norm full.
Figure 4
Hirshfeld surface of the title compound plotted over d norm.
Special details
Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å 2 ) | v3-fos-license |
2018-06-23T17:06:24.825Z | 2014-11-28T00:00:00.000 | 49425391 | {
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} | pes2o/s2orc | Heterogeneous Pd catalysts supported on silica matrices
Palladium catalysts deposited over different types of silica (amorphous silica, mesoporousmolecular sieves, solids obtained by co-condensation of silicate precursors and many others) modified with suitable donor moieties have gained enormous importance due to their wide application as catalysts for cross-coupling and other synthetically useful organic reactions. This work provides an overview of the chemistry of silica-supported palladium catalysts in different types of organic transformations in order to present the major features, advantages and limitations of various supports and immobilised ligands.
Introduction
Palladium-catalysed cross-coupling reactions such as Heck, Suzuki-Miyaura, Stille, Sonogashira etc. have gained enormous importance during the last few decades since many valuable products can be efficiently synthesised using these reactions with high efficiency. 1 In line with current challenges arising from the demands of industrial and ne chemistry, catalysts should exert not only high activity and selectivity to the target products, but they also have to be easily accessible (in terms of their price and synthesis), tolerant to the environment, stable (robust) and recyclable. In view of these requirements, solid catalysts appear to be more suitable for large-scale applications than their homogeneous counterparts. Consequently, supported palladium catalysts represent promising targets in catalyst design not only due to their wide applicability in practically important reactions 2 but also due to their chemical robustness, recyclability, environmental issues as well as economic aspects.
With this regard, silicas full many of the required criteria as suitable solid supports for deposited palladium catalysts. Moreover, silica materials are accessible in a broad variety differing in their structural and textural properties. Besides the commonly encountered amorphous representatives (such as conventional silica), one of the most important and relevant group of siliceous materials relevant to catalysis are mesoporous molecular sieves possessing large surface areas and void volumes. [3][4][5] Pore diameters in most of such materials are in the range between two and few tens of nanometres allowing for anchoring of bulky catalytically active species onto the support. The majority of mesoporous materials possess structures of hexagonal (MCM-41, 6 SBA-15, 7 FSM- 16 (ref. 8)) or cubic (MCM-48, 9 KIT-6, 10 SBA- 16 (ref. 11)) symmetry. In terms of porosity, the materials include those with disordered structures but uniform pore sizes (e.g. HMS, 12 TUD-1, 13 MSU 14 etc.), mesoporous solids with (SBA-2, SBA-12, SBA- 16) or without (SBA-15, MCM-48) large cages, and materials that also contain micropores in the walls of the mesopores (e.g. in SBA-15, 15 KIT-6 (ref. 16)). Several mesoporous materials with similar symmetry can be viewed as smallmesopore and large-mesopore analogues, although they have been synthesised using different procedures. The examples are hexagonal MCM-41 (pore size in the range 1.5-10 nm (ref. 17)) and SBA-15 (3.6-30 nm (ref. 18 and 19)), or cubic MCM-48 (1.6-4.2 nm (ref. 20 and 21)) and KIT-6 (8-10 nm (ref. 22)). Yet another group of silica-based materials, which are potentially applicable as supports, is constituted by delaminated zeolitic materials, such as ITQ-2 (ref. 23) and ITQ-6, 24 representing the so-called two-dimensional zeolites. 25,26 This structural diversity together with favourable chemical properties (stability, chemical inertness, accessibility to functional modications etc.) render these materials very attractive for catalyst design as the basic critical parametersoverall structure and diameter of the porescan be adjusted on purpose.
Mesoporous molecular sieves based on silica or alumina are hydrothermally unstable at high reaction temperatures and also lack active sites, which in turn limit their potential for direct catalytic applications. On the other hand, the possibility to modify silica supports with different reagents allows the anchoring of various functional moieties and thus the preparation of active catalysts for a number of reactions. [27][28][29][30][31][32] Furthermore, textural features of mesoporous molecular sieves offer a large potential for the direct or post-synthetic modication, such as the incorporation of active centres, graing or immobilization of active species including metal complexes or organometallic substrates.
Since silica-based catalysts are mainly utilised in liquidphase reactions, covalent bonding between the active species and the support surface is favoured in order to minimise the undesired leaching of the anchored groups and metal. There have been established several approaches towards the preparation of supported Pd-catalysts (Scheme 1). In general, the functional modication can be performed in one or several separate steps. In the one-step procedure, the substrate containing both the anchoring group (able to participate in the condensation with the surface silanol groups) and functional group linked to the metal must be assembled prior to the graing onto the support. This method usually provides better dened materials with well-distributed active centres but can be complicated by undesired processes, e.g., interactions of the anchoring moiety with the precursor of the active component or, simply, by a difficult preparation of the precursor to be immobilised. If the multi-step procedure is employed, the selected support is rstly covalently modied with a functional reagent possessing the combination of an anchoring moiety and a functional group able to interact with the active catalyst component or another reagent used to construct metal-binding groups. Aer the modication of the support surface (and possible creation of the suitable functional group), the material is loaded with the respective metal precursor. The main drawback of this approach lies in the lack of control of the interaction between the active component and the modied support. The preparation of silica supports containing functional groups (either with the metal or not) can be performed either postsynthetically (graing) or by direct synthesis using cocondensation method (Scheme 1).
The most frequently used modifying agent for either postsynthetic modication or co-condensation is (3-aminopropyl) triethoxysilane (APTES), which is commercially available and comprises both the anchoring (alkoxysilyl) and functional (amino) groups. However, it should be born in mind that the condensation reactions of APTES as well as other trialkoxysilanes need not necessarily proceed completely (e.g., for steric reasons) and the resulting materials thus typically contain nonhydrolyzed SiOR moieties and unreacted SiOH groups at the support surface. 46 Importantly, the terminal amine group makes APTES a good entry towards other functional moieties (Fig. 1). In particular, the simplest set of these APTES-derived (directly of formally) modiers is represented by linear or branched aliphatic aminocontaining groups. Because of its simplicity and versatility, such substances have been widely used for immobilization of palladium species. Already in 1974, Capka et al. prepared immobilised metal (Rh, Pt and Pd) catalysts by treatment of donorfunctionalised inert supports (in particular, silica) with [3-(dimethylamino)propyl]triethoxysilane and other anchoring groups. 47 Later, Sharf et al. 48 reported the preparation of u-aminoalkyl modied supports by treatment of the conventional silica gel with H 2 N(CH 2 ) n Si(OEt) 3 (n ¼ 1, 3), and their subsequent metalation with Na 2 [PdCl 4 ]. Even polymeric aminoalkyl chain can be graed onto the silica support as it was recently reported. 49,50 Nowadays, the materials containing linear or branched aliphatic amino-containing groups ( Fig. 1) with anchored Pd are mainly utilised as catalysts for the Heck, Suzuki-Miyaura and other cross-coupling reactions. 1 Another type of successfully employed surface functional groups belongs to heterocyclic ones (N-heterocyclic in particular; Fig. 1). Supported catalysts possessing N-heterocyclic anchoring groups are mainly represented by materials bearing iminopyridyl (and similar heteroaryl-imino) substituents resulting by condensation of surface-bound 3-aminopropyl groups with 2-pyridinecarbaldehyde or other heterocyclic aldehydes. 51 In general, other N-heterocyclic moieties can be used for metal anchoring 52,53 but they are relatively less commonly used for the preparation supported Pd catalysts. A number of different immobilised N-heterocyclic carbene (NHC) donors ( Fig. 1) have been used for the anchoring of palladium resulting in the formation of highly active NHC-Pd catalysts for C-C bond coupling and other types of reactions. [54][55][56] Recently, we have demonstrated that the aminopropyl groups at the surface are accessible also for conventional amidation reactions with functional carboxylic acids in the presence of peptide coupling agents. 57,58 The resulting functional supports containing thê Si(CH 2 ) 3 NHCOCH 2 Y (Y ¼ NMe 2 , SMe and PPh 2 ) mixed-donor ligating groups were used to immobilise palladium and the catalysts were evaluated in various C-C bond forming reactions. 59,60 Besides the N-based anchoring modiers, other donor groups (such as S-, P-, As-containing) can be also used. In this regard, sulphur-containing modifying groups, particularly those bearing terminal mercapto substituents, are among the most frequently utilised. Shimizu et al. 61 reported the preparation of an SH-functionalised mesoporous silica FSM-16 by treatment with (3-mercaptopropyl)triethoxysilane. A material based on amorphous silica was obtained by hydrolysis/ condensation reaction of the same mercapto precursor with tetraethyl orthosilicate (TEOS). The sulphur ligands in the sizerestricted mesopores of FSM-16 were the most effective for preventing aggregation of Pd species, which in turn resulted in a high durability and good recycling characteristics of the prepared supported catalysts. 61 The interest in new catalysts deposited on solid supports equipped with phosphine donor moieties was undoubtedly stimulated by analogies with highly efficient homogeneous (molecular) transition metal-phosphine catalysts. [62][63][64] However, the catalysts containing phosphine ligands are oen unstable at higher temperatures and the procedures for their preparation are rather complicated since the synthesis of the phosphine ligands requires multi-step reactions sequences and an exclusion of possible oxidants (or temporary protection of the phosphorus centre). 65,66 As an example of an As-containing catalyst can serve silica-supported poly- [3-(diphenylarsino)propyl]siloxane-palladium complex prepared via immobilization of (3-chloropropyl)triethoxysilane on fumed silica, followed by treatment with potassium diphenylarsenide and then a palladium source. 67 Most of the aforementioned supported materials bearing different types of donor groups exhibited high efficiency in cross-coupling reactions and could be recovered and reused. 68 In addition to examples mentioned before, there are many reports dealing with the preparation of supported catalysts modied with multidonor moieties (as an example, see Fig. 1) consisting of several donor atoms in positions allowing for chelate coordination. 69,70 Recent reports 71,72 have been devoted to the investigation of in situ transformations of palladium precatalysts before and during the catalytic reaction in order to elucidate the nature of the true active species in coupling reactions. It was established, that one of the most important parameters inuencing the nature of the true form of the catalyst is the temperature, at which the system operates. Donor ligands (N-, S-, P-based) can be indeed a component of truly active, soluble palladium complexes in reactions like Heck or Suzuki-Miyaura coupling. However, reaction conditions, under which such ligands can inuence the catalysis, are usually limited to temperatures below 80 C. 71 At temperatures above 120 C, the palladium catalyst is rapidly reduced to Pd(0) having a strong tendency to form soluble colloids. 72 Aer completion of the reaction cycle, Pd may participate in the next catalytic cycle or return to the colloidal system. When solid-supported Pd(0) particles were used, it was reported that the activity is associated with leached palladium species derived from the metal particles. 73 Aer reaction, the "soluble palladium" was oen observed to redeposit on the solid support. 74 In some cases, it has been suggested that the reaction occur on the palladium metal surface, 75,76 however no clear evidences have been presented that conrm the exceptional participation of palladium surface as true catalytic sites.
Although many catalytic systems were assumed to operate via mechanism involving the Pd(II)-Pd(IV) couple, there are no unambiguous data corroborating the involvement of this catalytic cycle in coupling reactions such as Suzuki-Miyaura, Heck or Sonogashira. [77][78][79] The various reactions that potentially could proceed through the Pd(II)-Pd(IV) mechanism have been consistently shown to proceed via a traditional Pd(0)-Pd(II) cycle. 71 The aim of this review is to provide an insight into the stateof-the-art in the eld of the application of silica-supported palladium catalysts in different types of reactions (crosscoupling, carbonylation/carboxylation, redox processes etc.). Features, advantages and limitations associated with the various supports and ligating groups are discussed. The article is organised with respect to the reactions in which the catalyst were employed so as to allow for a comparison of different approaches towards the design of catalysts for a particular type of organic transformation, covering the literature up to 2014.
Although many examples of C-N, C-O and C-S coupling reactions catalysed by Pd anchored to solid supports have been reported, [80][81][82][83] the C-C couplings still remain the main topic of interest at the moment. Hence, the main part of this review is devoted to C-C bond forming reactions though various C-X (X ¼ O, N, S) couplings are also mentioned as they result in the formation of valuable functional molecules such as amides, esters, heterocyclic compounds, etc.
Heck coupling
The reaction of alkyl-or aryl-substituted alkenes containing at least one hydrogen atom at the C]C double bond with aryl, benzyl, and vinyl halides or triates, which proceeds under the formation of new C-C bond and affords a substituted alkene, is known as the Heck coupling (or Mizoroki-Heck reaction; Scheme 2). It is generally catalysed by palladium species generated in situ from various Pd(II) salts or complexes and requires bases (organic or inorganic) to neutralize the acid HX formed during the reaction. 84 A wide range of functional groups, both in the alkene and in the halide, is compatible with the Heck coupling, 84 which renders the reaction synthetically robust and thus practically widely applicable. Mechanism of the Heck reaction is still a matter of debate because it is not yet fully evidenced whether the Pd(0)/Pd(II) or the Pd(II)/Pd(IV) redox couple is involved in the catalytic cycle. [85][86][87] One of the major drawbacks of the early catalytic systems was the precipitation of palladium black that limited the lifetime of the active species and, thereby, the overall catalytic efficacy. Consequently, numerous attempts have been made to circumvent these limitations by the development of new active molecular catalysts as well as through anchoring of the Pd species onto solid supports.
For instance, Zhu et al. 88 prepared a series of Pd(0) catalysts deposited over anilino-and pyridylamino-modied silica gel. These catalysts performed very well in Heck coupling of iodobenzene with styrene or acrylamide (TON up to ca. 17 000), being recyclable without a loss of activity.
Clark and co-workers reported the preparation of mesoporous silica gel modied by iminopyridine groups via the reaction of aminopropylated silica with 2-pyridinecarbaldehyde. These supports were subsequently metallated with a palladium(II) salt. The resulting materials proved to be active and reusable catalysts for Heck coupling of iodobenzene and methyl acrylate. 89 High catalytic activity of these catalyst generated considerable controversy, since the palladium was immobilised in the +II oxidation state, which raised questions about the mechanism of the coupling reaction and the possibility of the involvement of the Pd(II)/Pd(IV) couple in the catalytic cycle.
In order to obtain supported Pd(0) catalyst for the Heck reaction, Zhou and co-workers 90 graed mesoporous sieve MCM-41 by (EtO) 3 Si(CH 2 ) 3 NH 2 and then treated the modied support with PdCl 2 and reduced the deposited Pd(II) to Pd(0) Scheme 2 The Heck coupling reaction.
with Na [BH 4 ]. The resulting catalyst efficiently promoted Heck coupling of substituted iodobenzenes and activated alkenes with yields up to 98% (TOF was in the range of 60-160 h À1 ). The catalyst exhibited unchanged efficiency aer a prolonged exposure to air and could be recycled at least two times without any signicant loss of activity.
Later on, Zhou et al. used a series of amine-Pd(0) complexes deposited on fumed silica surface modied with thê Si(CH 2 ) 3 NR 2 groups (R ¼ H, Et, Bu) in the same reaction. 91 The catalysts exhibited comparably high yields, which quite signicantly depended on the nature of the solvent used. With the decrease in solvent polarity in the series DMF > EtOH > CH 3 CN > C 6 H 12 , the yield of the coupling product in the reaction of iodobenzene and acrylic acid over catalyst with R ¼ H decreased as follows 99.1 > 90.5 > 88.9 > 83.2%. However, for other catalysts this dependence was not monotonous indicating that the inuence of the polarity of solvent is not straightforward and general in this case.
Wang et al. 92 have shown that Pd(0) immobilised on conventional silica gel modied with^Si(CH 2 ) 3 NH 2 or Si(CH 2 ) 3 NH(CH 2 ) 2 NH 2 groups behaves as an active and recyclable catalyst in the Heck reaction of iodoarenes, bromoarenes, and even activated chloroarenes.
Demel et al. studied the relation between the nature of the nitrogen modifying group and the catalytic activity as well as the amount of leached metal in the Heck reaction over Pd(0) immobilised on mesoporous molecular sieves SBA-15 and MCM-48 performed under conventional (solvothermal) conditions as well as under microwave irradiation. 93 Among the catalysts modied with different alkylamino groups, the best properties in terms of activity and stability were exerted by those possessing the^Si(CH 2 ) 3 NH 2 and^Si(CH 2 ) 3 NH(CH 2 ) 2 NEt 2 groups. In contrast, material possessing the branched multi-donor^Si(CH 2 ) 3 NH(CH 2 ) 2 N((CH 2 ) 2 NH 2 ) 2 groups proved to be catalytically inactive and could be even used as an efficient catalytic poison (metal scavenger). It was concluded that not only the number of nitrogen atoms in the anchoring group but rather their type is the decisive factor controlling the stabilization (and availability) of the metal component in the reaction system.
A follow-up study on the inuence of the metal loading (and Pd/N molar ratio) in SBA-15 sieve modied with thê Si(CH 2 ) 3 NH(CH 2 ) 2 NEt 2 groups (Fig. 2) revealed an increase in the yield of n-butyl cinnamate in the Heck reaction of bromobenzene and n-butyl acrylate upon decreasing the Pd/N ratio in the catalysts. 94 As the Heck reaction over supported palladium catalysts typically takes place in solution with the metal rstly leaching out from the support and then returning back to the solid matrix, the observed trend was explained by a lower accessibility of the palladium for the organic reagents when the donor nitrogen groups are present in a large excess.
In contrast, Wang et al. 95 found a non-monotonous dependence of the catalytic activity and Pd leaching on the Pd/N molar ratio in SBA-15 sieve modied with the^Si(CH 2 ) 3 NH(CH 2 ) 2 NH 2 groups using the model coupling reaction of iodobenzene and acrylic acid. Catalyst having Pd/N ratio of 2 : 1 showed moderate leaching (0.1 ppm) at the highest yield of the target product (cf. the yields of 98% vs. 59 and 86% for catalysts with Pd/N ratio 2 : 1, 4 : 1 and 1 : 1, respectively). 95 More recently, it was demonstrated that the nature of the support (amorphous silica vs. mesoporous molecular sieve SBA-15) only insignicantly affects the catalytic performance of Pd(0) supported catalysts modied with^Si(CH 2 ) 3 NH(CH 2 ) 2 NEt 2 groups in the Heck reaction of n-butyl acrylate with bromobenzene. 96 Slightly higher conversions achieved over amorphous silica-based catalysts were due to a better availability of the active metal in this support. The role of mesopores was assumed to be limited only to altering the rate of diffusion of palladium species into the liquid phase (leaching).
Vallribera et al. have proposed the method of preparation of silica aerogels doped with Pd nanoparticles based on cohydrolysis of tetraethoxysilane (TEOS) and organofunctional alkoxysilanes of the type (CH 3 O) 3 Si(CH 2 ) 3 A, where A is a potential donor group, subsequent reaction with a Pd(II) salt. 97 The obtained materials were tested in the Heck reaction of iodobenzene with ethyl acrylate, styrene and methyl vinyl ketone and compared with carbon aerogels loaded with Pd. In general, silica-based catalysts were found to be more active and stable. For example, the reaction of iodobenzene with ethyl acrylate was complete aer 8 h using Pd/SiO 2 aerogel catalyst, which could be reused three times. Reactions in the presence of Pd-carbon aerogel catalyst required longer reaction times (24 h) to achieve similar results (up to 100% conversion). A better dispersion of the metal through silica matrix achieved during sol-gel process rendering the aggregation of the Pd particles more difficult was proposed as possible explanation of these results.
Mandal et al. 98 entrapped Pd and Pt nanoparticles on the surface of micrometer-sized zeolite NaY particles functionalised with the^Si(CH 2 ) 3 NH 2 groups. The synthesised materials were shown to be highly active and recyclable catalysts for the Heck coupling of iodobenzene with styrene to give a mixture of cis-and trans-stilbene (conversions ca. 95% in 1-3 runs; TOF z 1900 h À1 ; cis/trans ratio ca. 10 : 90). The high activity of the catalysts was attributed to an efficient stabilization of the metal nanoparticles on the zeolite surface preventing their aggregation and providing sufficient accessibility of the active centres for the reactants.
In the eld of catalyst containing sulphur donor moieties, Martra et al. 99 found that thiourea groups PhNHC(S) NH(CH 2 ) 3 Si^tethered to silica can reduce Pd(II) ions to afford Pd nanoparticles of about 2 nm in size that behave as a good catalyst in the Heck reaction of iodo-and bromoarenes with nbutyl acrylate. However, a progressive decrease in the yield of the coupling product took place upon recycling of this material due to a signicant leaching. Improvement of the catalytic performance, namely a higher activity at a negligible leaching of active species, was achieved through calcination at 500 C that altered not only the size of the particles and the removal of organic residua but, more importantly, changed the surface structure of the supported particles.
High and reproducible catalytic activity and signicantly lower leaching of metal species from SBA-15 functionalised witĥ Si(CH 2 ) 3 SH groups in comparison with aminopropylated counterparts (3 ppb vs. 35 ppm of Pd) was observed by Crudden et al. 100 The authors noticed an excellent metal scavenging effect of the mercaptopropylated support overcoming that of the related aminopropylated material. 101 Supportive information on the structure of these catalysts, e.g., about the oxidation state of the deposited palladium and its electronic environment, was inferred from X-ray photoelectron and X-ray Auger spectroscopic measurements. 102 Increasing leaching of Pd species with increasing S : Pd ratio in SBA-15 functionalised witĥ Si(CH 2 ) 3 SH groups in the Heck reaction of styrene with bromoacetophenone was described by the same authors, 103 being rationalised by increased mobility of the Pd species at lower concentrations of the ligating groups.
MCM-41-supported poly[(3-mercaptopropyl)siloxane palladium(0)] complex was shown to be highly active and stereoselective catalyst for the Heck arylation of conjugated alkenes with less reactive bromoarenes; the catalyst was reused at least ve times without a loss of activity. 104 Similar MCM-41-based materials obtained by functionalization of silica support with the^Si(CH 2 ) 3 SH groups, followed by treatment with palladium chloride and reduction using K[BH 4 ] were tested in the Heck reaction of aryl iodides with acrylic acid, methyl acrylate and styrene, achieving yields higher than 80% and long-term stability. 105 Cai and co-workers 106 rstly synthesised MCM-41-supported thioether-palladium(0) complex by immobilization of [3-(2cyanoethylsulfanyl)propyl]triethoxysilane, reacting with palladium chloride and subsequent reduction with hydrazine hydrate. The prepared material showed high activities and stereoselectivity in the Heck coupling of conjugated alkenes with aryl iodides and activated aryl bromides. It also efficiently catalysed the arylation of conjugated alkenes with non-activated bromoarenes. Increasing amount of palladium catalyst was shown to shorten the reaction time, but did not increase the yield of the target alkenes.
A uorous organic-inorganic hybrid material (Fig. 3) was prepared by sol-gel condensation between a monosilylated uorous precursor and a large excess of TEOS and used as a support for Pd(0) nanoparticles (diameter 4 AE 1 nm). The resulting material proved to be active in the Heck reaction of iodobenzene with butyl acrylate. 107 However, the authors did not investigate whether the Pd(0) nanoparticles on the solid surface are the actual catalysts or just represent the source of catalytically active species for the reaction occurring in the liquid phase. The same authors demonstrated that the amount of catalytically active Pd(0) nanoparticles depends on the number of the long peruoroalkyl chains in the uorous organic-inorganic matrix. The bis-silylated compound possessing two anchoring groups, 2,4-bis[3-(triethoxysilyl)propylamino]-6-S(CH 2 ) 2 C 8 F 17 R-1,3,5-triazine, allowed to increase the Pd(0) content from 4 to 13% and to carry out the catalytic Heck reaction with a lower quantity of solid material in the reaction mixture. 108 Pd(0) deposited on MCM-41 silica matrix bearing amino and phosphinoalkoxy groups was prepared by immobilization of APTES on the solid support, subsequent reactions with (diphenylphosphino)methanol and palladium chloride and reduction with hydrazine hydrate. Similarly to MCM-41supported poly[(3-mercaptopropyl)siloxane palladium(0)] complex obtained earlier, this Pd catalyst deposited over phosphino-functionalised MCM-41 was an active and stereoselective catalyst for the Heck arylation of conjugated alkenes with bromoarenes. 109 Similar approach was employed by Singh et al. 110 for immobilization of Pd(0) on silica gel. The prepared materials catalysed C-C coupling reactions of substituted aryl bromides and iodides with acrylic acid or styrene in yields up to 92%. The catalysts could be reused 15-16 times without any signicant decrease in the yield of the coupling product. Their further reuse up to 30 times was also possible but the yield was reduced by 10-15%.
A Pd(II)-SCS-pincer complex (A in Fig. 4) was covalently immobilised on mesoporous silica SBA-15 and evaluated as a precatalyst in Heck reaction. 111 Kinetic experiments and poisoning studies indicated that the pincer complexes decompose under reaction conditions via rupture of the palladium-ligand bonds to liberate active Pd(0) species catalysing the reaction of iodobenzene with n-butyl acrylate. 112 The same conclusion was made for the structurally related Pd/PCP-pincer complex (B in Fig. 4) graed onto the same support. 113 A saturated Pd-NHC complex was immobilised on amorphous silica by Artok and co-workers (Fig. 5). 114 The complex itself was found thermally stable. However, TEM observations, hot ltration, reusability and poisoning tests revealed that the complex acted only as a precursor of active Pd species in the Heck reactions when immobilised. The complex appears more stable when used in homogeneous system (TON in the range 10 4 to 10 5 for the reaction of bromobenzenes with styrene to give trans-stilbenes). Karimi and Enders 115 prepared a Pd-NHC complex on a silica support (via immobilised ionic liquids) and tested this material in the Heck reaction of aromatic halides with styrene and acrylic esters. Aryl iodides and bromides afforded good conversions (78-99%), while chlorides did not react under the conditions used. The catalyst was recycled four times with only a minor loss of its activity.
Suzuki-Miyaura reaction
Suzuki-Miyaura reaction (Scheme 3) is a Pd-catalysed crosscoupling reaction between organic boron compounds (usually, boronic acids or boronic esters) and aryl or alkyl halides/ triates proceeding under relatively mild reaction conditions. 116 Similarly to the Heck coupling, this reaction is believed to proceed through the sequence of oxidative addition, transmetalation and reductive elimination, and requires a base for the activation of the boron component. Suzuki-Miyaura coupling represents a relatively simple, condition-tolerant and versatile C-C bond forming reaction, which can be extended to various substrates and nds therefore wide applications in the synthesis of various simple and complex organic molecules, pharmaceuticals and natural products. 117 Mubofu et al. prepared a highly active (TONs in the range of 10 3 based on ten reuse experiments from batch reactions), recyclable and robust supported palladium catalyst for Suzuki reaction between phenylboronic acid and bromobenzene. The catalysts were obtained by interaction of aminopropylated silica with pyridine carbaldehyde and subsequent complexation of palladium acetate. 118 A series of silica supported palladium catalysts bearing N-N, N-S and N-O chelating ligands ( Fig. 6) have been prepared by Clark et al. 119,120 using three-step procedure consisting of modication of activated silica with (3-aminopropyl)trimethoxysilane, Schiff base condensation with 2-acetylpyridine (A), 2-pyridinecarbaldehyde (B), 2-thiophenecarbaldehyde (C), 2-furancarbaldehyde (D), 2-acetylfuran (E), 2-hydroxyacetophenone (F), 2-aminoacetophenone (G) or 2-pyrrolecarbaldehyde (H) and, nally, metallation with palladium acetate. A clear but not linear relationship between the Pd binding energies from XPS measurements and the structure of the chelating ligands or rate constants in the Suzuki reaction between benzeneboronic acid and bromobenzene was found. The activity of prepared catalysts decreased with increasing binding energy in the following sequence: A < E ¼ F < G < C < D < B < H (Fig. 6).
MCM-41-supported, possibly terdentate nitrogen ligand and its palladium(II) complex ( Fig. 7) was synthesised and tested in Suzuki-Miyaura coupling between aryl bromides and arylboronic acids in ref. 121 Various electron-donating and electronwithdrawing groups such as CH 3 , OCH 3 , Ph, Cl, CN, NO 2 , CF 3 , COCH 3 , and CO 2 CH 3 in both aryl bromide and arylboronic acid components were found to be well tolerated and the reaction gave the desired substituted biaryls in good to excellent yields (up to 99%). Even the reactions of bulky 1-naphthylboronic acid with aryl bromides proceeded satisfactorily, providing the coupling products in 81-85% yields. The catalyst was found to be reusable at least 10 times with a negligible loss in activity. The high stability and excellent reusability of the catalyst was Silica supports modied with ligands of three different types (containing amino, diamino and pyridyl moieties) were used to prepare supported Pd(II) complexes for Suzuki coupling reactions by Vassylyev et al. 122 The activity of the immobilised catalyst was found to be signicantly higher in comparison with the respective molecular complexes, which was regarded as an evidence that heterogeneous and not homogeneous Pd contributed to the activity of the supported catalysts. Different catalyst required different base additives in order to achieve optimal results, which was explained by variation in the energy of transition complex formation upon changing the coordination sphere of Pd.
Another series of supported catalysts containing different amounts of palladium was prepared by treatment of SBA-15type mesoporous molecular sieve bearing thê Si(CH 2 ) 3 NH(CH 2 ) 2 NEt 2 groups at the surface with palladium(II) acetate. 94 These catalysts were studied in Suzuki biaryl couplings to establish the inuence of metal loading and "innocent" surface modications (trimethylsilylation). It was found, that Suzuki reaction proceeds efficiently with both model and practically relevant substrates. The catalyst performance increased with an increasing degree of metalation (decreasing N/Pd ratio). Furthermore, catalyst poisoning tests revealed that the reaction takes place in the liquid phase with the catalyst serving as a reservoir of active metal species and also as a stabilizing support.
Pleixats and co-workers 123 described the preparation of organic-inorganic hybrid mesostructured worm-like and hexagonally organised silica materials containing imidazolium and dihydroimidazolium salts from mono-and disilylated monomers, loaded these materials with Pd(II) and investigated the activity and recyclability of the resulting catalysts in Suzuki-Miyaura cross-coupling. While all prepared materials showed efficient and fast reactions between the activated 4-bromoacetophenone and phenylboronic acid giving almost quantitative isolated yields of the target product in 0.5-1 h, Pd(II) supported on worm-like hybrid support showed slightly better reusability than hexagonally ordered material, as no decrease in the activity was observed aer ve cycles and almost quantitative yields of the coupling product were isolated during these runs. Only Pd(II) catalysts supported on worm-like hybrids were tested as a recyclable catalyst for the Suzuki coupling of phenylboronic acid with the less reactive 4-chloroacetophenone. 123 Although the yields were modest (34%), the reusability of these catalysts was established with this rather challenging substrate. Highresolution transmission electron microscopy analysis of the Pd(II) catalyst supported on worm-like hybrids recovered aer the 5th catalytic run revealed the formation of Pd nanoparticles (diameter ca. 3 nm).
Gruber-Woeler et al. 124 applied a two-step procedure to prepare a Pd-catalyst from silica gel-tethered bis(oxazoline) (BOX) ligand. In particular, the covalent immobilization of 2,2 0 -(1-methyl-11-dodecenylidene)bis(4,5-dihydrooxazole) on 3mercaptopropyl-functionalised silica gel, followed by metalation with Pd(OAc) 2 provided a stable and active catalyst. The superior behaviour of the prepared catalyst in comparison with homogeneous (BOX/Pd(OAc) 2 ) and in situ immobilised catalysts in Suzuki coupling reaction under various conditions was rationalised by self-deactivation of Pd(OAc) 2 in liquid phase and by the poisoning effect of thiol groups in the in situ prepared catalyst. Using hot ltration test, three phase test, and ICP/OES analysis, the authors showed that there is virtually no Pd leaching into the reaction solution under the applied reaction conditions. Furthermore, the catalyst was found to be chemically stable and can be reused for at least 10 times.
Tyrrell and co-workers 125 studied the activity of Pd-supported catalysts prepared via two complementary methodologies (i.e., by complexation of graed imidazolium moieties and by immobilization of dened complexes). A comparison of catalyst prepared by immobilization of the imidazolium salt to silica before the formation of the NHC complex (route A, Scheme 4), and catalyst resulting via graing the NHC complex directly onto silica (route B, Scheme 4), revealed that the former one provides an 84% yield of the coupling product at the 0.2 mol% metal loading, while second one gave a 90% yield under the same conditions. These data suggest that the catalysts prepared by immobilizing the pre-formed palladium complexes is slightly more active in the Suzuki coupling of aryl bromides than that prepared by the reaction of palladium acetate with the tethered imidazolium salt. Furthermore, the use of sterically bulky NHCs (such as the 2,6-(diisopropyl)phenyl-substituted ligand) resulted in an increased catalytic activity (conversions of >99% under the same reaction conditions), suggesting that bulkiness of the ligand plays an important role in controlling the catalytic activity of tethered palladium NHC catalysts.
Gude and Narayanan investigated the catalytic activity for three types of nanocatalystscolloidal supported metal nanoparticles (CSMNs) prepared with silica colloids in solution, 126 CSMNs prepared from dry silica colloids re-suspended in doubly deionised water, and palladium nanoparticles loaded onto bulk silica dispersed in doubly deionised waterin the Suzuki-Miyaura reaction between phenylboronic acid and iodobenzene. These three types of catalysts were prepared with and without the addition of APTES. The CSMNs obtained from non-functionalised wet silica colloids and palladium nanoparticles surprisingly showed the highest catalytic activity as compared to their counterparts prepared using the APTES linker. The authors assumed that APTES could act as a catalyst poison resulting in a lower catalytic activity in the Suzuki reaction. 126 In an attempt to clarify the effectiveness of a tripodal linker unit (e.g. 3-bromopropyltris[3-(dimethylisopropoxysilyl)propyl]silane), which can be bound to a silica surface via three independent Si-O-Si bonds, silica-immobilised palladium amine complex catalysts employing this linker unit were prepared and applied in the Suzuki coupling reaction of 4-bromobenzoic acid ethyl ester and phenylboronic acid. 127 Pd(II) supported on functionalised silica containing 3-(methylamino)propyl moiety, which comprises a secondary amine ligand, gave a higher yield than Pd(II) graed on support bearing tertiary amine modifying group (i.e. 3-(dimethylamino)propyl). Pd(II) catalyst resulting from the chelating ligandcontaining support exhibited a low catalytic activity. However, the hot ltration test suggested a substantial contribution from homogeneous catalytic reaction due to leached-out palladium species for the most active catalyst based on 3-(methylamino) propyl ligand. Thus, the authors concluded, that the order of activity among the catalysts with different amino ligands just reects the degree of palladium dissociation from the palladium complexes immobilised on the silica support. 127 Rossi et al. 128 reported the preparation of supported palladium nanoparticles stabilised by pendant phosphine groups by reacting a palladium complex [Pd(OAc) 2 L 2 ], where L is 2-(diphenylphosphino)benzaldehyde, with an aminopropylfunctionalised silica. The material proved to be an active catalyst in the Suzuki-Miyaura cross-coupling. Catalyst deactivation caused by silica etching and Pd nanoparticles aggregation was observed in the successive runs and demonstrated to be partly minimised upon following an alternative protocol consisting of addition of the base to the arylboronic acid solution prior to the contact with the catalyst. This approach allowed for catalyst reuse for up to ten recycles, while keeping similar catalytic activity during seven successive reactions.
A Pd(II) catalyst derived from MCM-41 functionalised with NNN-donor groups has been synthesised by Dhara et al. 129 by anchoring of APTS in the MCM-41 mesoporous molecular sieve, followed by reaction with 2,6-diacetylpyridine to give a N3-type Schiff base and metalation with Pd salt. The prepared material behaved as a highly active catalyst for the Suzuki-Miyaura cross-coupling reaction of both electron-poor (yields 92-98% aer 3 h TOS, TOF > 4400 h À1 ) and electron-rich (yields 90-95% aer 4-8 h TOS, TOF > 2200 h À1 ) aryl halides with phenylboronic acid. Hot ltration and three phase test excluded any signicant leaching of Pd species. 129 The organosilane-based NHC-Pd complex immobilised covalently on silica was shown to be efficient reusable heterogeneous catalyst in the Suzuki reaction of various aryl halide Scheme 4 Pd-supported catalyst prepared via complexation of grafted imidazolium moieties (A) and immobilization of defined complex (B). derivatives (except for aryl chloride derivatives) with phenylboronic acid under mild conditions. 130 Crudden et al. 100 demonstrated a high catalytic activity of Pd immobilised on mercaptopropyl-modied SBA-15 in Suzuki-Miyaura reactions of bromo-and chloroaromatics. Heteroaromatic substrates such as 3-bromopyridine and deactivated substrates such as 4-bromoanisole also underwent coupling reaction with good to excellent yields (82-99%). Reproducible high catalytic activity was observed in DMF, water or a mixture of the two solvents. In contrast to Pd immobilised on mercaptopropyl-modied silica, these materials could be reused several times with virtually no loss of activity, even in water. Later, it was shown that the method of introducing the alkylthiol groups inuences the stability of such Pd-supported catalysts. 131 A graing approach gave a signicantly more stable material, likely owing to protection of the siliceous surface from attack by the aqueous base, while the incorporation of the thiol by co-condensation provided a material with a minimal stability under the reaction conditions. Mesostructured mercaptopropyl-modied SBA-16 and KIT-6 materials were also shown to be viable solid supports for the immobilization of Pd(0) and Pd(II) complexes. 132 The behaviour of the last ones in the Suzuki-Miyaura coupling reaction of 4-bromoacetophenone and phenylboronic acid pinacol ester was comparable to that of Pd immobilised on mercaptopropyl-modied SBA-15.
Sullivan and co-workers 133 reported on Pd(II) complexes immobilised on porous silica (6 nm pore size) modied by disuldes (MeO) 3 Si(CH 2 ) 2 S(CH 2 ) n S(CH 2 ) 2 Si(OMe) 3 (n ¼ 2, 3), that showed high activity, reusability and resistance in the Suzuki-Miyaura reaction of aryl bromides with arylboronic acids at room temperature in isopropanol. The same authors 134 used co-condensation of TEOS with methyl[3-(triethoxysilyl)ethyl]thioglycolate to prepare^SiCH 2 CH 2 SCH 2 CO 2 Me modi-ed silica. Subsequent hydrolysis of the anchored ester group and reaction with palladium(II) acetate afforded a stable and recyclable supported Pd-catalyst (presumably with O,S-chelated Pd centres), which was successfully tested in Suzuki-Miyaura cross-coupling of aryl bromides with phenylboronic acid. 134 SBA-15 mesoporous support silylated at the outer surface with PhSi(OMe) 3 (prior to removal of the template) was functionalised with Ph 2 PC 6 H 4 CH 2 S(CH 2 ) 3 Si(OEt) 3 and then metalated with [Pd 2 (dibenzylideneacetone) 3 ] to afford an immobilised Pd-"triphenylphosphine" catalyst (Fig. 8). This recyclable catalyst was employed in Suzuki-Miyaura reaction of aryl bromides with arylboronic acids carried out in supercritical carbon dioxide, from which the reaction products separated upon cooling (isolated yields of biphenyls were in the range 83-99%). 135 MCM-41-supported bidentate phosphine Pd(0) complex synthesised by treatment of MCM-41 with APTES and then with (diphenylphosphino)methanol and palladium chloride and subsequent reduction with hydrazine hydrate mentioned above was shown to efficiently catalyse Suzuki-Miyaura reaction of aryl iodides and bromides with arylboronic acids (yields 84-98%). The negative hot ltration test was regarded by authors as an evidence of true heterogeneous nature of this catalyst. 136,137 Suzuki-Miyaura cross-coupling reactions of various aryl chlorides with arylboronic acids were examined also under different reaction conditions over Pd(0) immobilised on mesoporous-supported aryldicyclohexylphosphine, synthesised using a four-step approach (Scheme 5). 138 High conversions and yields (81-98%) were achieved for activated aryl chlorides.
Corma et al. 139 attempted to develop another heterogeneous Pd-supported catalyst by anchoring 2-dicyclohexylphosphino-2 0 ,6 0 -dimethoxy-1,1 0 -biphenyl (S-Phos) ligand on different solid supports such as non-soluble (cross-linked polystyrene) and soluble (non-cross-linked polystyrene and polyethyleneglycol) polymers, as well as high surface silica. The immobilised ligand was in situ converted to a palladium-phosphine complex. While the complete conversions and high selectivity to the expected trimethylbiphenyl was observed in Suzuki reaction of 2-chlorom-xylene and 2-tolylboronic acid for the two SPhos ligands anchored on soluble supports, SPhos anchored on silica proved to be completely inactive in this coupling reaction.
Jin et al. 140 reported the synthesis of hierarchical micromesoporous LTA zeolite-supported b-oxoiminatophosphinopalladium complex (Scheme 6) and its application as a catalyst for Suzuki-Miyaura reaction of aryl and heteroaryl chlorides. The catalyst achieved typically 80-90% conversions with 1 mol% Pd in aqueous ethanol in 5 h.
Sonogashira reaction
The Sonogashira reaction (Scheme 7) is a palladium-catalyzed sp 2 -sp cross-coupling reaction between a terminal alkyne and an aryl or vinyl halide/triate performed with or without the presence of a copper(I) co-catalyst. 141 It has become one of the most important methods to prepare arylalkynes and conjugated enynes, which represent reactive intermediates en route to many natural products and pharmaceuticals. [142][143][144][145][146] Palladium immobilised on silica modied with^Si(CH 2 ) 3 -NHCH 2 CH 2 NH 2 pendant groups was studied in the Sonogashira reaction of terminal alkynes with aromatic iodides. Reactions performed at 1 mol% Pd loading in reuxing ethanol with potassium carbonate as the base generally provided good yields of the coupling products (>90%). Reactions with aryl bromides typically required a higher temperature (110 C in DMF) to achieve similar conversions. The catalyst proved to have excellent stability, being used during as much as 30 consecutive runs without any decrease in the activity. 147 Asefa and co-workers 148 Sugi et al. investigated the catalytic activity of an quinoline-2carboimine palladium complex immobilised on MCM-41 ( Fig. 9) in Sonogashira reaction of various terminal and aliphatic alkynes with aryl and halides. 149 The catalyst provided excellent yields (>93% in most of the cases) of the corresponding cross-coupling products in reactions of terminal alkynes containing aryl substituents. On the other hand, only moderate catalytic activity was observed in reactions of an aliphatic alkyne due to a lower acidity of its terminal proton. The supported Pd catalyst was reusable without signicant loss of catalytic activity until the fourth recycle under aerobic conditions. Its catalytic performance was ascribed to effective catalysis by the quinoline-2-carboimine palladium complex immobilised on MCM-41 support with high BET area and to a high air-stability of the catalyst.
Analogous Pd catalysts obtained from imine-modied supports resulting by reactions of conventional aminopropylated silica with 2-pyridinecarbaldehyde, 2-acetylpyridine, or 2-(diphenylphosphino)benzaldehyde were efficient catalysts for Sonogashira reaction of ethynylbenzene (phenylacetylene) with different aryl iodides affording the respective substituted tolanes. 76 Possible beneciary effect of the phosphine donor groups arising from 2-(diphenylphosphino)benzaldehyde was partly eliminated by their oxidation to the corresponding phosphine oxide occurring during the catalyst preparation as evidenced by solid-state 31 P NMR measurements.
Trilla and co-workers 150 rstly synthesised hybrid silica materials with anchored di(2-pyridyl)methylamine-dichloropalladium(II) (Fig. 10). These catalysts were prepared by sol-gel co-gelication and found to behave as efficient and recyclable catalysts for the Sonogashira reaction of phenylacetylene and aryl bromides or iodides. 150 Notably, no Pd nanoparticles were formed during the course of the Sonogashira coupling over this catalyst. However, hot ltration test evidenced that both homogeneous and heterogeneous pathways participate in the catalytic process.
Hankari et al. investigated the inuence of the structure of the thiol precursor on the morphology of the mesoporous silica resulting by template-directed condensation or by postsynthetic graing. 151 Catalytic properties of the resulting Pdimpregnated materials were compared with the "classical" mercaptopropyl-functionalised silica obtained from (3-mercaptopropyl)triethoxysilane by direct hydrolysis/cocondensation with TEOS and subsequent impregnation with a Pd(II) salt. Compared with conventional thiol-functionalised materials obtained from mercaptopropyl-trialkoxysilane precursors, the new reported thiol-functionalised silica materials showed signicantly improved recyclability. This result was attributed to a higher hydrophobicity of these materials, imparted by the bulkier bis-silylated amide-thiol precursor. 151
Carbonylation/carboxylation
Oxidative carbonylation of phenol with carbon monoxide and oxygen (Scheme 9) is considered as one of the most practically relevant methods for the preparation of diphenyl carbonate from cheap and simple feedstock reagents. 152,153 Using the solgel technique, Li et al. prepared Pd complexes anchored on silica previously functionalised by silylpropropylated 1,2-diaminocyclohexane and tested these materials as catalysts in this reaction. 154 The highest yield of diphenyl carbonate (12%) was obtained with Cu 2 O as a co-catalyst while THF was the optimum solvent in terms of the yield and selectivity to diphenyl carbonate as well as low palladium leaching. Under the optimal conditions, the TON reached 60 mol of diphenyl carbonate/mol Pd and the metal leaching did not exceed 3%. The heterogeneous catalysts revealed higher catalytic activity compared with the homogeneous counterparts (Pd(OAc) 2 and PdCl 2 ). It was also found, that the Pd loading signicantly affects the reaction rate and the optimum Pd loading was found at ca. 5 wt%. The intermediacy of Pd(0) in the catalytic cycle was veried, while the recovered catalyst was found to contain a mixture of Pd(II) and Pd(0) species.
Chaudhari et al. 155 immobilised Pd and Pt nanoparticles on 3-aminopropylated zeolite NaY. In the presence of iodide promoters, the obtained materials exhibited high catalytic activities in oxidative carbonylation of aliphatic and aromatic amines to give symmetrically disubstituted ureas (Scheme 10; conversions in the range of 69-98%, selectivities > 90%). It was reported, that the immobilised Pd nanocatalyst exerts a higher activity (TOF of 157 h À1 ) than the previously reported materials, which is maintained even at relatively lower temperatures and pressure. [156][157][158] Palladium species immobilised inside functionally modied mesoporous materials (MCM-41, MCM-48, SBA-15) have been also synthesised by Chaudhari et al. 159 The starting material containing 3-aminopropyl functional groups were prepared via different synthesis strategies (viz. post-synthesis graing and one-pot co-condensation). Characterization (in particular by 13 C, 29 Si and 31 P solid-state CP-MAS NMR spectroscopy) conrmed that the anchoring of the complex [Pd(pyridine-2carboxylate)(PPh 3 )(OTs)] occurred as anticipated. The materials showed high activity (TOF 350-750 h À1 ), chemo-and regioselectivity of >99%, stability and recyclability in low-pressure carbonylation of olens and alcohols to carboxylic acids (Scheme 11), with the catalyst based on SBA-15 synthesised by post-synthetic graing approach showing the highest activity.
Lagasi and Moggi prepared triethoxysilyl-substituted amines by Schiff condensation of APTES with 2-acetylpyridine, 2,6-Scheme 8 Sonogashira-Henry tandem reaction. diacetylpyridine, or 2-acetylpyrazine and subsequent reduction using Na [BH 4 ]. The amines were employed in co-condensation with TEOS to afford functionalised silicas. 160 Materials obtained aer treatment with a Pd salt were probed as reusable catalysts for methoxycarbonylation of iodobenzene (Scheme 12). Catalyst prepared from 2-acetylpyridine reached TON of 2300 aer 20 cycles in the presence of triethylamine as a base promoter and also showed the highest stability during recycling tests: 83% conversion of iodobenzene and 97% selectivity to methyl benzoate was achieved aer 20th run without deactivation through leaching of palladium.
Double carbonylation of aryl iodide derivatives with secondary and primary amines to produce a-ketoamides (Scheme 13) over a series of silica supported palladium complexes with varying electronic properties and steric hindrance was described by Genelot et al. 161 Conversions up to 80% and selectivities up to 96% for the double carbonylated aketoamide products (the other product being the respective amide) were achieved in reactions of different aryl iodides with secondary and primary amines. It was demonstrated that two palladium complexes (designed with monodentate phosphine or chelating diphosphine linkers) supported on mesoporous SBA-15 material can be used repeatedly without a notable loss of activity and selectivity.
Lu and Alper 162 reported the synthesis of various dendrimers immobilised on silica support via Michael-type addition and amidation reactions, followed by the phosphinylation with (diphenylphosphino)methanol. The resulting phosphineterminated dendrimers were then reacted with a palladium salt to give the dendrimer Pd-complexes, and the resulting materials were tested in the synthesis of oxygen, nitrogen, or sulfur-containing medium ring fused heterocycles by intramolecular carbonylation reactions (Scheme 14). The dendritic catalysts exhibited very good activities in these transformations, affording the desired heterocycles in high yields (>90% in most of the cases). Importantly, a wide variety of functional substituents were tolerated in this process, including chloro, methoxy, triuoromethyl, cyano, acetyl, and methoxycarbonyl groups, and the catalysts could be used in eight successive cycles with only a slight decrease of their activity (total decrease in the yield was only 5%).
Carbonylative cross-coupling reactions, namely the synthesis of substituted ketones by reaction of arylboronic acids 163 or terminal alkynes 164 with aryl iodides under an atmospheric pressure of carbon monoxide (Scheme 15), have been studied by Cai and co-workers. In the rst case, 3-(2-aminoethylamino) propyl-functionalised MCM-41-immobilised palladium(II) catalyst was applied, while a MCM-41-supported bidentate phosphine palladium(0) complex was used for the carbonylative Sonogashira coupling reaction. These heterogeneous catalysts proved useful in the synthesis of diaryl and aryl-alkynyl ketones as they exhibited higher activity and selectivity than homogeneous catalyst [PdCl 2 (PPh 3 ) 2 ], and showed only negligible decrease in the activity aer several catalytic cycles (cf. 91 vs. 89% yield and 45.5 vs. 44.5 TON in the 1st and 10th run, respectively, in the reaction of arylboronic acids with aryl iodides).
Oxidation of alcohols
Selective oxidation of alcohols represents an attractive method of the production of aldehyde or ketone intermediates for ne chemistry. In particular, aromatic and allylic oxo compounds can be synthesised with high selectivity through selective oxidation of the corresponding alcohols and used as Scheme 11 Carbonylation of olefins and alcohols to carboxylic acids.
Scheme 12
Methoxycarbonylation of iodobenzene to methyl benzoate. components of pesticides, perfumes or avours. 165,166 However, such reactions traditionally utilise toxic and hazardous stoichiometric reagents and generate large quantities of contaminated waste. 167,168 During a search for alternative catalytic routes, Pd-supported heterogeneous catalysts emerged as attractive surrogates facilitating selective transformation of alcohols to aldehydes or ketones under moderate conditions via oxidative dehydrogenation mechanism. 169 Recently, Lee et al. 170 reported about graing of ultrathin alumina monolayers onto SBA-15 silica framework followed by impregnation of the resulting Al-SBA-15 with palladium. The formed Pd nanoparticles showed a good dispersion and surface oxidation characteristic of pristine aluminas with high active site densities typical for thermally stable, high-area mesoporous silicas. This combination allowed for signicant rate enhancements in the aerobic selective oxidation of cinnamyl alcohol to cinnamic aldehyde (Scheme 16) over Pd/Al-SBA-15 as compared with Pd on mesoporous alumina or silica supports investigated by the same group earlier. 171,172 Although Pd/Al-SBA-15 afforded a higher reaction rate in this selective oxidation than a Pd/meso-Al 2 O 3 catalyst, their corresponding TOFs per surface active sites were almost identical suggesting the involvement of similar active species and palladium-support interaction. It was proposed that the higher activity and TOFs observed for alumina (13 500 h À1 ) than silica (6100 h À1 ) supported palladium catalyst reect a superior ability of the former material to disperse and preserve smaller PdO nanoparticles during the oxidation. In addition, operando XAS measurements revealed strong correlations between the palladium oxidation state and its activity, which identify rapid on-stream PdO reduction as the major deactivation pathway that can be partly eliminated by an increase of the oxygen pressure.
Wilson et al. 172 reported similar materials employing amorphous silica as well as SBA-15, SBA-16 and KIT-6 molecular sieves as supports for the Pd particles. The catalysts were tested in selective oxidation of crotyl alcohol and cinnamyl alcohol. The relative activities for both alcohols were dependent on the degree of mesopore connectivity and palladium dispersion/ oxidation state, which corresponds with the previous studies demonstrating the benets of interconnected pore architectures. 173 Three-dimensional interconnected KIT-6 and SBA-16 conferred the highest initial reaction rates (24 000 mmol g À1 h À1 ), that proved to be superior to the two-dimensional SBA-15 (14 000 mmol g À1 h À1 ), which in turn outperformed commercial amorphous silica (6000 mmol g À1 h À1 ). Rate enhancements observed with interconnected mesoporous supports were also explained by their ability to stabilise dispersed palladium nanoparticles (1-2 nm) and thus provide a material with a high palladium oxide surface. Rate analysis and controlled experiments using in situ reduction/oxidation conditions conrmed surface palladium oxide as the active species in selective oxidation of allyl alcohol. On the other hand, slow reduction of palladium nanoparticles under oxygen-limited reaction conditions was shown to reduce the reaction rates and favour undesired hydrogenolysis of cinnamyl alcohol to trans-b-methylstyrene.
Yang et al. 174 used mesoporous molecular sieve SBA-16 modied with (3-aminopropyl)triethoxysilane and hexamethydisilazane to prepare deposited Pd and Pd/Au catalysts, which were tested in solvent-free oxidation of benzyl alcohol to benzaldehyde with molecular oxygen. It was found that aminefunctionalization remarkably improves the selectivity towards the desired product. Au/Pd bimetallic catalysts showed better catalytic performance than the respective monometallic catalysts (Au and Pd). The highest turnover frequency (about 8700 h À1 ) was achieved over a bimetallic catalyst with Au : Pd molar ratio equal to 1 : 5. A similar catalyst has been tested in aqueous phase. 175 In oxidation of different benzylic (Scheme 17) and allylic alcohols in the absence of a base performed under air or in O 2 atmosphere in water, the prepared bimetallic catalysts exhibited conversions up to 99%. The selectivities to the corresponding aldehydes and ketones were better than 99% in all the cases. Importantly, the catalyst could be recovered and reused twelve times without signicant changes in the reaction outcome (conversion and selectivity).
Other Au/Pd bimetallic catalysts supported on layered double hydroxide have been prepared and characterised by Hou et al. 176 These bimetallic catalysts showed much higher activity than the corresponding monometallic catalysts in selective alcohol oxidation (Scheme 18) in water. Authors suggested that synergetic interaction between Au and Pd is responsible for the high efficiency of these Au/Pd catalysts. The leaching test and elemental analysis showed that only a small amount of Pd (0.7 ppm) and Au (traces) were leached out from the catalysts during the reaction.
Li and co-workers 177 utilised mesoporous silica nanoparticles as a support for the preparation of deposited bimetallic Pd-Au catalyst by the conventional impregnationhydrogen reduction method. The resultant materials were evaluated in base-free oxidation of benzyl alcohol with oxygen (see Scheme 17 above). It was found that addition of a small amount of Pd (the Pd/Au atomic ratio was as low as 0.05/1) signicantly decreases the size of the gold particles and thereby remarkably enhance the catalyst activity. At the optimal Pd/Au atomic ratio of 0.2/1, the respective bimetallic catalyst showed 8-times and 3-times higher activity than the respective monometallic Au-and Pd-containing catalysts.
Scheme 17 Catalytic oxidation of benzyl alcohol to benzaldehyde. and (EtO) 3 SiBu-t. The modied supports were loaded with PdCl 2 , reduced with hydrogen and then tested in aerobic oxidation of neat benzyl alcohol and other alcohols to the corresponding aldehydes. The catalytic results revealed that the type and the amount of functional groups used for functionalization of the TUD-1 support greatly affect the catalytic performance. TUD-1 appropriately surface-covered by APTES coverage displayed the best results (TOF about 18 500 h À1 for benzyl alcohol oxidation) owing to the enhanced metal-support interaction and nely tuned local surface basicity around the Pd nanoparticles.
Lee et al. 179 reported the synthesis of hierarchically ordered (270 nm macropores and 5 nm mesopores) materials with SBA-15 architecture with further post-synthetic impregnation with palladium. Bulk and surface characterization revealed that incorporation of complementary macropores into mesoporous SBA-15 improve the dispersion of sub-2 nm Pd nanoparticles and thus increase the degree of their surface oxidation. The catalysts were employed in aerobic selective oxidation of sterically different allylic alcohols, in particular bulky phytol (Scheme 19). Kinetic proling suggested a relationship between nanoparticle dispersion and oxidation rate, identifying surface PdO as the catalytically active phase. Rates of selective oxidation of crotyl and cinnamyl alcohols (relatively smaller representatives) over these hierarchical catalysts were superior to those observed for analogous Pd catalyst deposited over mesoporous SBA-15 at equivalent Pd loading, irrespective of whether the micropores were present within the latter support. However, the hierarchical and purely mesoporous supports exhibited identical TOFs with each of these small alcohols. This contrasts with the trends observed for long chain allylic alcohols, where the excellent activity of Pd supported on hierarchical SBA-15 was evidenced by increased TOFs with respect to Pd/mesoporous SBA-15 (1502 vs. 480 h À1 for farnesol, and 1258 vs. 252 h À1 for phytol). Therefore, authors assumed that hierarchical nanoporous Pd-catalyst outperforms mesoporous analogues in allylic alcohol selective oxidation by stabilizing PdO nanoparticles and by improving in-pore diffusion and access to active sites for relatively bulky aliphatic substrates such as farnesol or phytol.
Karimi et al. 180 modied SBA-15 with bis(2-pyridyl)amide groups and loaded this support with palladium(II) acetate. The resulting material was utilised as a catalyst for oxidation of primary and secondary alcohols to the respective carbonyl compounds with air or pure oxygen. The catalyst showed high efficiency and could be recovered without any decrease in activity. A following study 181 compared this catalyst with analogous supported catalyst resulting by condensation of 3-aminopropyl silica with 2-acetylpyridine or bis(2-pyridyl)ketone and palladation of the respective 3-iminopropylated supports. Using TEM analysis of the samples before and aer catalysis, authors demonstrated that the nature of the ligand can affects the generation of nanoparticles in palladium catalyst deposited on hybrid amorphous silica. In the case of aerobic oxidation reactions using supported palladium catalyst on hybrid SBA-15, the combination of organic ligand and ordered mesoporous channels synergistically enhanced the overall catalytic activity, prevented aggregation of Pd nanoparticles and thus enabled the generation of a durable catalyst. Catalysts prepared with different types of bidentate ligands exhibited high efficiency (e.g., in the oxidation of 1-phenylethanol to acetophenone at 150 C under solvent-free conditions they reached TOFs of more than 18 000 h À1 ).
A similar terminal modifying group was employed by Leitner and co-workers, 182 who prepared [3-(bis(2-pyridyl)amino)propyl] triethoxysilane and condensed this reagent with TEOS in the presence of n-decylamine. The obtained functional siliceous material was loaded with palladium(II) acetate and reduced with benzyl alcohol (110 C) or hydrogen (40 bar, 110 C) to give a deposited Pd(0) catalyst, which was carefully characterised and found to be active in aerobic oxidation of alcohols in supercritical CO 2 . The observed high activity of Pd nanoparticles supported on mesoporous organic-inorganic hybrid materials was explained by the presence of small primary crystallites (approx. 2 nm) that conglomerate to assemblies of about 25 nm in size, thereby leading to metal particles with a larger number of high-indexed planes in small volume units.
A Pd-catalyst deposited on a guanidine-modied support was obtained using mesoporous molecular sieve SBA-16, 1,1,3,3tetramethyl-2-[3-(trimethoxysilyl)propyl]guanidine, (Me 2 N) 2 -C]N(CH 2 ) 3 Si(OMe) 3 , and a palladium(II) source. 183 Catalytic properties of the resulting material were evaluated in aerobic oxidation of benzylic alcohols and cinnamyl alcohol, affording the products in 94-99% yields and >99% selectivity. The catalyst could be reused 15 times without any notable decrease in its activity and selectivity. A related catalyst based on periodic mesoporous hybrid material with a palladium-guanidine complex was synthesised in the presence of CTAB as a structure-directing agent. 184 The resulting material exhibits a 2D-hexagonal structure with well-developed porous system, into which Pd-guanidine complex was covalently integrated. The resulting mesoporous catalyst was similarly tested in oxidation of benzylic alcohols under atmospheric pressure of O 2 , exhibiting high conversion and selectivity (up to 99%).
Reduction of nitro compounds
Aminoaromatics produced by the hydrogenation of corresponding nitro precursors are important commodity chemicals employed in the synthesis of numerous industrially important products such as agrochemicals, pharmaceuticals and dyes. 185,186 Therefore, a number of researchers have focused on the selective reduction of nitro-compounds to the respective amines and other reduced derivatives mediated by deposited palladium catalysts.
Recently, the treatment of MCM-41 with (MeO) 3 Si(CH 2 ) 3 -NHCH 2 CH 2 NH 2 and, subsequently, with [PdCl(h 3 -C 3 H 5 )] 2 were used to prepare a catalyst, which was successfully tested in hydrogenation of isomeric chloronitrobenzenes to chloroanilines (Scheme 20), showing only a low dehydrohalogenation. 187,188 The prepared catalyst was found to be highly active with reasonable selectivities, performing better than its commercial analogue (Pd supported on Al 2 O 3 ).
El-Sheikh et al. 189 reported the synthesis of nanocomposite Pd-catalysts based on SBA-15 using two different reduction routes (under H 2 and using sodium citrate) and compared them with Au and Pt-analogues. In all the catalysts, metal nanoparticles with size < 30 nm were produced inside the SBA-15 pores as well as at the external walls. Catalytic efficiency of the synthesised nanocomposites was tested in the reduction of 4-nitrophenol to 4-aminophenol (Scheme 21). It was found, that the catalytic activity strongly depends on the synthetic route. Two efficiency trends were observed: Pd > Pt > Au for the H 2reduced materials, and Au > Pt > Pd for those obtained via the sodium citrate route. The highest reaction rate was observed in the case of Pd nanoparticles prepared by reduction with H 2 with a rate constant of 0.715 s À1 .
3-Aminopropyl and 3-mercaptopropyl groups were used to modify surface of various composite catalyst, e.g., silica-coated magnetic Fe 2 O 3 nanoparticles. 190 Aer metalation with palladium(II) acetate, these particles were used as magnetically separable and recyclable catalyst for catalytic hydrogenation of nitrobenzene to aniline (Scheme 22). Pd nanoclusters were shown to be deposited with high dispersion and stability, especially when [N-(2-aminoethyl)-3-aminopropyl]trimethoxysilane was used as the anchoring agent. The catalyst prepared with this N-containing modier showed the highest conversion rate of 0.39 mmol s À1 while its S-analogue bearing 3-mercaptopropyl groups and commercial Pd carbon-supported catalyst exhibited conversion rates of only 0.12 and 0.08 mmol s À1 , respectively.
Further examples of the use of silica-coated magnetic supports were provided by Ma et al., who described a method to stabilise Pd(0) particles either on the surface of hollow magnetic mesoporous spheres with Fe 3 O 4 nanoparticles embedded in the mesoporous shell 191 or on amine-functionalised magnetite nanoparticles. 192 The hydrogenation of a set of nitro compounds was performed under mild conditions (1 atm H 2 /25 C) with 96-99% yields aer 60 min. It was shown, that such catalysts can be easily magnetically separated and reused for up to ve successive hydrogenation reactions without change in substrate conversion. The recyclability of the catalyst was attributed to the efficient stabilization of the active Pd metal by the amine groups on the surface of hollow magnetic mesoporous spheres. Commercial 10% Pd/C catalyst required signicantly longer reaction time under the same conditions.
Qazi and Sullivan 193 employed (MeO) 3 SiCH 2 CH 2 S(CH 2 ) 3 -S(CH 2 ) 2 Si(OMe) 3 to modify porous silicas with different pore sizes (5, 7 and 14 nm average diameter) and to prepare Pdcatalysts for hydrogenation of nitrobenzene to aniline (see Scheme 22). In contrast to expectations based on diffusion issues, conversion decreased with increase of the average pore size while it simultaneously increased with the growth of surface area. Catalyst with the smallest average pore size (5 nm), the narrowest pore size distribution and the highest surface area displayed the best kinetic prole, achieving complete reduction within 60 min at 10 atm H 2 and 50 C, while reactions over catalysts with 7 and 14 nm channels reached 100% conversion in 80 and 120 min, respectively. The palladium surface loadings (1.3 mmol m À2 for material with 5 nm pores, 2.1 mmol m À2 for 7 nm pores, and 2.3 mmol m À2 for 14 nm pores) suggest a greater degree of site isolation in the smallestpore material, which could be play an important role in controlling the conversion efficiency. The use of commercial Pd/ C (10%) also resulted in complete conversion to aniline. However, leaching tests of the ltrate revealed that 24% of the Pd had leached from Pd/C in contrast to the negative leaching tests for all prepared catalysts as measured by the standard hot ltration tests and analysis for Pd in ltrate by ICP-OES measurements.
Yasuda et al. 194 prepared a set of Pd catalysts supported on various silicas including mesoporous and non-porous ones aiming at an elucidation of activity-controlling factors in selective hydrogenation of 1-nitrohexane to N-hexyl hydroxylamine (Scheme 23). The Pd catalysts supported on non-porous silica or silicas that possessed large pores efficiently produced Scheme 20 Hydrogenation of isomeric chloronitrobenzenes to the corresponding chloroanilines.
Scheme 21 Reduction of 4-nitrophenol into 4-aminophenol.
Scheme 22 Catalytic hydrogenation of nitrobenzene to aniline. the target product in high yields (89-96%). Based on this observation, the authors concluded that the inuence of pore diffusion was small. On the other hand, the catalytic activity was strongly depended on the Pd dispersion, decreasing generally with the increased Pd dispersion (or the decreased Pd particle size).
A heterogeneous catalytic assembly consisting of Pd nanoparticles on silica support and ionic liquid multi-imidazolium brushes has been developed by Chen et al. 195 The uniform Pd particles size distribution (from 0.5 to 1.5 nm) has been found in these materials, which mediated the hydrogenation of various nitroarenes to arylamines with almost 100% yield and selectivity under solvent-free conditions at room temperature and atmospheric pressure. Notably, the catalyst even facilitated the transformation of solid water-insoluble nitrobenzenes with complete conversions albeit at a lower rate. The performance of these material was compared with those reported in the literature, 196,197 indicating much higher efficiency of the catalyst combining the advantages of an ionic liquid, Pd nanoparticles and a heterogeneous catalyst.
Hydrogenation of unsaturated compounds
Hydrogenation of unsaturated double and triple bonds is among the most important processes in laboratory synthesis as well as in petrochemical, pharmaceutical and food industries. 198,199 These reactions typically require control of the selectivity and, hence, careful catalyst design. 200 In order to prepare a series of supports surface-functionalised witĥ Si(CH 2 ) 3 NHCH 2 CH 2 NH 2 groups, Shimazu and co-workers 201 treated mesoporous molecular sieve MCM-41 or silica (Aerosil 200) with (EtO) 3 Si(CH 2 ) 3 NHCH 2 CH 2 NH 2 . MCM-41-based support was further trimethylsilylated and all solid supports were loaded with palladium(II) acetate. The resulting materials were tested as catalysts for regioselective hydrogenation of nonconjugated terpenic dienes (Scheme 24). The best results were obtained with Pd-catalysts based on aminated MCM-41 and silica; catalyst prepared from the silylated MCM-41 support exerted a lower activity. Furthermore, dienes possessing hydroxyl groups in their structure were hydrogenated faster than limonene, which was attributed to supportive interactions of the OH group in the substrate and those present on the support surface.
Repeated Michael addition of methyl acrylate to aminopropyl-SBA-15 and amidation reactions of the ester groups with 1,2-diaminoethane were used to construct poly(amino-amine) dendrimers at the solid support and the resulting hybrid materials were used for immobilization of in situ generated Pd nanoparticles. The nanoparticle catalysts were tested in hydrogenation of allyl alcohol to 1-propanol (Scheme 25), showing higher activities (TOF up to 2300 h À1 , > 99% conversion, selectivity up to 91%) than catalysts based on dendrimer-encapsulated Pd nanoparticles. Selectivity of the reaction increased with increasing size (generation) of the hyperbranched modier. 202 Another composite catalyst was prepared by Wu et al. 203 via deposition of Pd nanoparticles onto aminopropylated silica and subsequent covering of these particles with polystyrene. As catalysts, the resulting materials showed good activities in hydrogenation of 2,4-dimethyl-1,3-pentadiene to 2,4-dimethyl-2-pentene (Scheme 26) and allyl alcohol to 1-propanol (Scheme 25) performed in supercritical CO 2 . The authors suggested that the polymer coating in the catalysts suppresses the rate of isomerization of allyl alcohol during the hydrogenation reaction by altering both the environment of active sites and access to these active sites, similar to the function of dendrimers 204 or polyelectrolyte multilayers. 205 This positively affects the selectivity of the reduction while supercritical CO 2 increases the reaction rates.
Han et al. 206 synthesised a Pd(II)-loaded material from mesoporous silica modied by N-heterocyclic carbene ionic liquids with different alkyl chain lengths and tested them in hydrogenation of alkenes and allyl alcohol. The activity of the catalysts in the reduction of allyl alcohol was similar to that of commercial Pd/C catalyst, while in hydrogenation of 1-hexene and cyclohexene, the efficiency was lower than that of Pd/C. Nonetheless, the selectivities to 1-propanol were in the range 80-85%, which is higher than those obtained using the commercial Pd/C catalyst (74%). This may indicate that the ionic liquids can not only immobilize the metal catalyst, but also enhance the selectivity of the reaction. The steric hindrance of the prepared catalysts was expected to increase with the length of the alkyl chain present in the ionic liquid residua leading to an increase in reaction selectivity, but when the chain length was longer than C 10 , the selectivity to 1-propanol was not changed any further, presumably because the effective steric hindrance reaching a sort of limiting value.
Ying et al. 207 reported the synthesis of palladium nanoparticles supported on siliceous mesocellular foam using (MeO) 3 Si(CH 2 ) 3 NHCONH 2 or (MeO) 3 Si(CH 2 ) 3 NHCONH(CH 2 ) 3 -Si(OMe) 3 as the modiers. The resulting materials were tested in hydrogenation reaction of activated olens under relatively low hydrogen pressure (2.5 atm) and room temperature. The target products were obtained in excellent yields (quantitatively) and the catalyst was successfully recycled and reused for 10 runs without loss in activity.
complexes tethered on Pd-loaded silica were evaluated as catalysts for hydrogenation of arenes. 209 More recently, Mou and co-workers 210 synthesised and characterised silicasupported Pd "single-atom alloy" by decreasing the loading of Pd to the ppm level when alloying Pd with gold nanoparticles at about 3 nm and applied it to selective hydrogenation of acetylene in the presence of an excess of ethylene. The conversion of acetylene was found to decrease while the selectivity to ethylene increased with decreasing Pd loading. When the temperature was raised from 80 C to 160 C, the selectivity for ethylene increased by about 10-times as compared to the monometallic Pd/SiO 2 catalyst with similar, ppm-level Pd loading. This was explained by a weaker adsorption of ethylene on the Pd "singleatom alloy" compared with the monometallic Pd/SiO 2 catalyst. The Pd in the fabricated structure was hold responsible for the increased reactivity, while the Au is believed to play a key role in isolating the Pd atoms and preventing the over-hydrogenation of acetylene.
The preparation of Pd(0) nanoparticle catalyst supported by an aminopolymer-silica composite was reported by Jones et al. 211 Small palladium nanoparticles with a narrow distribution were generated through reduction of Pd(II) species and loaded into a mesoporous silica material functionalised with branched poly(ethyleneimine) (PEI) polymers. The resulting catalysts exhibited high activity in hydrogenation of diphenylacetylene to selectively produce cis-stilbene (Scheme 27) under mild conditions. It was found that the rate of over-hydrogenation could be signicantly reduced by increasing the support porosity and by using a highmolecular-weight polymer. These single-component catalysts could be easily recovered and recycled with no leaching of palladium detectable, retaining their high activities and selectivity over several cycles.
Rhee and co-workers 212 prepared Pd particles (2-4 nm in size) dispersed on two types of solid supports (3-aminopropyl functionalised silica gel and cross-linked poly(4-vinylpyridine-co-styrene) gel). The reactivity of obtained catalysts was evaluated in hydrogenation of various a,b-unsaturated carbonyl compounds. Both catalysts exhibited high yields of the C]C bond hydrogenation products (95-100% in 0.5-6 h), although Pd dispersed on silica was more efficient than Pd dispersed on the polymer. The lower reactivity of latter catalyst was attributed to its structure, assuming that the pyridyl units of the polymer surrounded the Pd making it less accessible for catalysis.
Albonetti et al. 215 prepared a series of bimetallic mesostructured Pd/Cu MCM-41 catalysts by impregnation with Pd precursors and direct hydrothermal synthesis using different silica sources. The catalysts were tested in hydrogenative dechlorination of a uorinated substrate (Scheme 29). The incorporation of Pd and Cu during the course of MCM-41 synthesis, regardless of the hydrothermal treatment, did not destroy the typical hexagonal channel array and ordered pore system of the parent MCM-41. However, the calcination for the removal of the template led to a segregation of metal oxides, and thus large Pd/Cu bimetallic particles were obtained aer reduction. The impregnation led to pore occlusion, more pronounced for the sample obtained from silicates as silica source. Aer the reduction, both isolated monometallic Cu particles and large bimetallic Pd/Cu particles were found to coexist on the external surface of the support. A lower conversion of CF 3 OCFClCF 2 Cl was achieved with catalyst possessing larger metallic particles, while the presence of monometallic particles decreased the selectivity to the target uorinated ether.
Another active catalyst obtained by depositing palladium(II) acetate over SBA-15 type sieve equipped with the^SiCH 2 CH 2 -CH 2 NHCH 2 CH 2 NEt 2 groups 216 was used in reductive dehalogenation of aryl halides with in situ generated trimethylammonium formate (Scheme 30). The reaction proceeded efficiently with aryl bromides and iodides (better than with Pd/C) and with only a minor leaching of Pd. Chlorobenzenes reacted at considerably slower reaction rates while aromatic uorides remained unaffected, which allowed for selective removal of the more reactive (i.e., heavier) halide from unsymmetrical dihaloarenes.
Other reactions
Catalyst obtained by deposition of palladium(II) acetate onto SBA-15 type support modied by (MeO) 3 Si(CH 2 ) 3 -NHCH 2 CH 2 NH 2 and silylated with chloro-trimethylsilane was employed in addition of allyl chlorides to aldehydes and ketones to give homoallylic alcohols (Scheme 31). 217 The reactions proceeded well with various substrates at 2 mol% Pd loading in the presence of SnCl 2 as a stoichiometric additive.
Alkylation reaction of 3-chloropropylated SBA-15 with 1,4diazabicyclo[2.2.2]octane (dabco) afforded another support bearing 3-(1-azonia-4-azabicyclo[2.2.2]octane)propyl groups at the surface. Catalyst obtained aer deposition of palladium(II) acetate and combined with CuI co-catalyst (both 1 mol%) efficiently mediated oxidative coupling of various terminal alkynes to the corresponding 1,3-butadiynes (Scheme 32). 218,219 Tsai and co-workers prepared a Pd(II)-2,2 0 -bipyridine complex possessing two anchoring triethoxysilyl groups and graed this complex onto nanosized MCM-41. The resulting anchored complex was successfully applied as a highly efficient and recyclable catalyst for Kumada coupling of arylmagnesium halides with aryl halides to give biaryls (Scheme 33) 220 and in arylation of acyl chlorides with triarylbismuth reagents leading to ketones (Scheme 34). 221 A rather specic class of immobilised catalytic systems was introduced by Alper, 222,223 who constructed 224 amido-amine dendrimers (up to the fourth generation) at silica and modi-ed them with metal-binding donor groups at the terminal positions (typically -N(CH 2 PPh 2 ) 2 ). The resulting anchored ligands were extensively evaluated in various Pd-catalysed processes such as hydroesterication of alkenes (Scheme 35). 225 A similar catalyst was prepared also with pincer-type ligating moieties and Pd/PCP terminal groups. 226 Li et al. prepared a series of Pd-catalysts deposited over periodic mesoporous organosilica modied with 2-(diphenyphosphino)ethyl groups by surfactant-directed co-condensation of 1,4-bis(triethoxylsilyl)benzene (or 4,4 0 -bis(triethoxysilyl) biphenyl) with the pre-formed Pd(II) complex [PdCl 2 {Ph 2 PCH 2 -CH 2 Si(OEt) 3 -kP} 2 ] in the presence of P123 triblock copolymer or, alternatively, via post-synthetic palladation of a periodic mesoporous organosilica support prepared by co-condensation of [2-(diphenylphosphino) The resulting materials were tested in allylation of benzaldehyde (Scheme 36) using water as the reaction medium. Catalysts based on periodic mesoporous organosilica typically showed higher activities than those derived from SBA-15, oen comparable with [PdCl 2 (PPh 3 ) 2 ] as their homogenous counterpart. [228][229][230] A series of mesoporous siliceous materials with different pore structures and the donor 2-(diphenylphosphino)ethyl substituents was obtained by hydrolysis and condensation under solvothermal conditions of a mixture containing TEOS, [2-(diphenylphosphino)ethyl]triethoxysilane (ca. 3-13% of total Si in the reaction mixture) and various structure-directing agents. 231 Upon treatment with palladium(II) acetate, these supports were converted to deposited Pd-catalysts, which were tested in allylation of styrene oxide (Scheme 37). The reaction performed in the presence of indium(I) chloride (2.2 equiv.) afforded the desired product in ca. 60-80% yields.
Furthermore, Pd catalysts deposited over mercaptopropylated MCM-41 were evaluated in Stille reaction (Scheme 39) of organostannanes with aryl halides to give the respective biaryls or arylalkanes in reasonable to very good yields (70-90% aer 5 h, TOFs up to 30 h À1 ). 233 A series of mesocelular MCF-type silica supports functionalised by treatment with (MeO) 3 SiCH 2 CH 2 CH 2 X, where X is NH 2 , SH and NHCONH 2 , and loaded with palladium(II) acetate were evaluated in Pd-catalyzed decarboxylation of stearic acid to n-heptadecane (Scheme 40). 234 These materials reached 80-95% conversion aer 6 h at 300 C with 100% selectivity to the main product. Similar reaction with ethyl stearate resulted in considerably lower conversion (ca. 15%) and selectivity (n-heptadecane 87%, stearic acid 13%).
Pd-catalysts deposited on silica and 3-sulfonatopropyl modied silica were tested as catalysts for the direct synthesis of hydrogen peroxide from hydrogen and oxygen, with the latter exerting better activities and selectivity. 235 More recent studies were focused on similar catalysts deposited on 3-sulfonatopropyl modied mesoporous silica supports (MCM-41, MCM-48, MCF, MSU-1 and SBA-15). 236,237 In this case, the catalytic results were shown to be inuenced by the amount of acidic groups at the support surface and by calcination temperature used during the preparation of the MCF support.
Reek et al. 239 reacted silica with different trialkoxysilanefunctionalised phosphines and then silylated the unreacted OH groups with Me 2 SiCl 2 /NEt 3 . The obtained phosphinylated supports were metalated by [Pd(dibenzylideneacetone) 2 ] and employed as catalysts in allylic substitution of allylic substrates with sodium diethyl 2-methylmalonate (Scheme 42). In the reactions with the cinnamyl substrate, all catalysts afforded preferentially the linear alkylation product (linear : branched z 95 : 5-98 : 2) but with different overall activities. Johnson et al. 240 immobilised a chiral ferrocene diphosphine 241 on mesoporous molecular sieve MCM-41 and high-area non-porous silica gel (Carbosil) via introduction of 3-bromopropyl groups followed by subsequent quarternization with the ligand's amino group. Catalysts obtained aer palladation with PdCl 2 were tested in asymmetric allylic amination of cinnamoyl acetate with benzylamine (Scheme 43). A comparison with the corresponding molecular catalyst showed that immobilization and the type of support affect both the regioselectivity (linear vs. branched product) and enantioselectivity of the allylation reaction. Whereas the molecular catalyst produced only linear product, the immobilised ones afforded ca. 50 : 50 mixture of the amination products (linear and branched; see Scheme 43). The catalyst prepared from Carbosil achieved a lower enantioselectivity (ee 43%) than that based on MCM-41 (ee 93-99%).
Summary and outlook
Palladium catalysts deposited over different types of silica (amorphous, mesoporous molecular sieves, solids obtained by co-condensation and many others) modied with suitable donor groups (most oen with N-, S-and/or P-donor moieties) have been extensively studied due to their wide application eld and favourable catalytic properties. Many catalysts reported to date show performance similar to or even superior to their homogeneous counterparts and can be reused in several reaction runs without any notable decrease of their activity and selectivity. The most oen investigated processes in which these catalysts were employed are rather expectedly cross-coupling reactions that are of great practical importance due to their frequent applications in organic synthesis, typically as the crucial molecule-assembling steps. Notwithstanding, research into other reaction types such as redox processes continues apace, bringing new interesting and practically applicable results.
The possibility of ne-tuning the catalytic properties by means of the anchoring group, metal additive (in bimetallic catalysts) or through the morphology of the support makes the deposited palladium catalysts extremely versatile and thus allows for the design of purpose-tailored materials. Despite the enormous progress in the area and numerous reports published in the recent past, the catalyst design still remains far from predictable. Although the simple anchoring groups in which -SH, -NH 2 or -PPh 2 are covalently attached to the support by means of an alkyl group typically perform well or are used as starting materials for further modications of the support surface, the search for new, more complicated modifying "substituents" still appears desirable in view of sought-for improvements in the catalytic efficacy (see, for instance, the promising properties of Pd catalyst deposited over supports with immobilised ionic liquids or dendrimers). Attention also should be paid to newly emerging siliceous materials with specic properties (internal structure, density etc.) that have been studied less than the already well-established ones such as conventional silica, mesoporous molecular sieves and zeolites. Alongside with the experiments aimed at the design of new catalysts and physicochemical studies into their properties, new applications (i.e., new organic processes employing these materials as catalysts) are sought for in order to fully exploit the multifaceted catalytic chemistry of immobilised palladium species. | v3-fos-license |
2020-01-30T09:15:28.308Z | 2020-01-29T00:00:00.000 | 210953724 | {
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} | pes2o/s2orc | Alisol A-24-acetate promotes glucose uptake via activation of AMPK in C2C12 myotubes
Background Alisol A-24-acetate (AA-24-a) is one of the main active triterpenes isolated from the well-known medicinal plant Alisma orientale (Sam.) Juz., which possesses multiple biological activities, including a hypoglycemic effect. Whether AA-24-a is a hypoglycemic-active compound of A. orientale (Sam.) Juz. is unclear. The present study aimed to clarify the effect and potential mechanism of action of AA-24-a on glucose uptake in C2C12 myotubes. Method Effects of AA-24-a on glucose uptake and GLUT4 translocation to the plasma membrane were evaluated. Glucose uptake was determined using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake assay. Cell membrane proteins were isolated and glucose transporter 4 (GLUT4) protein was detected by western blotting to examine the translocation of GLUT4 to the plasma membrane. To determine the underlying mechanism, the phosphorylation levels of proteins involved in the insulin and 5′-adenosine monophosphate-activated protein kinase (AMPK) pathways were examined using western blotting. Furthermore, specific inhibitors of key enzymes in AMPK signaling pathway were used to examine the role of these kinases in the AA-24-a-induced glucose uptake and GLUT4 translocation. Results We found that AA-24-a significantly promoted glucose uptake and GLUT4 translocation in C2C12 myotubes. AA-24-a increased the phosphorylation of AMPK, but had no effect on the insulin-dependent pathway involving insulin receptor substrate 1 (IRS1) and protein kinase B (PKB/AKT). In addition, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and the AKT substrate of 160 kDa (AS160), two proteins that act downstream of AMPK, was upregulated. Compound C, an AMPK inhibitor, blocked AA-24-a–induced AMPK pathway activation and reversed AA-24-a–induced glucose uptake and GLUT4 translocation to the plasma membrane, indicating that AA-24-a promotes glucose metabolism via the AMPK pathway in vitro. STO-609, a calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) inhibitor, also attenuated AA-24-a–induced glucose uptake and GLUT4 translocation. Moreover, STO-609 weakened AA-24-a-induced phosphorylation of AMPK, p38 MAPK and AS160. Conclusions These results indicate that AA-24-a isolated from A. orientale (Sam.) Juz. significantly enhances glucose uptake via the CaMKKβ-AMPK-p38 MAPK/AS160 pathway.
Background
Type 2 diabetes mellitus (T2DM) has become one of the most serious global health issues and is mainly attributed to excess body weight and inactivity. According to the International Diabetes Federation, 425 million people worldwide are currently suffering from T2DM [1]. T2DM is characterized by over-nutrition and insulin resistance, which lead to high blood glucose levels and vascular complications that include cardiovascular diseases, diabetic nephropathy and diabetic retinopathy [2][3][4]. Lowering blood glucose levels reduces the incidence of such complications. Thus it is pivotal to T2DM research that we find ways to promote glucose uptake in skeletal muscle and lower glucose production in the liver.
Glucose uptake in skeletal muscle is regulated by two distinct pathways: 1) stimulation by insulin through IRS1 and phosphatidylinositol 3 (PI3)-kinase [5]; and 2) stimulation by muscle contraction and exercise through the activation of AMPK [6]. AMPK has long been regarded as a promising therapeutic target for metabolic syndrome. AMPK belongs to a family of serine/threonine kinases, acting as a cellular energy sensor that monitors the AMP: ATP ratio to maintain cellular homeostasis [7]. The Thr 172 site on the α subunit of AMPK is crucial to AMPK regulation [8]. In its activated state, AMPK can phosphorylate multiple kinases and other downstream target proteins to exert its various functions. AMPK can phosphorylate and inhibit acetyl-CoA carboxylase (ACC), promoting fatty acid transportation and βoxidation [9]. AMPK also phosphorylates and inhibits the AS160, which ultimately promotes the translocation of GLUT4 [6]. Translocation of GLUT4 from vesicles to the plasma membrane is a critical step in cellular glucose uptake. Furthermore, studies have revealed that AMPK can also phosphorylate and activate p38 MAPK [10], whose role in promoting cell glucose uptake has already been clarified [10,11]. AMPK is regulated by various upstream kinases, including but not limited to CaMKKβ, transforming growth factor (TGF) β-activated kinase 1 (TAK1) and liver kinase B1 (LKB1) [12].
Alisma orientale (Sam.) Juz., a well-known medicinal plant, is mainly found in China, Russia, Japan, Mongolia and North India. Its dried rhizome, Rhizoma Alismatis, is a well-known traditional Chinese medicine that has been widely used in China for more than 1000 years. Pharmacological research has revealed that it has multiple biological activities that include diuretic, anti-inflammatory, antitumor, hepatoprotective, hypolipidemic and hypoglycemic effects [13][14][15][16][17][18].
Alisol A-24-acetate (AA-24-a) is one of the main active triterpenes that have been isolated from Rhizoma Alismatis. While it has been reported that AA-24-a can lower cholesterol [19] and prevent hepatic steatosis [20], its potential effect on glucose metabolism has not been investigated. Glucose uptake by peripheral tissues such as skeletal muscles and adipocytes is important for the maintenance of glucose homeostasis [21], and is one mechanism for prevention or amelioration of hyperglycemia and T2DM. Because the skeletal muscles are responsible for approximately 75% of glucose uptake, we chose to use myotubes from a murine cell line, C2C12, to evaluate the effect of AA-24-a on glucose metabolism. While our preliminary study revealed that AA-24-a significantly promoted glucose consumption in C2C12 myotubes (unpublished results), not much is known about its effect on glucose uptake in myotubes. We hypothesized that triterpenes AA-24-a isolated from Rhizoma Alismatis might improve glucose metabolism by promoting glucose uptake via the IRS1/PI3-kinase pathway or the AMPK pathway. To test this hypothesis, we examined the expression of key components of the IRS1/PI3-kinase and AMPK pathways. And then, specific kinase inhibitors were used to investigate the mechanism of AA-24-a on glucose uptake in C2C12 myotubes.
Cell culture and differentiation
The C2C12 mouse myoblasts were obtained from The National Center for Drug Screening (Shanghai, China). C2C12 mouse myoblasts were maintained in DMEM supplemented with 10% (v/v) FBS, streptomycin (100 U/ mL), and penicillin (100 U/mL) (37°C; 5% CO 2 ). Cells were seeded into cell culture plates at a density of 5 × 10 4 cells/mL. After 24 h (about 70% confluence), the medium was switched to DMEM supplemented with 2% (v/v) horse serum and replaced after 2, 4 and 6 days of culture. Experiments were initiated on day 7, when myotube differentiation was complete. Cells were serum-starved for 6 h before being subjected to any experimental treatment.
CCK-8 assay
Fully differentiated C2C12 mouse myotubes were cultured in 96-well plates and treated with AA-24-a at varying concentrations and a range of time periods. CCK-8 reagents (included in Cell Counting Kit, 10 μL) were added to each well 1 h before harvesting. Cells were maintained in an incubator (37°C; 5% CO 2 ) for 1 h, then absorbance was measured at 450 nm. Cell viability was calculated by the following formula:
2-NBDG uptake
Cell glucose uptake was determined as a measure of 2-NBDG uptake using the following procedure. Fully differentiated C2C12 mouse myotubes were cultured in black 96-well plates and treated with AA-24-a at varying concentrations and a range of time periods. At 1 h before harvest, cells were washed twice with warm sterile phosphate buffered saline (PBS) then the medium was switched to glucose-free DMEM supplemented with 0.2% fatty-acidfree bovine serum albumin (BSA). After 1 h, cells were washed once with sterile PBS (warmed up at 37°C) and then incubated with the same medium containing 80 μM 2-NBDG for 30 min. Cells were then washed once more with warm sterile PBS before measuring the fluorescence intensity of each well (Ex485nm, Em520nm). Cell glucose uptake was calculated by following formula:
Western blotting
After treatment cells were washed twice with ice-cold PBS then harvested in radioimmunoprecipitation assay lysis buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS] and 50 mmol/L Tris; pH 8.0) containing protease and phosphatase inhibitors. Cell membrane proteins were extracted using a Mem-PERa Plus Membrane Protein Extraction Kit according to the manufacturer's protocol. The protein concentrations were determined using a bicinchoninic acid (BCA) Protein Assay Kit (Yeasan) according to the manufacturer's protocol. Equal quantities of protein were separated on 10% SDSpolyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to nitrocellulose membranes. The membranes were blocked in 5% non-fat drymilk solution at room temperature for 1 h and incubated overnight at 4°C with primary antibodies. After three washes in tris-buffered saline with 0.1% Tween 20, the membranes were incubated for 1 h with secondary antibodies at room temperature. The blots were washed and then visualized on an Odyssey CLx Imaging System. All blots were analyzed by Image-Pro Plus Software.
Statistical analysis
Results are expressed as mean ± standard deviation (SD). Statistical significance among different experimental groups was determined by one-way analysis of variance followed by Duncan's multiple-comparisons test using SPSS software (IBM; Armonk, NY, USA). Differences between two groups were identified using Student's t-test. P values of < 0.05 and < 0.01 were considered to be statistically significant and extremely significant, respectively.
AA-24-a promotes glucose uptake and GLUT4 translocation in C2C12 myotubes
To determine the effects of AA-24-a treatment on the viability of C2C12 myotubes, we performed a CCK-8 assay. AA- 24-a had no effect on cell viability in cultures of differentiated C2C12 myotubes treated with 0-40 μM AA-24-a for 12 h (Fig. 1a, P > 0.05) and 24 h (Additional file 1: Figure S1). Next we investigated the effects of AA-24-a on glucose uptake and GLUT4 translocation. Differentiated C2C12 myotubes were treated with 0-40 μM AA-24-a for 12 h or insulin (100 nM, 15 min) as a positive control. As shown in Fig. 1b, treatment with 10, 20 and 40 μM AA-24-a significantly promoted cell glucose uptake by factors of 1.28-, 1.57-and 1.70-fold, respectively, compared with the control group (P < 0.01 or 0.05). After treatment cells with 40 μM AA-24-a for 12 h, the level of GLUT4 protein in the plasma membrane was significantly increased by 2.97-fold compared with those of control cells (Fig. 1c, d, P < 0.01). These results indicated that AA-24-a promoted GLUT4 translocation and cell glucose uptake in C2C12 myotubes.
To confirm whether the promotional effect of AA-24a on glucose uptake and GLUT4 translocation was mediated through AMPK activation, we pretreated the myotubes with compound C (15 μM), an AMPK-specific inhibitor, for 1 h prior to AA-24-a (40 μM) treatment for 12 h (in the presence of compound C). As shown in Fig. 3, AA-24-a-stimulated glucose uptake (Fig. 3b) and GLUT4 translocation (Fig. 3c, d)
AA-24-a activates AMPK pathway via CaMKKβ in C2C12 myotubes
Multiple kinases can activate AMPK, including CaMKKβ and LKB1 [12]. It has also been reported that CaMKKβ acts as an upstream effector of AMPKa2 in the activation of glucose uptake [22]. To investigate the possible role of CaMKKβ in AA-24-a-mediated glucose uptake, a CaMKKβ inhibitor (STO-609) was employed. As results shown in Fig AA-24-a treatment alone). These results indicated that AA-24-a promotes glucose uptake through the CaMKKβ-AMPK-p38 MAPK/AS160 pathway.
Discussion
Because muscles play a key role in the regulation of energy balance and are considered the most important tissue for glucose disposal [23], we used C2C12 myotubes in this pilot study demonstrating the stimulation of glucose uptake by AA-24-a from Rhizoma Alismatis. GLUT4 translocation is central to glucose metabolism. The translocation of GLUT4 from intracellular vesicles to the plasma membrane is the most important step in regulating glucose uptake, and can be promoted by insulin as well as AMPK [24]. By extracting membrane proteins from cell lysates, we found that treatment of AA-24-a led to significantly increased GLUT4 levels in the plasma membrane, which indicating that AA-24-a enhanced GLUT4 translocation. This result was in agreement with the upregulation of glucose uptake induced by AA-24-a treatment in C2C12 myotubes.
As mentioned above, skeletal muscle glucose uptake is regulated by two distinct pathways: the insulin-dependent IRS1/PI3K pathway [5] and the AMPK pathway, which is activated by muscle contraction or exercise [6]. We found that AA-24-a did not have effects on IRS1 or AKT but strongly activated AMPK, suggesting that AMPK pathway probably involved in AA-24-a stimulates glucose uptake. AMPK plays important roles in maintaining cell energy and glucose homeostasis [7]. Once activated, it accelerates ATP-generating catabolic pathways, including those for lipid metabolism, glucose uptake and fatty acid oxidation, by directly regulating the key metabolic enzymes [9,12]. Because of its involvement in the metabolic syndrome, AMPK has been extensively studied. Metformin and AICAR are powerful AMPK agonists, with the former widely used in T2DM treatment [25]. In this study, we observed that AA-24-a increased the phosphorylation of AMPK on Thr172, which is the most important regulatory site of AMPK. AA-24-a also increased the phosphorylation of ACC, a downstream target of AMPK that is commonly used as proof of AMPK activation. Moreover, AMPK inhibitor compound C attenuated AA-24-a-induced AMPK activation, glucose uptake and GLUT4 translocation. Thus, our data indicate that AA-24-a increases cell glucose uptake and GLUT4 translocation via the AMPK pathway. A previous study that demonstrated significant effects of AA-24-a on the AMPK pathway in terms of amelioration of hepatic steatosis and inhibition of inflammation in HepG2 cells [20] accords with our results. Another study in C57BL/6 mice and WRL-68 liver cells also indicated that AA-24-a inhibits oxidative stress and stimulates autophagy by activating AMPK [26].
AS160 is an important downstream protein of the insulin pathway [5,6]. Recent research efforts have revealed that AMPK phosphorylates AS160 to inactivate it, eventually leading to upregulation of glucose uptake and GLUT4 translocation [6]. Our study demonstrated that the AA-24-a-mediated phosphorylation of AS160 was blocked by AMPK inhibitor compound C, indicating that AA-24-a-mediated inactivation of AS160 by AMPK is one of the underlying mechanisms of upregulation of glucose uptake and GLUT4 translocation.
Recent studies have provided evidence that p38 MAPK plays a pivotal role in glucose uptake in skeletal muscles [10,11]. Many studies have investigated the connection between AMPK and p38 MAPK. Activation of p38 MAPK is almost completely abolished in various cells expressing the dominant-negative AMPK mutant [27,28]. Therefore, it seems clear that p38 MAPK is a downstream protein of AMPK that participates in AMPK-dependent regulation of glucose uptake. In the current study, we found that AA-24-a-induced p38 MAPK activation was completely blocked by the AMPK inhibitor compound C, which suggests that AA-24-a-induced glucose uptake occurs via the AMPK-p38 MAPK pathway in C2C12 myotubes. AMPK is regulated by LKB1, CaMKKβ and TAK1 among other upstream kinases [12]. We found that preincubation of STO-609, an established CaMKKβ inhibitor, reversed the AA-24-a-induced upregulation of glucose uptake and GLUT4 translocation, as well as activation of the AMPK pathway, demonstrating that AA-24-a promotes glucose uptake and activates AMPK through CaMKKβ. However, Ca 2+ ionophores, which increase cellular Ca 2+ levels to activate CaMKKβ, also activate AMPK phosphorylation in the same cell line. Whether AA-24-a exerts its effect by regulating the Ca 2+ ionophore concentration in cells remains to be discovered.
The above results demonstrated that AA-24-a, one of the main active triterpenes of Rhizoma Alismatis, significantly enhances glucose uptake via the CaMKKβ-AMPK-p38 MAPK/AS160 pathway in C2C12 myotubes. Then, this effect need be validated in vivo, the following studies will attempt to investigate whether AA-24-a decrease blood glucose through activation of AMPK in diabetic animal models.
Conclusions
In summary, this pilot study has shown that AA-24-a promoted glucose uptake and GLUT4 translocation in C2C12 myotubes through a mechanism involving the CaMKKβ-mediated phosphorylation of AMPK and its downstream proteins p38 MAPK and AS160. These findings provide insight into the hypoglycemic functions of AA-24-a and raise the possibility that AA-24-a could be developed as an anti-diabetic agent.
Additional file 1: Figure S1. Effects of AA-24-a on cell viability in C2C12 myotubes treated with AA-24-a for 24 h. | v3-fos-license |
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} | pes2o/s2orc | JARID2 and the PRC2 complex regulate the cell cycle in skeletal muscle
JARID2 is a noncatalytic member of the polycomb repressive complex 2 (PRC2) which methylates of histone 3 lysine 27 (H3K27). In this work, we show that JARID2 and the PRC2 complex regulate the cell cycle in skeletal muscle cells to control proliferation and mitotic exit. We found that the stable depletion of JARID2 leads to increased proliferation and cell accumulation in S phase. The regulation of the cell cycle by JARID2 is mediated by direct repression of both cyclin D1 and cyclin E1, both of which are targets of PRC2-mediated H3K27 methylation. Intriguingly, we also find that the retinoblastoma protein (RB1) is a direct target of JARID2 and the PRC2 complex. The depletion of JARID2 is not sufficient to activate RB1. However, the ectopic expression of RB1 can suppress cyclin D1 expression in JARID2-depleted cells. Transient depletion of JARID2 in skeletal muscle cells leads to a transient up-regulation of cyclin D1 that is quickly suppressed with no resulting effect on proliferation, Taken together, we show that JARID2 and the PRC2 complex regulate skeletal muscle proliferation in a precise manner that involves the repression of cyclin D1, thus restraining proliferation and repressing RB1, which is required for mitotic exit and terminal differentiation.
JARID2 is a noncatalytic member of the polycomb repressive complex 2 (PRC2) which methylates of histone 3 lysine 27 (H3K27). In this work, we show that JARID2 and the PRC2 complex regulate the cell cycle in skeletal muscle cells to control proliferation and mitotic exit. We found that the stable depletion of JARID2 leads to increased proliferation and cell accumulation in S phase. The regulation of the cell cycle by JARID2 is mediated by direct repression of both cyclin D1 and cyclin E1, both of which are targets of PRC2-mediated H3K27 methylation. Intriguingly, we also find that the retinoblastoma protein (RB1) is a direct target of JARID2 and the PRC2 complex. The depletion of JARID2 is not sufficient to activate RB1. However, the ectopic expression of RB1 can suppress cyclin D1 expression in JARID2-depleted cells. Transient depletion of JARID2 in skeletal muscle cells leads to a transient up-regulation of cyclin D1 that is quickly suppressed with no resulting effect on proliferation, Taken together, we show that JARID2 and the PRC2 complex regulate skeletal muscle proliferation in a precise manner that involves the repression of cyclin D1, thus restraining proliferation and repressing RB1, which is required for mitotic exit and terminal differentiation.
Cell proliferation and differentiation are mutually exclusive but closely coordinated and highly regulated processes. In skeletal muscle, the process is marked by a large number of gene expression changes as cell cycle genes go from highly expressed to permanently silenced while another set of genes that are normally repressed during proliferation are activated during differentiation (1).
Two essential protein families that regulate the cell cycle are cyclins and cyclin-dependent kinases (CDKs). 2 These complexes guide the transition of cells through the different phases of the cell cycle. In the mammalian system, cyclins D and E govern the progress through the G 1 /S phases whereas the mitotic cyclins A and B promote progression through the S/G 2 /M phases of the cell cycle (2). Mitogenic signaling stimulates the synthesis and assembly of the short-lived D-type cyclins with CDK4 or CDK6 (3). Cyclin D/CDK complexes are active as long as mitogenic stimulation continues. Cyclin D/CDK acts directly as a kinase to phosphorylate cellular substrates critical for cell cycle progression. D-type cyclins also act to sequester the CDK inhibitors p27 and p21 in a kinase-independent role (2). The protein level of cyclin E is maximum at the G 1 /S transition and is followed by an increase in cyclin A levels during S phase. Both cyclin A and E can form complexes with CDK2, although CDK1 can only form a complex with cyclin A. Rising levels of cyclin B result in the induction of CDK1 at the G 2 /M phase. Coordinated cell cycle progression is a result of the highly regulated fluctuation in the expression of cyclins and activation of cognate CDKs (reviewed in Ref. 1).
Skeletal muscle determination and differentiation are controlled by four highly related basic-helix-loop-helix transcription factors known as the myogenic regulatory factors (MRFs) which include Myf5 (Myf5), MyoD (Myod), myogenin (Myog), and MRF4 (Myf6) (4 -6). MyoD and Myf5 have been shown to promote the proliferation of muscle progenitor cells whereas myogenin appears to possess the intrinsic activity required to mediate cell cycle exit as the exogenous expression of myogenin in proliferating myoblast causes premature exit of myoblast from the cell cycle (7). MyoD can also promote cell cycle arrest by induction of the cyclin-dependent kinase inhibitor p21 (8,9) along with other Cip1/Kip1 family members, p57 and p27, which inhibit a wide range of CDKs essential for cell cycle progression (10). During differentiation, the induction of p21 is followed by the expression of myogenin, which marks a "point of no return" for mitotic exit. High levels of p21 are required for maintenance of the post-mitotic state in differentiated myoblasts and MyoD can also induce RB1 expression (11).
In skeletal muscle, the retinoblastoma protein (pRB, RB1) has a critical role in negative regulation of cell cycle progression by promoting exit from the cell cycle and maintaining the differentiated state (12)(13)(14). RB1 maintains cell cycle arrest, which is required for skeletal muscle differentiation, in part by regulating the activity of the E2F family of transcription factors. Pocket proteins, a family that includes RB1 as well as related p107 and p130 proteins, bind and repress E2F activity (12). Activation of cyclin D and CDK4/6 proteins by mitogen-rich conditions allows monophosphorylation of pocket proteins which releases bound activator E2F transcription factors from hypophosphorylated RB1 proteins. Thus, cyclin D/CDK expression results in activation of E2F target cell cycle genes including cyclin E. Cyclin E, in complex with CDK2, further hyperphosphorylates RB1 proteins into the hyperphosphorylated inactive form of RB1 which allows cells to progress through the G 1 /S restriction point (reviewed in Refs. 15 and 16).
Polycomb repressor proteins have an important developmental role and play essential roles in skeletal muscle. The two major polycomb complexes, Polycomb Repressive Complex-1 (PRC1) and Polycomb Repressive Complex-2 (PRC2), are responsible for the ubiquitination of lysine 119 of histone H2A (H2AK119) (17) and the methylation of lysine 27 on histone H3 tail (H3K27), respectively (18 -20). The methylation of H3K27 by PRC2 requires one of the SET domain-containing enhancer of zeste proteins, EZH1 or EZH2. Promoters and enhancers of many lineage-specific genes contain dimethylated and trimethylated H3K27, and the methylation profile of many of these genes is lost upon differentiation (reviewed in Ref. 21).
JARID2, a founding member of Jumonji family of histone demethylases, has no detectable histone demethylase activity and yet is evolutionarily conserved from Drosophila to human (Ref. 22,reviewed in Ref. 23). Recently, it has been shown to be a substoichiometric component of PRC2 which aids in targeting PRC2 and is required for embryonic stem cell differentiation (24). In mice, JARID2 was found to be essential for heart development (25). JARID2 mutant mice exhibit various neural tube, cardiac, and hematopoietic defects and die in utero because of cardiac and hematopoietic defects (26). In cardiac cells, JARID2 has been shown to control the cell cycle through regulation of cyclin D1 (25,27,28). However, the role of JARID2 in skeletal muscle development and regeneration is not well-understood.
We have recently shown that loss of JARID2 blocks differentiation in C2C12 cells through the modulation of the canonical Wnt signaling pathway (29). Here, we examined the effect of JARID2 on the proliferation of skeletal muscle cells. We found that depletion of JARID2 increased the proliferation rate and caused de-repression of positive cell cycle regulators like cyclin D1 and cyclin E1, whereas the negative cell cycle regulators like p21 and RB1 were down-regulated. JARID2 has been shown to recruit the histone methyltransferases G9a and GLP to directly regulate cyclin D1 in cardiomyocytes (30). We show here that JARID2 recruits the PRC2 complex to repress cyclin D1 and cyclin E1 in skeletal muscle and results in H3K27 methylation of target promoters. In a related study, we also have found that the modest depletion or chemical inhibition of EZH2 promotes proliferation through the JARID2-directed regulation of cyclin D1 and cyclin E1. 3 We also show that Rb1 is a direct target of JARID2 and the PRC2 complex, but loss of repression by PRC2 is not sufficient to de-repress gene expression. The differentiation block with consequential increase in proliferation could be restored by exogenous expression of JARID2. Together, we show that JARID2 regulates the cell cycle in PRC2-dependent manner in skeletal muscle cells.
JARID2-depleted cells have increased proliferation
To understand the function of JARID2 in skeletal muscle proliferation, we established cell lines depleted for JARID2 with three independent shRNA constructs in C2C12 cells (29), a well-established in vitro differentiation model. mRNA and protein confirmation of one JARID2 depletion line derived from each shRNA construct is shown in Fig. S1. All described experiments were performed in two cell lines depleted with independent shRNA constructs (shJarid2-1 and shJarid2-3), and the results were consistent. For clarity, only the results for one shRNA construct (shJarid2-3) are shown. We assayed for the proliferation rate of these cells, and we found that C2C12 cells depleted for JARID2 proliferated significantly faster than cells with the scrambled control (Fig. 1A). To determine whether increased DNA synthesis was correlated with the enhanced proliferation observed, we performed an EdU incorporation assay for DNA synthesis and found that the number of actively proliferating cells marked by EdU incorporation was significantly higher in JARID2-depleted cells (Fig. 1B). To correlate the enhanced proliferation with changes in the cell cycle, the cell cycle state was assessed by flow cytometry following propidium iodide staining of the cells. We found that JARID2depleted cells were much more highly enriched in the S phase at the expense of cells in the G 1 phase than the cells with a scrambled control, indicating that JARID2 plays a role in regulating the G 1 /S transition ( Fig. 1C and Fig. S2).
JARID2 represses the positive cell cycle genes cyclin D1 and cyclin E1 to regulate the G 1 /S transition
To understand how JARID2 was regulating the G 1 /S transition, we examined the expression of factors that mediate this transition, including the gene encoding cyclin D1 (Ccnd1). Ccnd1 is methylated at histone H3 lysine 27 in C2C12 cells in a PRC2-dependent manner upon differentiation (13). We found that Ccnd1 mRNA was up-regulated in cells depleted for JARID2 ( Fig. 2A), and this result was confirmed at the protein level as well (Fig. 2B). We next examined cyclin E1 (Ccne1), which functions in concert with cyclin D1 for the cells transition from G 1 to S phase. We found that cyclin E1, like cyclin D1, was also up-regulated at the level of mRNA (Fig. 2C) and protein (Fig. 2D). CDK4/6 and CDK2 are the CDKs that partner with cyclin D1 and cyclin E1, respectively, to phosphorylate various cellular substrates including the retinoblastoma protein (RB1) to regulate cell cycle progression. Thus, we also examined the expression of CDK2 and CDK4 and found that they were up-regulated at the mRNA level (Fig. 2, E and G) as well as the protein level (Fig. 2F).
Stable loss of JARID2 results in repression of negative cell cycle regulators
The cyclin-dependent kinase inhibitor p21 and retinoblastoma proteins (RB1) are known negative regulators of cell cycle progression (G 1 /S transition) and are key regulators of cell cycle exit. Thus, we assayed for the expression of RB1 (Rb1) and found that Rb1 was severely down-regulated at the level of both mRNA (Fig. 3A) and protein (Fig. 3B). We also examined the expression of p21 (Cdkn1a), which is also required for cell cycle exit. We found that Cdkn1a expression was inhibited at the mRNA level upon JARID2 depletion (Fig. 3C). The protein level was less severely affected, but the results show that Cdkn1a is not up-regulated upon the depletion of JARID2 (Fig. 3D).
Exogenous expression of JARID2 restores cell proliferation defect in JARID2-depleted C2C12 cells
To confirm the dependence on JARID2, we asked if restoration of JARID2 could rescue the observed effects on the cell cycle regulators. Scr control and JARID2-depleted cells were transfected with an expression construct for JARID2 (pEF-Ja-rid2) or vector control (pEF). The expression of JARID2 was confirmed by mRNA ( Fig. 4A) and protein (Fig. 4B) analysis. We found that restoration of JARID2 rescued the increased proliferation defect observed on the JARID2 depletion (Fig. 4C). We also found that restoration of JARID2 down-regulated cyclin D1 at the mRNA (Fig. 4D), and protein ( Fig. 4E) level, thus confirming that JARID2 controls the expression of cyclin D1 in skeletal muscle cells. Restoration of JARID2 also suppressed the expression of Ccne1 (Fig. 4F). Cdk4 and Cdk2 mRNA expression were restored by JARID2 expression as well (Fig. 4, G and H). This effect could also be observed at the protein level (Fig. 4I). Interestingly, expression of the negative cell cycle regulators, RB1 and p21, were also rescued, resulting in an up-regulation of expression (Fig. 4J).
JARID2 directly represses cyclin D1 in PRC2-dependent manner
In cardiac cells, JARID2 represses Ccnd1 though alterations in histone H3 lysine 9 (H3K9) methylation (30). To determine whether JARID2 directly repressed Ccnd1 in skeletal muscle, chromatin immunoprecipitation (ChIP) assays were used to probe JARID2 recruitment to the Ccnd1 promoter. We found that JARID2 was present on the Ccnd1 promoter and that depletion of JARID2 reduced this association (Fig. 5A). We next examined the methylation of H3K9 (H3K9me) in cells with or without JARID2 and found no change in the H3K9me profile (Fig. 5B). The antibody used in these experiments was panmethyl H3K9 that recognizes all three states of H3K9 methylation. We next examined the methylation of H3K27 as JARID2 has been shown to direct the PRC2 complex to target genes (31,32) and found that depletion of JARID2 led to a significant depletion of this modification (Fig. 5C). To confirm that this reduction was not because of a loss of total histone 3 (H3) at this promoter, we also examined H3 levels in this region and found that H3 levels were unaltered (Fig. 5C). We also assayed for the presence of the catalytic subunit of the PRC2 complex, EZH2, and we found that EZH2 was associated with the Ccnd1 promoter and that the association was reduced when JARID2 was depleted (Fig. 5D). EZH2 has previously been shown to bind to the Ccnd1 promoter in myotubes (13), but we show here that this can also be detected in myoblasts and importantly, the recruitment of EZH2 depends on JARID2. As a positive control
JARID2 regulates the cell cycle in skeletal muscle
for these experiments, we also examined the H3K27me3 status of the Hoxb7 promoter, a well-established target of the PRC2 complex (33). H3K27me3 was highly enriched on the Hoxb7 promoter and the depletion of JARID2 did not significantly disrupt this modification (Fig. 5E). Total H3 levels were also unaltered (Fig. 5E). The percentage input calculation shows that the Hoxb7 promoter is more highly enriched for H3K27me3 than Ccnd1, which may explain how the methylation of the Ccnd1 and Ccne1 promoters could have been overlooked in previous genome-wide studies. As a negative control, we also examined a promoter proximal region of Tnni2 which we have previously shown is not a target of the PRC2 complex in proliferating cells panel). B, right panel, blots were quantified and plotted. Plots were normalized to GAPDH loading control. C and D, JARID2 also represses cyclin E1. As in A, cells were assayed for cyclin E1 by qRT-PCR (C) and Western blotting (D, left panel). D, right panel, blots in C were quantified and plotted. Plots were normalized to GAPDH loading control. E-G, JARID2 also represses cyclin-dependent kinases. Cells in A were assayed for Cdk2 and Cdk4 by qRT-PCR (E and G) and Western blotting (F, left panel). D and F were performed simultaneously and the same GAPDH blot was used. Blots in F were quantified and plotted. Plots were normalized to GAPDH (F, right panel). Error bars, mean Ϯ S.E. (Student t test; *, p Ͻ 0.05 and ***, p Ͻ 0.001; n ϭ 3-6 (qRT-PCR) and 3 (Western blots) biological replicates.) JARID2 regulates the cell cycle in skeletal muscle (32). We found that JARID2 and EZH2 were not recruited to this region and H3K27me3 and H3K9me were not altered upon JARID2 depletion (Fig. 5F).
Cyclin D1 is an essential cell cycle regulator and is activated by several factors, including -catenin, the effector of the canonical Wnt pathway. We have recently found that JARID2 directly represses an antagonist of the canonical Wnt signaling pathway, Sfrp1, which leads to an inhibition of the nuclear translocation of -catenin in JARID2-depleted cells (29). To determine whether an alteration in -catenin recruitment contributed to the activation of cyclin D1, we performed ChIP assays for -catenin on the Ccnd1 promoter. We found that -catenin levels were reduced on the Ccnd1 promoter upon JARID2 depletion (Fig. 5G). This result is consistent with our study showing the JARID2-dependent reduction in the nuclear translocation of -catenin (29). This result also shows that the up-regulation of cyclin D1 observed is because of the loss of the JARID2/PRC2-dependent H3K27 methylation and not an enhanced recruitment of the activator -catenin.
We next asked if Ccne1 was also a direct target of JARID2 and the PRC2 complex. JARID2 was found to be associated with the Ccne1 promoter, and JARID2 depletion reduced this association (Fig. 5H). Like Ccnd1, we found that H3K9 methylation on the Ccne1 promoter was unchanged upon JARID2 depletion (Fig. 5I). We found that H3K27 methylation at the Ccne1 promoter was reduced upon JARID2 depletion and again, total H3 levels were unaltered (Fig. 5J). The H3K27me3 enrichment in the JARID2-depleted cells was below the IgG control signal; therefore, the value was zeroed. We also observed a decrease in EZH2 enrichment on the Ccne1 promoter upon JARID2 depletion (Fig. 5K), showing that JARID2 is required to recruit PRC2 to the Ccne1 promoter. Together, these data establish that Ccnd1 and Ccne1 are direct targets of the PRC2 complex and that JARID2 directs the PRC2 complex to these target genes.
RB1 is also a direct target of JARID2 and the PRC2 complex
Cyclin D1 and cyclin E1 function to prevent cell cycle exit by inhibiting the function of the RB1. RB1 is required for the irreversible cell cycle exit associated with the arrest of myoblasts and myogenic differentiation (13,14). We showed that RB1 was down-regulated upon JARID2 depletion (Fig. 3, A and B), but RB1 has also been shown to cooperate with PRC2 in the methylation of cell cycle genes (13). To understand if Rb1 itself was a target of H3K27me3, we asked if the Rb1 gene was a direct target of JARID2 and the PRC2 complex. We found that JARID2 was recruited to the Rb1 promoter and depletion of JARID2 reduced this association (Fig. 6A). We next examined the histone methylation of the Rb1 promoter. We found that the H3K9 methylation profile was significantly low and was not altered upon JARID2 depletion (Fig. 6B), but H3K27 methylation was significantly reduced (Fig. 6C). Total H3 was not reduced and, in fact, was modestly enhanced upon JARID2 depletion (Fig. 6C). We also examined the association of EZH2 and found that EZH2 was associated with the Rb1 promoter and the depletion of JARID2 reduced this binding (Fig. 6D). Thus, Rb1 is a direct target of a JARID2-guided PRC2 complex. However, our data clearly show that the relief of H3K27me inhibition is not sufficient to overcome the cyclin D1-mediated repression of Rb1.
We next asked if the Cdkn1a was directly regulated by JARID2. ChIP assays were performed for JARID2, EZH2, H3K27me3, and H3K9me. However, we did not see any signif-
JARID2 regulates the cell cycle in skeletal muscle
icant change in the enrichment of H3K9me, H3K27me3, JARID2, and EZH2 for the Cdkn1a promoter upon JARID2 depletion (Fig. S3), suggesting that Cdkn1a is not a direct target of the JARID2-guided PRC2 complex. We do, however, note that H3K27me3 was enriched on the Cdkn1A promoter and this association was lost upon EZH2 depletion, indicating that JARID2 regulates the cell cycle in skeletal muscle EZH2 does directly regulate the Cdkn1a locus. 3 Our results indicate that this regulation may be independent of JARID2.
To understand how the suppression of Rb1 contributed to the effects observed in JARID2-depleted cells, we used expression construct for RB1-WT and a constitutively active form of RB1 which contains 14 mutated phosphorylation sites (RB-NPC) (34). These constructs were transiently transfected in scr and JARID2-depleted cells. Cells were harvested both before and after differentiation conditions. The known up-regulation of RB1 upon differentiation and the loss of RB1 expression in JARID2-depleted cells could be observed in this experiment (Fig. 6E). We found that RB1-WT could efficiently silence cyclin D1 expression in JARID2-depleted cells (Fig. 6E). The nonphosphorylatable form of RB1 (NPC) did not effectively suppress cyclin D1 (Fig. 6E). Phosphorylation of RB1 releases E2F and our data suggest that phosphorylation of RB1 is required for cyclin D1 repression. We have also shown that the depletion of JARID2 inhibits the expression of myogenin through the loss of -catenin-mediated expression of MyoD (29). Although RB1 could effectively suppress cyclin D1, it could not restore myogenin expression (Fig. 6E). Thus, the effects of JARID2 on the cell cycle and Wnt pathway signaling are independent and show that JARID2 and the PRC2 complex regulate myogenesis through multiple pathways.
Transient loss of JARID2 transiently up-regulates cyclin D1
Our results showing that JARID2 is required to restrain proliferation through the PRC2 complex were surprising given that ablation of EZH2 has been shown to lead to reductions in the muscle stem cell pool (35). In our companion manuscript with EZH2, 3 we show that EZH2 restrains cell proliferation through precise regulation of both positive and negative cell cycle genes in skeletal muscle and that the resulting phenotype of enhanced proliferation or cell death correlated to the level of EZH2 depletion or inhibition. To understand if JARID2 had similar effects, we asked if we could observe enhanced proliferation and the up-regulation of cyclin D1 using a transient depletion approach. JARID2 was transiently depleted in C2C12 cells using the same shRNAs used to generate the stable cell lines, and cells were harvested 24 and 48 h following the transfection. Quantification of JARID2 mRNA following the depletion is shown in Fig. 7A. Unlike EZH2 depletion or loss, we did not see any discernible difference in cell proliferation (Fig. 7B). However, we also did not observe enhanced proliferation, indicating that stable depletion of JARID2 is required to see this effect. Myogenin is a known target of the PRC2 complex in skeletal muscle (33) and we found that an immediate up-regulation of Myog could be observed in the transient approach (Fig. 7C). However, the expression quickly returned to normal levels by 48 h post transfection (Fig. 7C). We next asked how cyclin D1 expression was affected in a transient depletion of JARID2. Like myogenin, we saw an immediate burst of mRNA expression 24 h post transfection. However, by 48 h post transfection the expression was lower than the scrambled control (Fig. 7D). At the protein level, no up-regulation of cyclin D1 could be detected (Fig. 7E). To confirm that JARID2 was depleted in this approach, protein expression of JARID2 was examined as well, and we saw that JARID2 was down-regulated, consistent with the mRNA analysis (Fig. 7E). The depletion efficiency of JARID2 correlated closely with the burst of Ccnd1 mRNA expression detected in this approach.
C2C12 cells are known to have mutated p16/p19 locus (36), and it was not clear what impact the mutated locus had on the results we observed with C2C12 cells. To answer this question, we next used freshly isolated primary myoblasts to determine whether we could recapitulate the results we saw with C2C12 cells. JARID2 was transiently depleted in primary myoblasts using the shRNA constructs used for stable depletion and cells harvested 24 or 48 h post transfection. We found that JARID2 was depleted at both time points (Fig. 8A), but as we saw in C2C12 cells, an up-regulation of cyclin D1 expression could be observed at 24 h post transfection, but cyclin D1 levels quickly returned to baseline 48 h post transfection (Fig. 8B). Again similar to what we observed in C2C12 cells using a transient approach, we observed no changes in cell cycle progression upon on the transient loss of JARID2 in primary myoblasts (Fig. 8C). We also saw no significant increase in DNA synthesis as assayed by an EdU incorporation assay (Fig. 8D). At the 48 h time point, all positive cell cycle genes assayed were down-regulated including Ccne1 (Fig. 8E), Cdk4 (Fig. 8F), and Cdk2 (Fig. 8G). Down-regulation of CYCLIN D1, CDK4, and CDK2 were observed at the protein level as well (Fig. 8H). As we observed with a transient EZH2 depletion, 3 the negative cell cycle regulator Cdkn1a (p21) was up-regulated (Fig. 8I), and this effect could also be seen at the protein level (Fig. 8H). The Cdkn2a (p16/p19) locus was down-regulated (Fig. 8J), indicating that the loss of this locus is not implicated in the proliferation defect observed in C2C12 cells upon stable depletion of JARID2. Intriguingly, although a significant effect on cell viability was not observed upon transient JARID2 depletion, we did observe increased mRNA expression of the pro-apoptotic gene Bax (Fig. 8K) and decreased expression of the anti-apoptotic factor Bcl2 (Fig. 8L)
JARID2 regulates the cell cycle in skeletal muscle
Together, our results confirm that JARID2 directly regulates cyclin D1 through the PRC2 complex, but because of the complex effects on both positive and negative cell cycle genes, sustained depletion is required to see a sustained activation of cyclin D1 and enhanced proliferation.
Discussion
In this work, we show that JARID2, along with the PRC2 complex, restrains proliferation by repressing the pro-proliferative cell cycle regulators cyclin D1 and cyclin E1. When expressed, these factors then phosphorylate RB1, thus releasing activator E2F transcription factors that further up-regulate various cell cycle genes including cyclin E1. This feed-forward loop forms a switch that decides whether the cell proceeds to S phase surpassing the G 1 /S restriction point or exits the cell cycle in a RB1-dependent manner.
Our finding that JARID2 regulates cell proliferation is in agreement with the previous finding in heart muscle which showed that JARID2 regulates cell proliferation through repression of the Ccnd1 gene (25,27). However, unlike in cardiomyocytes where JARID2 has been shown repress Ccnd1 by H3K9 methyltransferases (30), we show that JARID2 represses
JARID2 regulates the cell cycle in skeletal muscle
Ccnd1 by recruiting a H3K27 methyltransferase, the PRC2 complex, in skeletal muscle. This finding is in agreement with previous findings that showed no significant contribution of H3K9 methylation during skeletal muscle differentiation (33). JARID2 has also been shown to regulate various target genes in a PRC2-dependent manner during cardiac development as well (38). Clearly, the role of JARID2, an inactive histone demethylase, and its regulatory partner complexes is evolutionarily diverse depending on cell type. It is interesting that JARID2/PRC2 directly regulates both cyclin D1 and cyclin E1 to maintain control of the cell cycle. The dual regulation serves to maintain control of two central regulators of the G 1 /S transition phase. Our results also suggest that the PRC2 complex has many divergent roles in skeletal muscle cells that require a fine balance of JARID2 and EZH2 expression.
Intriguingly, JARID2 and the PRC2 complex also methylate Rb1 in proliferating cells, the repression of which prevents mitotic exit and terminal differentiation. Thus, the normal function of JARID2 and the PRC2 complex in skeletal muscle appears to be restraining the rate of proliferation while also preventing mitotic exit. In the JARID2 depletions characterized here, the loss of inhibition of cyclin D1 dominates the potential de-repression of Rb1. However, when JARID2 expression was restored, it was interesting that the normal expression of both negative and positive cell cycle regulators was restored. In this case, the repression of cyclin D1 is clearly sufficient to restore RB1 expression, irrespective of H3K27 methylation. Figure 6. Rb1 is directly regulated by JARID2 but not Cdkn1a (p21). A-D, RB1 is a direct target of JARID2 and the PRC2 complex. ChIP assays were performed on proliferating scr and JARID2-depleted (shJarid2) C2C12 cells with antibodies against JARID2 (A), H3K9me (B), H3K27me3 and H3 (C) EZH2 (D), and primers spanning the Rb1 promoter. IgG background signals were subtracted from immunoprecipitation signals. E, restoration of RB1 can inhibit cyclin D1, but not activate myogenin. C2C12 cells with scrambled (scr) and JARID2 depletion (shJarid2) were transiently transfected with expression constructs for RB1 (RB1-WT), and RB1 mutated at phosphorylation sites (RB1-NPC). UD represents the proliferative, undifferentiated time point and D2Ј represents 2 days in differentiation media. Cells were analyzed for total RB1, cyclin D1, and myogenin. GAPDH was used as a loading control. # represents the band of interest for RB1 protein as determined by molecular weight standards. Error bars, mean Ϯ S.E. (Student's t test; ns represents not significant, *, p value Ͻ 0.05; **, p value Ͻ 0.01; ***, p value Ͻ 0.001; n ϭ 3 biological replicates.)
JARID2 regulates the cell cycle in skeletal muscle
JARID2 has also been shown to regulate cell proliferation through repression of Ccnd1 in leukemia (28). JARID2 has been identified as an oncogene that is often overexpressed in multiple cancers including rhabdomyosarcoma (39). In rhabdomyosarcoma, JARID2 is essential for cell viability and loss of JARID2 rescues partial differentiation in rhabdomyosarcoma (39). However, JARID2 has recently been shown to act as tumor sup-pressor in myeloid neoplasm through restriction of hematopoietic progenitor cells self-renewal (40).
Importantly, in our study we found that JARID2 could be efficiently depleted in C2C12 cells without a significant impact on viability, unlike EZH2. We did observe changes in apoptotic cell marker expression upon transient JARID2 depletion in primary myoblasts, so it remains to be determined if JARID2 could Recent studies have shown that the expression Cdk4/cyclin D1 expands neural stem cells and increases neurogenesis (41). It has also been shown that regulation of the cell cycle through reintroduction of cyclin D1 and CDK4 in cardiac cells can increase cardiac cell regeneration and repair post injury (42). Our work suggests that inhibiting the function of JARID2 could potentially be used to expand the muscle stem cell pool and enhance muscle repair and regeneration. Clearly, the roles of JARID2 and the PRC2 complex are complex in skeletal muscle and serve to both inhibit and activate several steps of the myogenic program. Characterization of each of these roles aid in the understanding of how these factors are involved in normal myogenesis, repair, and disease.
Experimental procedures
Cell culture C2C12 cells (ATCC) were grown in DMEM, supplemented with 10% FBS (HyClone Laboratories) according to standard protocols. Proliferating C2C12 myoblasts were grown in DMEM supplemented with 10% FBS (HyClone Laboratories), and 2% horse serum (Gibco Laboratories) in DMEM was used to induce differentiation. Primary myoblasts were isolated according to standard protocols (43). Briefly, hind limb muscles of neonatal mice were isolated and digested with Collagenase Type II (Worthington Biochemical Corp.). Cells were filtered through a sterile 70-micron filter, plated on gelatin-coated plates in 20% FBS in F-10 basal media with 1ϫ penicillin-streptomycin (Corning) and 2.5 ng/ml bFGF (gift of D. Cornelison, University of Missouri). Primary myoblasts were enriched by pre-plating cells onto uncoated plates for 30 min before transferring myoblast suspensions to collagen-coated plates and repeating until the majority of the cells were myoblasts. Myoblast identity was confirmed by expression analysis of MRFs, differentiation assays, and myosin heavy chain immunofluorescence. All mouse procedures were approved by the Southern Illinois University Institutional Animal Care and Use Committee.
Plasmid constructs
JARID2 was depleted with shRNA constructs designed by the RNAi Consortium in the pLOK.1 plasmid (Open Biosystems) as described (29). Three constructs targeting murine Jarid2 and one scrambled control were linearized using the ScaI restriction enzyme (New England Biolabs), transfected into C2C12 cells using TurboFect (Thermo Scientific) following manufacturer's protocol, and selected with puromycin (2 g/ml). Individual clones were selected, propagated, and confirmed by mRNA and protein analysis. For transient transfections, circu-lar (uncut) plasmids were transfected using TurboFect. mRNA and protein were extracted at indicated time points. Drug selection was not used for transient transfection experiments.
The coding sequence of murine Jarid2 was PCR amplified from cDNA reverse transcribed from the total RNA extracted from proliferating C2C12 cells. The PCR amplified Jarid2 insert was cloned into the pEF6/V5 His TOPO TA expression vector according to manufacturer's protocol (Invitrogen). The clones were confirmed by sequencing. For rescue of JARID2 expression, pEF6-Jarid2 plasmid was stably transfected in C2C12 cells previously depleted of JARID2 using shRNA construct (mature antisense sequence: 5Ј-AAATTGCACATGGATGACAGG-3Ј) that targets 3Ј-untranslated region (UTR) of Jarid2 mRNA. The stable clones were selected with blasticidin (10 g/ml), confirmed by mRNA and protein analysis.
Expression plasmids for WT and a nonphosphorylatable form (NPC) of retinoblastoma protein (RB1) were generated in the lab of Sibylle Mittnacht (University College London Cancer Institute) (34). Colin Goding (University of Oxford Ludwig Cancer Research) used these constructs to characterize the interaction of Mitf and RB1 (37) and generously gifted these constructs to our laboratory. The constructs were confirmed by restriction enzyme mapping and sequencing. The plasmids were transfected using TurboFect as described previously.
Western blot analysis
Cell extracts were made by lysing PBS washed cell pellets in radioimmune precipitation assay buffer (RIPA) supplemented with protease inhibitors (cOmplete Protease Inhibitor, Roche Diagnostics). Following incubation on ice, clear lysates were obtained by centrifugation. Protein concentrations were determined by Bradford's assay (Bio-Rad). For each sample, 30 g of protein was loaded on each gel unless otherwise specified. Proteins were transferred onto a PVDF membrane using a tank blotter (Bio-Rad). The membranes were then blocked with 5% milk in 1ϫ Tris-buffered saline plus Tween 20 (TBST) and incubated with primary antibody overnight at 4°C. Membranes were then washed with 1ϫ TBST and incubated with the corresponding secondary antibody. Membranes were again washed with 1ϫ TBST, incubated with chemiluminescent substrate according to manufacturer's protocol (SuperSignal, Pierce) and visualized by an iBright Imaging System or developed by autoradiography. The antibodies used include anti-JARID2 (ABclonal), anti-cyclinD1 (DCS-6, Invitrogen), anticyclin E1 (Santa Cruz Biotechnology), anti-CDK2 (ABclonal), anti-CDK4 (ABclonal), anti-pRB1 (LabVision) (Fig. 4I), anti-pRB1(Cell Signaling Technology) (Figs. 3B and 6E), anti-p21 (ABclonal), anti-tubulin (E7, Developmental Studies Hybridoma Figure 8. Transient loss of Jarid2 does not decrease cell viability in primary myoblasts. A, freshly isolated primary myoblasts were transiently transfected with scrambled (scr) control or shRNA against Jarid2 (shJarid2) without drug selection. Jarid2 expression was assayed by qRT-PCR at the indicated time points. B, cells in A were assayed for cyclin D1 expression by qRT-PCR. C, transient loss of Jarid2 in primary myoblast does not have significant change cell cycle progression. Cells in A were ethanol fixed, propidium iodide stained, and analyzed for cell cycle phase distribution using flow cytometry. D, transient loss of Jarid2 did not result in a significant increase in DNA synthesis in primary myoblasts. Cells in A were assayed for DNA synthesis by EdU incorporation assay (left panel). Five random fields were counted for EdU ϩ nuclei and plotted (right panel). DAPI was used to stain nuclei. Scale bar, 50 m; n ϭ 2 biological replicates. E-J, cell cycle genes are deregulated upon transient loss of Jarid2 in primary myoblasts. Cells as in A were analyzed for the expression of cyclin E1 (Ccne1) (E), Cdk4 (F), Cdk2 (G), p21 (Cdkn1a) (I), and Cdkn2a (p16/19) (J) by qRT-PCR and Western blotting (H). GAPDH was used as a loading control. # represents the band of interest for CDK2 based on molecular weight standards. K and L, Jarid2 depletion increased apoptotic marker expression in primary myoblasts. Cells in A were assayed for the pro-apoptotic marker Bax (K) and anti-apoptotic marker Bcl2 (L) by qRT-PCR. Error bars, mean Ϯ S.E. (Student's t test; ns represents not significant; ***, p Ͻ 0.001; n ϭ 3 biological replicates.) Bank), and anti-GAPDH (Millipore). Protein expression levels were quantified using iBright analysis software or ImageJ (National Institutes of Health) software on at least three independent experiments unless otherwise mentioned. Representative images are shown.
Quantitative real-time PCR
RNA was isolated from cells by TRIzol extractions (Invitrogen). Following treatment with DNase (Promega), 2 g of total RNA was reversed transcribed with MultiScribe™ MuLV Reverse Transcriptase (Applied Biosystems). cDNA equivalent to 40 ng was used for quantitative PCR amplification (Applied Biosystems) with SYBR green PCR master mix (Applied Biosystems). Samples in which no reverse transcriptase was added (no RT) were included for each RNA sample. Quantitative PCR data were calculated using the comparative Ct method (Applied Biosystems). Standard deviations from the mean of the [⌬] Ct values were calculated from three independent RNA samples. Primers are described in Table S1. Where possible, intron-spanning primers were used. All quantitative PCR was performed in triplicate, and three independent RNA samples were assayed for each time point. For measurements of relative gene expression, a -fold change was calculated for each sample pair and then normalized to the -fold change observed at hypoxanthine guanine phosphoribosyl transferase (HPRT) and/or 18S rRNA.
ChIP assays
ChIP assays were performed as described previously (37). The following antibodies were used: anti-Jarid2 (Cell Signaling Technology), anti-Ezh2 (Cell Signaling Technology), anti-H3K27me3 (Cell Signaling Technology), anti-H3K9me (pan) (Cell Signaling Technology), anti-H3 (Cell Signaling Technology) and anti--Catenin (Santa Cruz Biotechnology). Rabbit IgG (Santa Cruz Biotechnology) was used as a nonspecific control. Primers are described in Table S1. The real-time PCR was performed in triplicate. The results were represented as the percentage of immunoprecipitation over input signal (% Input) with IgG background signal subtracted. All ChIP assays shown are representative of at least three individual experiments unless otherwise stated. Standard error from the mean was calculated and plotted as the error bar.
EdU incorporation assay
Cells were assayed using a Click-iT EdU Alexa Fluor 488 Imaging Kit according to the manufacturer's protocol (Life Technologies). C2C12 and primary myoblast cells were grown in the presence of EdU for 2 and 3 h, respectively. EdU positive nuclei were counted in at least five random fields on microscopic images taken at 200ϫ and 400ϫ magnification using a Leica microscope.
Proliferation assay
An equal number of cells were seeded, and on the indicated days, cells were harvested using trypsin, resuspended and counted under a light microscope using a hemocytometer. Cell viability was determined by Trypan Blue staining. Cell counting was performed in duplicate for at least three blinded biological replicates.
Cell cycle analysis by flow cytometry
Cells were split in 100-mm diameter plates at 1:5 dilution, grown for 48 h in duplicates in 10% FBS in 5% CO 2 incubator. The cell plates were washed twice with sterile 1ϫ PBS, pH 7.4, harvested using trypsin, and resuspended in 1ϫ PBS, pH 7.4. Cells were pelleted down at 600 ϫ g for 5 min at room temperature, washed twice with 1ϫ PBS and were fixed in chilled 70% ethanol overnight at Ϫ20°C. Cells were washed with 1ϫ PBS, pH 7.4, treated with RNase (10 g/ml), and stained with propidium iodide (50 g/ml) for 2 h at room temperature before running through a BD Accuri C6 flow cytometer (BD Biosciences). Data were analyzed using FlowJo (FlowJo LLC) software.
Statistics
Data are presented as mean Ϯ S.E. Statistical comparisons were performed using the unpaired two-tailed Student's t tests and analysis of variance (ANOVA) test followed by Tukey's multiple comparison test. A probability value of Ͻ 0.05 was taken to indicate significance. All statistical analyses and graphs were made in GraphPad Prism 8.0 software. | v3-fos-license |
2014-10-01T00:00:00.000Z | 1987-04-01T00:00:00.000 | 13609189 | {
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} | pes2o/s2orc | Nerve membrane ion channels as the target site of environmental toxicants.
There are many environmentally important chemicals which exhibit potent effects on the nervous system. Examples include insecticides such as pyrethroids, DDT, cyclodienes, organophosphates and carbamates, and heavy metals such as mercury and lead. Since nerve excitation takes place in a fraction of a second, electrophysiological methods provide us with the most straightforward approach to the study of the mechanisms of action of environmental toxicants on the nervous system. Aquatic animals such as crayfish, lobster, squid, and marine snails represent extremely useful materials for such electrophysiological studies, because much of our knowledge of nerve excitation is derived from those animals. Nerve excitation takes place as a result of opening and closing of ion channels of the membrane. These functions are independent of metabolic energy, and can be measured most effectively by voltage clamp techniques as applied to the giant axons of the crayfish and the squid. Patch clamp techniques developed during the past 10 years have added a new dimension to the electrophysiological investigation. These techniques allow us to measure the activity of individual ion channels, thereby making it possible to analyze the interaction of toxic molecules directly with single ion channels. Examples are given summarizing electrophysiological studies of environmental neurotoxicants. The abdominal nerve cords and neuromuscular preparations isolated from the crayfish are convenient materials for bioassay of certain environmental toxicants such as pyrethroids, chlorinated hydrocarbons, and other insecticides. Detailed voltage clamp and patch clamp analyses have revealed that pyrethroids and DDT modify the sodium channel to remain open for an extended period of time. This change causes an increase in depolarizing afterpotential which reaches the threshold for repetitive discharges to be produced.(ABSTRACT TRUNCATED AT 250 WORDS)
Introduction
Numerous environmental toxicants are known to cause serious damage to the nervous system. These neurotoxic substances include insecticides, heavy metals, and hexanes. Indeed, most of the insecticides currently in use for agricultural, medical, and veterinary purposes are very potent neuropoisons as exemplified by pyrethroids, DDT, cyclodienes, organophosphates and carbamates. The environmental hazards of heavy metals such as mercury and lead are due primarily to damage, both acute and chronic, to the nervous system. Therefore, to prevent and manage intoxication by environmental neurotoxicants, it is imperative to understand the mechanisms of toxic action of these agents on the nervous system.
Various approaches and methods have been used to accomplish this goal, including electrophysiological, neurochemical, histological, and behavioral techniques. Since the major function of the nervous system is to generate and transmit excitation in the form of an elec-*Department of Pharmacology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611 trical signal, or impulse, electrophysiological techniques provide us with the most straighforward and powerful approach to elucidate the mechanisms of action of neurotoxicants at the cellular and molecular level.
Aquatic animals represent an important group with respect to environmental neurotoxicology for several reasons. First, some of them provide us with very unique and important materials for the study of the mechanism of action of neurotoxicants. For example, the nervous systems of the crayfish, lobster, squid, and marine snails have been used very widely to study the mechanisms of nerve excitation in general. Almost all aspects of our present knowledge of nerve excitation are derived from study of aquatic animal models. These nerve preparations have been used extensively for the study of neurotoxicants as well. Second, because aquatic animals are very sensitive to certain environmental neurotoxicants such as cyclodienes, they are important for gaining an understanding of differential sensitivity. This paper summarizes some of our recent environmental studies on aquatic animals. Major emphasis is on the study of neuroactive insecticides on the nervous system of the crayfish and squid. Similar approaches can be easily applied to the study of other environmental neurotoxicants. It is important to realize that information gained through these studies utilizing aquatic animals can be easily applied to the human. Therefore, such studies can contribute immensely to our health care with respect to the environmental toxicants.
Mechanisms of Nerve Excitation
The nerve membrane generates impulses or action potentials which are transmitted from the sensory cells to the central nervous system and then to the motor system. The action potential is generated as a result of changes in membrane permeabilities to ions such as sodium, potassium and calcium. The resting membrane potential (RP), inside negative with respect to the outside by 50 to 100 mV, assumes a value close to the equilibrium potential for potassium (EK), because the resting membrane is almost exclusively permeable to potassium ( Fig. 1). When the membrane is depolarized (stimulated), the membrane permeability (conductance) to sodium (gNa) increases rapidly, so that the membrane potential approaches a value close to the equilibrium potential for sodium (ENa); this is the rising phase of the action potential (AP). However, the increased gNa starts decreasing quickly and at about the same time the potassium permeability (conductance) gK starts increasing beyond its resting value. These changes in gNa and gK bring the membrane potential back toward the potassium equilibrium potential; this is the falling phase of the action potential.
During the rising phase of the action potential, sodium Passive Fluxes Na-K Pump FIGURE 1. Diagram of the mechanism of resting and action potential production. RP, resting potential; AP, action potential; EK, POtassium equilibrium potential; ENa, sodium equilibrium potential; gNa, sodium conductance; gK, potassium conductance (4).
ions enter the cell according to their electrochemical gradient, and during the falling phase of the action potential, potassium ions leave the cell according to their electrochemical gradient. The increases in gNa and gK are the result of opening of "sodium channels" and "potassium channels," respectively, so that the sodium influx and potassium efflux occur through these open channels. The resultant increase in intracellular sodium concentration and decrease in intracellular potassium concentration are very small indeed, being calculated to be approximately 1/1000 oftheir initial concentrations for a nerve fiber 1 ,um in diameter. However, no matter how small the internal ionic concentration changes may be, they must be restored in order for the nerve fiber to continue to generate many action potentials. This is accomplished by a mechanism called the Na-K pump, which pushes out extra sodium and absorbs potassium. This pump is operated by metabolic energy. By contrast, changes in gNa and gK, or opening and closing of sodium and potassium channels, take place normally in the absence of metabolic energy, and therefore are metabolism-independent processes.
When an action potential arrives at the nerve terminal, a neurotransmitter is released. The transmitter in turn binds to the postsynaptic receptor, resulting in changes in ionic permeabilities in the postsynaptic membrane. These changes in permeabilities generate action potentials in the postsynaptic neurons or effector cells, thus completing synaptic transmission. A large number of neurotransmitters have been identified in various animals and synapses, including acetylcholine, norepinephrine, glycine, glutamate, y-aminobutyric acid, and certain peptides, to mention a few. Postsynaptic ionic permeabilities are due to various ion channels permeable to sodium, potassium, calcium, chloride, etc., depending on the synapse.
Electrophysiological Methods
Routine electrophysiological methods utilizing extracellular electrodes or intracellular microelectrodes are useful for recording action potentials from nerve fibers, sensory neurons, and synapses. However, these methods are far from sufficient to elucidate the mechanism of action of neurotoxicants on membranes, as they do not permit analysis in terms of ion channels. The function of ion channels can best be studied by voltage clamp techniques, which allow us to measure ionic permeabilities in the form of ionic conductances. Voltage clamp techniques were originally developed for the squid giant axons by Cole (1) and improved and extensively used by Hodgkin, Huxley, and Katz (2). It has now become a routine technique for the study of neurotoxicant effects on nerve membrane ion channels (3,4). The voltage clamp technique was also adapted to postsynaptic membranes such as end-plate membranes (5).
A dramatic advance in technology was made about 10 years ago by Neher and Sakmann (6), who successfully developed a patch clamp technique to record opening and closing of individual ion channels. The technique has been much improved in the interim, and now it is possible to measure single channel activity of practically any cell, including inexcitable cells (7). We can now analyze interactions of toxic molecules with a single ion channel. Much progress has been made for the study of environmental neurotoxicants also (8)(9)(10).
Crayfish Nerve Cords
The abdominal nerve cord isolated from the crayfish is a convenient and sensitive material for simple bioassay of certain neurotoxicants (11). Spontaneous discharges, as recorded by a pair of wire electrodes, can serve as a measure of either stimulating or paralyzing effects of neurotoxicants. An example of an experiment for the pyrethroid allethrin is shown in Figure 2. Allethrin at a concentration of 1 I±M causes a drastic increase in spontaneous discharges within 5 min, followed by complete paralysis later. Even at a lower concentration of 100 nM. allethrin causes a larnre increase in the frequency of spontaneous disci insecticides such as carbofuran, I have also been found to stimulate
Crayfish Neuromuscular Preparations
Crayfish neuromuscular preparations have also been found to be a very useful material for bioassay of certain toxicants. These preparations have both excitatory and inhibitory innervation. L-Glutamate and y-aminobutyric acid are the respective transmitters. Excitatory transmission is very sensitive to pyrethroid and DDT-type compounds. An example is illustrated in Figure 4 in which muscle contractions evoked by nerve stimulation are first augmented and then blocked by 100 nM allethrin. It was later found that the augmented contraction produced by pyrethroids and DDT-type insecticides was due primarily to the stimulation of presynaptic nerves (12). Repetitive discharges were generated in the presynaptic nerve which caused repetitive and augmented activity of the muscle.
Crayfish and Squid Giant Axons iarges (Fig. 3). Other The giant axons isolated from the crayfish or the squid iDT, and toxaphene have proved to be among the most convenient materials the nerve cord.
to determine the mechanisms of action of neurotoxicants. First, the basic mechanism of nerve excitation has been studied extensively using these preparations. RN In fact, because of its large size the squid giant axon is 60 min the prototype nerve preparation from which most of our present knowledge of nerve excitation has been derived. Second, voltage clamp experiments to analyze the ion channel function can be performed with these giant axons with the highest degree of accuracy. Third, these giant axons can be perfused intracellularly as well as extracellularly, providing us with a high degree of flexibility for voltage clamp measurements. Fourth, the ma-mV terials are relatively readily available.
msec
In order to illustrate the usefulness of these giant axon preparations and to summarize our current knowledge of the mechanisms of action of toxicants, some of he spontaneous discharges our studies of DDT and pyrethroid insecticides will be 1).
briefly described. When applied to the isolated giant axon, some of these insecticides cause repetitive afterdischarges in response to a single stimulus (Fig. 5). A Allethrln Repetitive afterdischarges are produced when the depolarizing afterpotential is elevated to the level of threshold for excitation. Thus, the next question is how the depolarizing afterpotential is increased by DDT or pyrethroids. This can best be studied by voltage clamp experiments.
Voltage clamp experiments with crayfish and squid giant axons have clearly shown that the sodium current is greatly prolonged by pyrethroids and DDT (13)(14)(15)(16). An example of such an experiment is shown in Figure 6. The control record represents the transient sodium current as evoked by a step depolarizing pulse. After 60 , O .
, application of allethrin, the sodium current is greatly 50 60 70 80 prolonged while its peak amplitude remains unchanged. A prolonged sodium current increases the depolarizing n the frequency of sponta-afterpotential, which in turn generates repetitive afinal cord. Each point rep-terdischarges. Thus, the major effect of pyrethroids and i of 0.2 or 1 sec. DDT is to prolong the sodium current.
Single Channel Experiments
The sodium current recorded from a giant axon under voltage clamp conditions is a sum of individual sodium channel currents. Thus, it is difficult to analyze direct interactions of insecticide molecules with individual so- shown that pyrethroids cause a remarkable prolongation of sodium channel opening (8)(9)(10).
An example of a single channel experiment is illustrated in Figure 7. Single sodium channel currents were recorded from a neuroblastoma cell (N1E-115 line) by using the patch clamp technique (10). In the control cell, individual sodium channels open during a step depolarizing pulse and can be seen as downward square deflections on the record. After application of 60 puM tetramethrin, individual sodium channel currents are still observed with their amplitude unchanged. However The observed change by pyrethroids at the single sodium channel level can account for the symptoms of poisoning at the animal level. Calculations were made of the percentage of sodium channel population that must be modified by tetramethrin for the depolarizing afterpotential to reach the threshold for repetitive discharges. Only a very small fraction of sodium channels, less than 1%, needs to be modified for this change in depolarizing afterpotential which leads to severe symptoms of poisoning in animals (17). This is why the pyrethroids are very potent as insecticides. This situation also provides us with an excellent example of "toxicological amplification" from ion channel to animal. | v3-fos-license |
2018-12-15T05:08:04.037Z | 2013-08-21T00:00:00.000 | 56261649 | {
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} | pes2o/s2orc | Study on Channel Features and Mechanism of Clinoptilolite Modified by LaCl 3
Natural clinoptilolite was modified by LaCl3 at different ion concentrations, particularly focusing on the effects of LaCl3 on surface area, average pore width and pore volume distribution. And the structure was characterized by analyzing the X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, and Energy Dispersive Spectroscopy (EDS). The results showed that the surface area, total pore volume and micropore area decreased after modified by LaCl3, while the average pore width increased, among which the micropore area most significantly changed. The result of IR indicated that LaCl3 did not enter into silico-oxygen tetrahedron skeleton to participate in skeleton vibration. The result of XRD showed that the crystallinity of the modified clinoptilolite was slightly reduced, and the diffraction peak deviated to small angle, but the crystal structure kept invariant. The result of EDS, FT-IR and XRD demonstrated that LaCl3 can be loaded on the surface and channels of clinoptilolite, which led to degradation in absorption performance of clinoptilolite to ammonia nitrogen while for phosphorus increases.
Introduction
Clinoptilolite is a porous material with a regular structure and aperture less than 10 nm, composed of aluminosilicate framework, cavities, channels, cations, and water molecules (Krishnarao & Subrahmanyam, 2003).The specific surface area increases ten million times when the cube with side length of 10 -2 m is divided into small cubes with side length of 10 -9 m.Thus the nanometer-grade grain has an ultra-high specific surface area, with many unique surface effects (Dou et al., 2009).Pores in the framework and channels of the clinoptilolite have a large internal surface area, and thus leads to the potential of adsorption (Zhang & Shi, 2012;Chen, 2012).However, clinoptilolite has a poor adsorption performance to phosphate because its structure is negatively charged (Sun et al., 2010).Since rare earth metals have good affinity with phosphate ions, the application of rare earth metals in sewage treatment has become a new technical focusing point (Wu & Hu, 2011).Lanthanum is cheaper when compared with other rare earth elements and the effect of trace lanthanum on human health is not yet conclusive (Chen et al., 2010).Therefore lanthanum has been widely used to improve the performance of existing adsorbents and the phosphorus removal rate.
Currently, researchers often use lanthanum chloride and lanthanum nitrate for clinoptilolite modification, adjusting the pH by adding NaOH to make lanthanum hydroxide precipitate to the surface of clinoptilolite.Or roasting the clinoptilolite modified by lanthanum hydroxide in order to render lanthanum hydroxide decompose to lanthanum oxide (Zhang, Wan, & Chang, 2011;Zhang, Zhou, & Liu, 2012).Considering that the generation of lanthanum hydroxide needs to consume a lot of alkali, and if using lanthanum oxide to modify directly, the problems are that lanthanum oxide is insoluble in water and the pharmaceutical utilization is low.Furthermore, previous studies mostly concentrated on the performance of lanthanum-modified clinoptilolite to remove contaminants, but the pore characteristics of modified clinoptilolite particles and the action mechanism between lanthanum and clinoptilolite are little studied.Thus this article uses clinoptilolite as research subject, and uses LaCl 3 to modify clinoptilolite.Then through analyzing the surface area, average pore width and pore volume distribution changes of modified and unmodified clinoptilolite, the influence law of clinoptilolite pore characteristics caused by modification can be found.Using EDS, IR, XRD, and etc. to characterize its structure, and analyze the action mechanism of LaCl 3 and clinoptilolite particles.
Materials and Equipments
Sample materials: Natural clinoptilolite from Shenyang was chosen for sample materials.After grinding for a certain period of time and sieving, particle size of natural clinoptilolite between 0.12 to 0.16 mm was selected to be cleaned its surface impurities with deionized water, then it was oven-dried for preparation (Sun et al., 2012 ).The chemical concentrations of the sample were: SiO 2 (68.75%),Al 2 O 3 (12.26%),Fe 2 O 3 (1.21%),CaO (2.57%), K 2 O (2.83%), Na 2 O (1.08%), MgO (0.81%).XRD diffraction analysis showed that the clinoptilolite is calcium type clinoptilolite.During the test, the pharmacy used were of analytical pure, the water used was deionized water.
Test and analysis equipment: Rigaku DMAX-RB 12KW X-ray diffractometer, Nicolet 5MX Fourier Transform Infrared Spectrometer, V-Sorb 4800 Specific surface area and pore size analyzer, S250MK3 Scanning electron microscopy.
The Preparation of Modified Clinoptilolite
4 grams of natural clinoptilolite were dissolved in a series of solution with different concentration of lanthanum chloride, add deionized water to 200 ml.Natural clinoptilolite was dispersed in solution by prolonged stirring (2 h, 300 r•min -1 ) with a magnetic stirrer.After filtering, the sample was oven-dried at 60 o C for 3 h, place the samples in the dryer for preparation.
Characterization of Channel Characteristics Change
Using V-Sorb 4800 specific surface area analyzer and pore size distribution, with static volumetric method for measuring principle, according to the nitrogen adsorption isotherm, using the BET method to calculate the specific surface area of the natural clinoptilolite and the modified, using BJH method to calculate the pore width distribution of mesoporesand macropores, HK method for micropore, micropore volume is calculated using t-plot method.
Structural Characterization
EDS was used for testing the composition difference between natural and modified clinoptilolite, XRD was used for testing their crystal structure change, and IR spectroscopy was for testing their group change.
Pore Characteristics of LaCl 3 -Modified Clinoptilolite before and after the Modification
Physical adsorption method-BET is now recognized as one of the best ways to determine specific surface area of solid.Pore size distribution is a characterization of the relationship between pore radius and pore volume of porous materials, and the BJH method is one of the most widely used methods to determine pore size distribution.But BJH ignores the enhancement of adsorption potential in micropores, only suitable for describing pore size distribution of mesopores.HK model considers the enhancement of microscopic potential, resulting in a good description of pore size distribution to some certain extent (Zhang et al., 2006).
Table 1 shows the data of surface area and pore size distribution of unmodified and modified clinoptilolite.The BET specific surface area of unmodified clinoptilolite is 38.54 m 2 /g, and the total pore volume is 0.058 cm 3 /g.T-plot method shows that micropore area is 6.49 m 2 /g.After modification of LaCl 3 with different concentrations, the surface area, total pore volume, micopore area decreased, but the average pore width increased.Figures 1(a-c) showed pore volume distributions of micropore, mesopore and macropore of clinptilolite modificated by LaCl 3 with different concentrations, respectively.The results showed that different concentrations of LaCl 3 had different effects on clinoptilolite modification, and pore volume distributions of micropore, mesopore and macropore were also different.This data of pore volume distribution was obtained by combining BJH adsorption method and HK model.The overall tests showed that LaCl 3 -modification caused decrease in pore volume ratio of micropore and mesopore, but increase in pore volume ratio of macropore, compared with the unmodified clinoptilolite.This is mainly due to that the adsorption sites of La presented in the surface and pores of clinoptilolite, thus a proportion of LaCl 3 was loaded within channels in the clinoptilolite, and this part of La mainly existed in micropores and mesopores.
Through variations of specific surface area, average pore width, total pore volume, micropore surface area and pore volume distribution after modification by LaCl 3 with different concentrations, it can be inferred that the decrease in pore volume ratio of micropore and mesopore will inevitably result in a large increase in the pore volume ratio of macropore, which was not enough to prove that modification can widen the channels, but it can prove that LaCl 3 existed in the micropores and surface of the clinoptilolite, thereby causing reduction in the specific surface area and micropore area of modified clinoptilolite.As an adsorbent material, its structure feature has a great impact on its adsorption performance; so materials with large surface area and total pore volume have good adsorption performance.But the study found that phosphorus removal performance of the clinoptilolite is greatly improved after LaCl 3 modification (Lin et al., 2009), therefore the adsorption performance of clinoptilolite itself has not been improved after modification, while the phosphorus removal performance improvement is due to load of LaCl 3 , but the rich pores and large specific surface area of the clinoptilolite itself played a catalytic role in LaCl 3 modification.It also can be found that the ammonia nitrogen adsorption performance of the clinoptilolite decreased after LaCl 3 modification, it is due to ammonia adsorption effect are mainly ion exchange and physical adsorption (Hrenovic et al., 2008;Zhang et al., 2010;Widiastuti et al., 2011).Thus, LaCl 3 does exist in the surface and pores of the clinoptilolite, plugging channels of the clinoptilolite, and resulting in degradation in ammonia adsorption performance (Li et al., 2012).
Since inorganic acid can dissolve some impurities plugging in the channels of the clinoptilolite, so that dredging the cavities and channels, and increasing pore volume and surface area of the clinoptilolite (Wender et al., 2011).
To further prove that LaCl 3 has been loaded to the surface and channels of the clinoptilolite after LaCl 3 modification, the article first modified clinoptilolite with HCl (5%), and then further modified the clinoptilolite with LaCl 3 .Results are shown in Table 2 and Figure 1 (d), it can be found that the specific surface area of the clinoptilolite greatly increased after modified by HCl, from 38.54 m 2 /g to 52.15 m 2 /g.Total pore volume also increased from 0.058 m 2 /g to 0.075 m 2 /g.Average pore width reduced to 5.78 nm after acid modification, which is due to a large number of micropores appeared after modification and micropore area increased from 6.49 m 2 /g to 19.27 m 2 /g.Pore volume ratio of micropore increased dramatically, pore volume ratio of mesopore decreased, and pore volume ratio of macropore slightly increased, thereby resulting in an decrease in average pore width.
After treated by LaCl 3 , the pore characteristics of HCl-modified clinoptilolite also varied intensively.The specific surface area decreased from 52.15 m 2 /g to 41.54 m 2 /g, the total pore volume decreased from 0.075 m 2 /g to 0.064 m 2 /g, the micropore area decreased from 19.27 m 2 /g to 15.29 m 2 /g, and the average pore width increased to 6.16 nm.Pore volume ratio of micropore changed little, pore volume ratio of mesopore decreased from 67.20% to 64.73%.Thus proved the modification effect of LaCl 3 on clinoptilolite, and LaCl 3 could load to the surface and pores of the clinoptilolite.
Energy Dispersive Spectroscopy Analysis
According to different X-ray photon characteristic energy of different elements, energy spectrometer was used to analyze the composition of the material.Element distribution of substances can be characterized by EDS analysis.Although the contents and distributions of the elements or oxides within the whole sample can't be quantitatively analyzed by EDS, the type of the each element contained in the sample and the trend of variation of the element content can be qualitatively analyzed.Table 3 showed the energy spectrum of the natural and modified clinoptilolite.The results showed that La did not exist in the natural clinoptilolite, and the composition of the clinoptilolite changed after LaCl 3 -modification, La accounted for 0.26% in the atomic ratio, the atomic ratios of other elements except Si all decreased.Proved that LaCl 3 had loaded to the clinoptilolite, thereby improved phosphorus removal performance of the clinoptilolite.
Infrared Spectrum Analysis
Infrared spectrum is a useful method in the study of the structure of clinoptilolite (Li et al., 2009).Infrared spectrum is based on the measurement of bond vibration or rotational frequency of the molecules, including stretching vibration of the bond and flexural vibration of the bond.Therefore, it has already been applied to determine the overall skeleton structure of clinoptilolite.Generally, the wave-number of absorption spectrum of clay minerals ranges from 400 to 4000 cm -1 and belongs to the middle infrared region (Shi et al., 2013).
Wavenumbles(cm -1 ) From Figure 2, it is revealed that there is no change in the FT-IR between the modified and natural clinoptilolite.
The bond peak and strength of Si-O, Si-O-Si (Ai) and Si-O-Si (Ai) are within the allowed error range.No new key band is observed in the clinoptilolites modified by LaCl 3 .If La enters the tetrahedron skeleton and joins the skeleton vibration, the flexural vibration of Si-O loaded with La will be stronger because the covalent radius of La is larger.Meanwhile, the flexural vibration of Si-O-Si (Ai) becomes more difficult after the modification and wave number decreases.Therefore, La is loaded only to the surface of clinoptilolites and does not enter its tetrahedron skeleton structure and joins the skeleton vibration.Figure 2 also shows that FT-IR of the modified clinoptilolites after phosphorus adsorption corresponds with that before phosphorus adsorption and no new key band is observed, which indicates that during the phosphorus adsorption process, phosphorus only reacts with La loaded on the surface and pore canal of clinoptilolites and does not enter its tetrahedron skeleton structure, neither react chemically with clinoptilolites, which may arise the new key band of P-O.
In conclusion, La is only loaded to the surface and pore canal of clinoptilolites and does not enter its tetrahedron skeleton structure and joins the skeleton vibration.Besides, the La loaded to the surface and pore canal of clinoptilolites constitutes the adsorption site for phosphorus, which enhances the adsorption ability of clinoptilolite for phosphorus.
XRD Analysis
XRD, which makes use of the diffraction phenomena of X-ray in the crystal, is used to analyze the crystal structure, parameters and defect of the materials.It is the most effective way to analyze the space structure of clinoptilolite (Castaldia et al., 2008).Comparing Figure 3 and Figure 4 with the standard maps, the diffraction peaks of this natural clinoptilolite are mainly composed of clinoptilolite (about 22.5°, 25° and 30°), SiO 2 (about 22° and 44.5°) and other silicon and aluminium oxides (about 39°, 58° and 68°) (Karapınar, 2009).Clinoptilolites before and after modification both show obvious crystal structure, with nearly same spectral lines and few changes in the number of diffraction peaks.This indicates that the overall skeleton structure and structural holes are not changed by the modification process.Comparing Figure 3 with Figure 4, there is a slight decrease in the intensity of the main diffraction peak, which means that the degree of crystallinity of the clinoptilolites modified by LaCl 3 decease slightly.There is not obvious change in the interplanar spacing between the main diffraction peaks.However, there is a trend that the diffraction peaks of modified clinoptilolite shift to small angles, which indicates that the aperture of modified clinoptilolite has the trend to increase.This is corresponded with the specific surface area and pore size analysis.The diffraction peak of La is not observed from the modified clinoptilolite, which indicates that La is only loaded to the surface of clinoptilolite and does not change its structure.
Conclusions
(1) After modified by LaCl 3 of different concentrations, the surface area, total pore volume and micropore area of clinoptilolites decreased equally and the average pore width increases.The pore distribution test shows that the volume proportion of micropore and mesopore decreases while the volume proportion of macropore increases.Combined with the EDS test, it showed that LaCl 3 is loaded on the surface and pore canal of clinoptilolites, so its adsorption for ammonia nitrogen decreases while for phosphorus increases as a result of the presence of La.
(2) After modified by hydrochloric acid, the surface area, total pore volume of clinoptilolites increase greatly, and the volume proportion of micropore increases greatly while the volume proportion of mesopore decreases and the volume proportion of macropore increases slightly.After modified by LaCl 3 , the surface area, total pore volume and micropore area of the hydrochloric acid-modified clinoptilolites change greatly.This verifies that LaCl 3 can be loaded on the surface and pore canal of clinoptilolites.
(3) The infrared spectrum shows that La is only loaded to the surface and pore canal of clinoptilolites and does not enter its tetrahedron skeleton structure and the skeleton shake.From another aspect, the loaded La forms the adsorption site for phosphorus which enhances the adsorption ability of clinoptilolite for phosphorus.
(4) XRD spectrums of modified and unmodified clinoptilolites show that the crystallinity of the clinoptilolites modified by LaCl 3 decease slightly, there is not obvious change in the interplanar spacing, and there is a trend that the diffraction peaks of modified clinoptilolite shift to small angles.However, the crystal structure is not changed.La is only loaded to the surface of clinoptilolite and does not change its overall skeleton structure and structural holes.
Figure 3 .
Figure 3. XRD of the natural clinoptilolite
Table 1 .
Change of surface area and distribution of pore width about clinoptilolite modified by LaCl 3
Table 2 .
Change of surface area and distribution of pore width about clinoptilolite modified by HCl | v3-fos-license |
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} | pes2o/s2orc | Integration of Fourier Transform Infrared Spectroscopy, Fluorescence Spectroscopy, Steady-state Kinetics and Molecular Dynamics Simulations of Gαi1 Distinguishes between the GTP Hydrolysis and GDP Release Mechanism*
Background: Multiple turnover GTPase assays of Gα are dominated by nucleotide exchange. Results: FTIR elucidates single turnover rates and individual phosphate vibrations. Conclusion: Gαi1-R178S is slowed down in single turnover hydrolysis by 2 orders of magnitude, Gαi1-Asp229 and -Asp231 are key players in Ras-like/all-α domain coordination. Significance: With FTIR on Gα established, detailed information on the reaction mechanism can be obtained. Gα subunits are central molecular switches in cells. They are activated by G protein-coupled receptors that exchange GDP for GTP, similar to small GTPase activation mechanisms. Gα subunits are turned off by GTP hydrolysis. For the first time we employed time-resolved FTIR difference spectroscopy to investigate the molecular reaction mechanisms of Gαi1. FTIR spectroscopy is a powerful tool that monitors reactions label free with high spatio-temporal resolution. In contrast to common multiple turnover assays, FTIR spectroscopy depicts the single turnover GTPase reaction without nucleotide exchange/Mg2+ binding bias. Global fit analysis resulted in one apparent rate constant of 0.02 s−1 at 15 °C. Isotopic labeling was applied to assign the individual phosphate vibrations for α-, β-, and γ-GTP (1243, 1224, and 1156 cm−1, respectively), α- and β-GDP (1214 and 1134/1103 cm−1, respectively), and free phosphate (1078/991 cm−1). In contrast to Ras·GAP catalysis, the bond breakage of the β-γ-phosphate but not the Pi release is rate-limiting in the GTPase reaction. Complementary common GTPase assays were used. Reversed phase HPLC provided multiple turnover rates and tryptophan fluorescence provided nucleotide exchange rates. Experiments were complemented by molecular dynamics simulations. This broad approach provided detailed insights at atomic resolution and allows now to identify key residues of Gαi1 in GTP hydrolysis and nucleotide exchange. Mutants of the intrinsic arginine finger (Gαi1-R178S) affected exclusively the hydrolysis reaction. The effect of nucleotide binding (Gαi1-D272N) and Ras-like/all-α interface coordination (Gαi1-D229N/Gαi1-D231N) on the nucleotide exchange reaction was furthermore elucidated.
Heterotrimeric G proteins are interaction partners of G protein-coupled receptors (GPCRs) 4 and deliver external signals into the cell (1). They are switched on by exchange of GDP for GTP induced by the GPCR as exchange factor and switched off by GTP hydrolysis. The nucleotide is bound between two domains of the G␣-subunit, namely the Ras-like domain, which is similar to the G-domain of small GTPases, and the all-␣ domain. In its inactive state G␣ i1 ␥ exists GDP bound in its heterotrimeric form. Activation by guanosine nucleotide exchange factors, like GPCRs or non-receptor guanosine nucleotide exchange factors (2)(3)(4), leads to nucleotide exchange in the ␣-subunit. Incorporation of GTP alters the protein conformation in the switch I-III regions (5), which causes separation of the G␣ i1 and ␥ subunits and signal transduction, e.g. by binding of G␣ i1 to adenylate cyclase isoforms that in turn inhibit the production of cAMP from ATP (6). GTPase activity of G␣ i1 leads to hydrolysis of GTP to GDP and P i , inactivation, and reassociation with its ␥ subunits. Heterotrimeric G proteins are equipped with an intrinsic arginine finger (Arg 178 in G␣ i1 ) usually provided in case of small GTPases by the GAP protein, which is known to function as a key residue for catalyzing the hydrolysis reaction. Therefore intrinsic GTPase rates of G␣ i1 are rather comparable with Ras⅐GAP than to Ras. The hydrolysis mechanism taking place in G␣ i1 thereby determines the duration of its active state, which can be pathogenic when hindered, e.g. by ADP-ribosylation catalyzed by pertussis toxin (7). As for Ras, the intrinsic hydrolysis activity of G␣ i1 can be further accelerated by GTPase activating proteins (GAPs), which are called regulators of G protein signaling (RGS), e.g. RGS4 in case of G␣ i1 (8). It is generally known that GDP/GTP exchange is the rate-limiting step in multiple turnover measurements of G␣ isoforms (9 -12). Therefore, beside steady-state assays using ␥-32 P labeling (13) or malachite green (14), pre-steady-state assays are used to characterize the hydrolysis reaction of G␣ isoforms (15)(16)(17)(18). We present here for the first time single turnover measurements of G␣ i1 using time-resolved FTIR spectroscopy, an ultrasensitive method that can be applied in solution and has been successfully used for photoactivable proteins like bacteriorhodopsin (19,20), channelrhodopsin (21), and other rhodopsins (22). Adenylyltransferases (23), ATPases (24 -27), and GTPases (28 -31) can also be investigated by usage of caged nucleotides (28). The resulting photolysis and hydrolysis difference spectra depict the label-free GTP and GDP states of G␣ i1 and are able to reflect environmental changes in the sub-Å range (32) and the dynamics at a time resolution of milliseconds. Bridging the gap between single turnover and steadystate kinetics, we also applied a multiple turnover GTPase assay using reversed phase HPLC and additionally measured the nucleotide exchange rate using tryptophan fluorescence of Trp 211 at 280/340 nm as extensively described elsewhere (15,18,33) and molecular dynamics simulations. GDP release and GTP uptake is a complex mechanism determined by the movement of the all-␣ domain, which was shown by structural studies (34). The Ras-like/all-␣ interface thereby depicts a complex interaction network including both amino acids and the nucleotide. By orchestration of different methods we were able to determine the effect of point mutations and distinguish their role in hydrolysis and nucleotide exchange.
Materials and Methods
G␣ i1 -WT and mutant proteins were expressed, isolated, and characterized by various biophysical and biochemical methods (Fig. 1).
Michaelis-Menten multiple turnover kinetics were monitored via reversed phase HPLC. The kinetics include both the catalytic reaction and the dissociation/association kinetics depicted as time per turnover. The isolated single turnover hydrolysis reaction was obtained via FTIR spectroscopy as half-life values of the global fit. The isolated nucleotide exchange kinetics were investigated via tryptophan fluorescence spectroscopy and depicted as half-life values of the intensity change. Experiments were accompanied by molecular dynamics simulations to decode the molecular reactions at the atomic level.
Cloning-The gene for the human GNAI1 (UniProtKB accession number P63096-1; kind gift from C. Wetzel, University of Regensburg, Germany (39)) was amplified by polymerase chain reaction using the oligonucleotide primers GCGC-CCATGGGCTGCACGCTGAGC and GCGCGGATCCTTA-AAAGAGACCACAATCTTTTAG (restriction sites for NcoI and BamHI are underlined). Resulting fragments were cut with NcoI and BamHI and ligated into the vector pET27bmod (kind gift from M. Engelhard, MPI Dortmund, Germany (40)) with a N-terminal ϫ10 histidine tag and tobacco etch virus (TEV) site. The plasmid was transformed into Escherichia coli DH5␣ for amplification. G␣ i1 mutants R178S, D229N, D231N, and D272N were created by overlap PCR using appropriate primers. Integrity of each construct was confirmed by sequencing. cDNA encoding human RGS4 was acquired from the Missouri cDNA Resource Center (Rolla, MO), tagged with a N-terminal ϫ10 histidine tag and TEV site, and also ligated into the vector pET27bmod. Amplification was performed similar to G␣ i1 .
Protein Expression-The plasmid encoding G␣ i1 was transformed into E. coli Rosetta 2 (DE3) (Novagen, Merck, Darmstadt, Germany) and incubated overnight at 37°C on LB agar plates containing 0.2% (w/v) glucose as well as 50 g/ml of kanamycin and 20 g/ml of chloramphenicol for plasmid and strain selection. A preculture (LB medium, 50 g/ml of kanamycin, 20 g/ml of chloramphenicol, 0.2% (w/v) glucose) was inoculated and incubated overnight at 37°C and 160 rpm. The plasmid encoding RGS4 was transformed into E. coli BL21(DE3) under identical conditions using only kanamycin for plasmid selection. For the main culture, 18 liters of LB medium supplemented with 50 g/ml kanamycin and 0.2% glucose were inoculated with the preculture and grown at 37°C, 100 rpm, and 20 liters/min airflow in a Biostat C20-3 Fermenter (Sartorius, Göttingen, Germany). At an A 600 of 0.5-0.6 the culture was cooled to 18°C and protein expression was induced by addition of 0.25 mM isopropyl 1-thio--D-galactopyranoside. After 15-18 h the cells were harvested by centrifugation at 5000 ϫ g and 4°C, suspended in buffer A (20 mM Tris, pH 8, 300 mM NaCl, 1 mM MgCl 2 , 0.5 mM EDTA, 5 mM D-norleucine for G␣ i1 or 50 mM Tris, pH 8, 150 mM NaCl, 0.5 mM EDTA, 5 mM D-norleucine for RGS4), flash frozen, and stored at Ϫ80°C.
Protein Isolation-Frozen cells were thawed, supplemented with 0.3 mM PMSF, 5 mM -mercaptoethanol, DNase (G␣ i1 containing cells were additionally supplemented with 0.1 mM GDP), and disrupted using a microfluidizer M-110L (Microflu-FIGURE 1. G␣ i1 is switched on by the exchange of GDP for GTP (k off /k on ), then GTP hydrolysis proceeds (k hyd ) and P i is released. Multiple turnover kinetics were measured via HPLC, which cannot distinguish between the three processes. Nucleotide exchange kinetics (k off /k on ) were monitored via tryptophan fluorescence spectroscopy. Single turnover kinetics (k hyd ) were measured via time-resolved FTIR difference spectroscopy.
idics Corp., Newton, MA) at 800 bar. To spin down cell fragments and not disrupted cells, the suspension was centrifuged for 45 min at 45,000 ϫ g and 4°C. RGS4 containing cells were centrifuged with an additional low-speed step for 15 min at 18,000 ϫ g and 4°C followed by high-speed centrifugation for 45 min at 75,000 ϫ g and 4°C. The supernatant was applied to a 25-ml nickel-nitrilotriacetic acid superflow (Qiagen, Hilden, Germany) column, equilibrated with buffer B (buffer A ϩ 0.3 mM PMSF ϩ 5 mM -mercaptoethanol ϩ 20 mM imidazole), using a Ä KTApurifier 100 system (GE Healthcare Life Sciences, Freiburg, Germany) at 6°C with a flow rate of 1-2 ml/min. After a washing step with buffer C (buffer B ϩ 4 mM MgCl 2 , 400 mM KCl, 1 mM ATP) for 8 -10 column volumes and a subsequent step with buffer B ϩ 50 mM imidazole for another 8 -10 column volumes, the proteins were eluted with buffer B ϩ 200 mM imidazole. The fractions containing G␣ i1 or RGS4 were selected after SDS-PAGE, pooled, supplemented with 5 mM DTT, and concentrated to 5 ml using a 10,000 MWCO concentrator (Amicon Ultra-15, Merck Millipore, Darmstadt, Germany). For gel filtration chromatography, the pool was applied to an illustra HiLoad 26/600 Superdex 200 pg column (GE Healthcare Life Sciences, Freiburg, Germany) equilibrated with buffer D (20 mM Tris, pH 8, 300 mM NaCl, 1 mM MgCl 2 , 2 mM DTT, 0.1 mM GDP for G␣ i1 or 50 mM Hepes, pH 8, 100 mM KCl, 2 mM DTT for RGS4). Peak fractions were analyzed by SDS-PAGE. Purest fractions containing G␣ i1 or RGS4 were mixed 1:2 with buffer E (20 mM Tris, pH 8, 1 mM MgCl 2 , 0.1 mM GDP for G␣ i1 or 50 mM Hepes, pH 8, 100 mM KCl for RGS4), pooled, and concentrated to ϳ20 mg/ml for G␣ i1 or ϳ10 mg/ml for RGS4 using a 10,000 MWCO concentrator. Protein concentration was determined using Bradford reagent as triplicate. The concentrated pool was aliquoted, flash frozen, and stored at Ϫ80°C until utilization. Coomassie-stained gels after SDS-PAGE of purified proteins are depicted in Fig. 2.
Multiple Turnover GTPase Measurements-For determination of the GTPase activity under multiple turnover conditions, the samples contained 10 M G␣ i1 in 20 mM Tris, pH 8, 150 mM NaCl, 0.5 mM MgCl 2 , and 0.1 mM DTT. After tempering for 5 min at 30°C, 0.1 mM GTP (G␣ i1 -WT, -R178S, -D229N, -D231N) or 2.5 mM GTP (G␣ i1 -D272N) was added and immediately the first aliquot of the sample was analyzed by reversed phase HPLC at 254 nm (Beckman Coulter System Gold, Pasadena CA) (mobile phase: 50 mM P i , pH 6.5, 5 mM tetrabutylammonium bromide, 7.5% AcN; stationary phase: ODS-Hypersil C18 column). After a 10-min incubation at 30°C, a second aliquot was analyzed by HPLC. The amount of GTP was chosen to guarantee a substantial excess of GTP during the whole time of the measurements. Evaluation of the data were done by integration of the GDP and GTP peaks followed by normalization (sum of the areas A GDP ϩ A GTP ϭ 1). Determination of the time for one turnover per molecule G␣ i1 , including exchange of GDP for GTP and GTP hydrolysis, was done according to the calculation of turnover times formula, with time points t 0 and t 1 [min], the areas of the GTP peak at the time points t 0 and t 1 A GTP , t0 and A GTP , t1 (normalized area), and the concentration of G␣ i1 and GTP c(G␣ i1 /GTP). Calculated values were averaged and the standard deviation was calculated from three experiments for each wild type and mutant protein.
Monitoring the Nucleotide Exchange Rate Using Fluorescence Spectroscopy-The nucleotide exchange rate of wild type and mutant G␣ i1 was measured via tryptophan fluorescence of Trp 211 (41, 42) using a Jasco FP 6500 Spectrofluorometer (Easton, MD). 500 nM wild type or mutant protein was supplemented with 20 mM Tris, pH 8, 150 mM NaCl, 1 mM MgCl 2 , and 1 mM DTT and tempered 5 min to 30°C. After monitoring the fluorescence baseline for another 5 min, nucleotide exchange was initiated by the addition of 2.5 M GTP␥S and the reaction was monitored with exc ϭ 280 nm and em ϭ 340 nm for at least 60 min. Mixing was ensured by constant stirring with a stirring bar and the temperature was controlled by an external water bath.
Data were fitted in OriginPro 9 (OriginLab Corp., Northampton, MA) using a monoexponential formula with a linear correction, which accounts for bleaching, with the rate constant k, the amplitude coefficient a, the slope m, and the offset n. Half-life values were calculated using (t1 ⁄ 2 ϭ ln(2)/k). Half-life values were averaged and the standard deviation was calculated from three experiments for each wild type and mutant protein.
Nucleotide Exchange of Wild Type and Mutant G␣ i1 to Caged GTP-The exchange of bound GDP to photolabile pHPcgGTP or NPEcgGTP was performed in the presence of alkaline phosphatase coupled to agarose beads, which is unable to hydrolyze caged compounds. Phosphatase beads were washed 5 times in buffer 1 (50 mM Tris, pH 7.5, 100 M ZnSO 4 ) to remove free phosphatase. Each washing step was followed by centrifugation at 10,000 ϫ g and the supernatant was checked for free phosphatase using a colorimetric assay with para-nitrophenylphosphate (43). 5 mg of wild type or mutant G␣ i1 were supplemented with 50 mM Tris, pH 7.5, 10 M ZnSO 4 and a 2ϫ molar excess of the caged nucleotide. Hydrolysis of free and proteinbound GDP to guanosine was monitored via HPLC. After 3 h at room temperature Ͼ95% of GDP was hydrolyzed. Samples were centrifuged at 10,000 ϫ g for 2 min and the supernatant was re-buffered through a Nap5 column (GE Healthcare Life Sciences) that was equilibrated with 10 mM Hepes, pH 7.5, 7.5 mM NaCl, 0.25 mM MgCl 2 , 1 mM DTT at 7°C. Protein fractions were pooled and concentrated in a 10,000 MWCO concentrator (Amicon Ultra-0.5, Merck Millipore, Darmstadt, Germany). Concentrations were determined using Bradford reagent as triplicate and the nucleotide exchange rate of bound caged nucleotide was again determined via HPLC (Ͼ95%). Samples were aliquoted into 107.5 g portions (5 mM final concentration in FTIR measurements), flash frozen in liquid nitrogen, and stored at Ϫ80°C. Subsequently samples were lyophilized for 3 h at Ϫ55°C/0.05 mbar in a Christ Alpha-1-2 LDPlus Lyophilizer (Martin Christ GmbH, Osterode am Harz, Germany) and stored light protected in parafilm and aluminum foil at Ϫ20°C.
FTIR Measurements on G␣ i1 -FTIR measurements were performed using 5 mM G␣ i1 ⅐cgGTP in 200 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM MgCl 2 , 20 mM DTT, 0.1% (v/v) ethylene glycol at 15°C. RGS4 catalyzed measurements were performed by the addition of 5 mM RGS4 to establish a 1:1 complex with G␣ i1 . Sample preparation was done under red light to protect the photolabile caged group. Composition of the required residual buffer depends on the protein concentration of the samples after nucleotide exchange to achieve the above named ion concentrations. FTIR samples were prepared between two CaF 2 windows (Ø 2 cm, 2 mm thickness, one of them with a 10-m deepened area 1 cm in diameter). One lyophilisate of G␣ i1 ⅐cgGTP was dissolved in 0.5 l of the appropriate residual buffer at the center of the deepened window and subsequently covered with the second window, whose rim had been lubricated with a thin ϳ1 mm wide silicon grease film. The windows were fixed in a metal cuvette and mounted in the spectrometer (Bruker IFS 66v/S or Vertex 80 v (Bruker, Ettlingen, Germany)). After sample equilibration, background spectra were taken (400 scans) and photolysis of the caged compounds was carried out with an LPX 240 XeCl excimer laser (Lambda Physics, Göttingen, Germany) by 12 flashes within 24 ms (pHPcgGTP) or 40 flashes within 80 ms (NPEcgGTP) at 308 nm (100 -200 mJ/flash, 20 ns pulse duration) (28). Measurements were performed in the rapid-scan mode of the spectrometer for 30 min (G␣ i1 -WT, -D229N, -D231N, -D272N) or 3 h (G␣ i1 -R178S) using a liquid nitrogen-cooled mercury cadmium telluride detector. Data between 1800 and 950 cm Ϫ1 was collected with a spectral resolution of 4 cm Ϫ1 using an aperture of 5 mm in the double-sided forward-backward data acquisition mode with a scanner speed of 120 kHz. Data were analyzed via global fit (44). The absorbance change (⌬A(,t)) was fit-ted with a sum of exponential functions n describing the apparent rate constants k 1 and amplitudes a 1 of the hydrolysis reaction and the amplitudes a 0 of the photolysis reaction for every wavenumber .
In the figures disappearing bands face downward and appearing bands face upward. Data were averaged over at least 3 measurements. Half-lives were calculated as arithmetic means, variation was calculated as standard deviations.
Molecular Dynamics Simulation and Evaluation-Molecular dynamics (MD) simulations were performed starting with the G␣ i1 ⅐Mg 2ϩ ⅐GTP␥S structure of Protein Data Bank (PDB) code 1GIA (5) that depicts the truncated (⌬1-32 ⌬345-354) active state of G␣ i1 . Structure preparation was performed in Moby (45) and included correction of dihedrals, angles, and bonds according to the UA amber84 forcefield (46), protonation of ionizable side chains using the PKA,MAX,UH,JAB3 algorithm as well as replacement of the GTP␥S for a GTP molecule (total charge: Ϫ4) and initial solvation by the Vedani algorithm (47). Point mutations were realized in Moby and were followed by a short headgroup optimization. Simulation systems were set up in GROMACS 4.0.7 (48 -52). The prepared structures were thoroughly solvated in a cubic simulation cell filled with 154 mM NaCl in explicit TIP4P water. Simulations were carried out in the all atom OPLS forcefield (53) with GTP parameters from T. Rudack (54) at 310 K using the berendsen thermo-and barostat and a time step of 1 fs. Long range electrostatics were calculated using PME (cutoff 0.9 nm), short range electrostatics were calculated using a VDW cutoff of 1.4 nm. Bonds were constrained using LINCS. Systems were energy minimized and equilibrated for 25 ps with restrained protein positions followed by three free MD runs, each to a simulation time of 100 ns (total simulation time 1.5 s).
Structure analysis was performed using the GROMACS evaluation tools and the contact matrix algorithm implemented in Moby. Pictures were created using PyMOL 1.7.1.1 (Schrödinger LLC, Portland, OR) and Gnuplot 4.4 (55).
Results
FTIR Measurements of G␣ i1 -Time-resolved FTIR spectroscopy enables label-free detection of the GTP/GDP vibrations as well as determination of the apparent kinetics of the hydrolysis reaction. The protein was loaded with caged GTP and the sample was excited at 308 nm with a laser flash to remove the caged group (28) that cleaves rapidly (10 7 s Ϫ1 for pHPcgGTP (36)). The resulting difference spectrum is referred to as photolysis spectrum. Subsequently the intrinsic hydrolysis reaction in G␣ i1 takes place (Fig. 3).
The reaction (Scheme 1) is observed in FTIR. Global fit analysis of the absorbance changes revealed a monoexponential function that describes the hydrolysis (Fig. 4). No intermediate enrichment was observed in the measurements of G␣ i1 -WT. Global fit analysis of five independent G␣ i1 -WT measurements at 15°C resulted in a half-life of 32.7 Ϯ 2.5 s (k hyd ϭ 0.02 s Ϫ1 ).
Data analysis according to Equation 3 resulted in photolysis and hydrolysis spectrums that represent the transition from the pHPcgGTP to the GTP bound active state of G␣ i1 and the transition from the active GTP bound state to the inactive GDP bound state, respectively. Bands facing downward represent the educt state, bands facing upward represent the product state. Both spectra show numerous highly reproducible bands in the protein (1680 -1350 cm Ϫ1 ) and the phosphate (1350 -950 cm Ϫ1 ) region (Fig. 5). Surprisingly a band at 1784 cm Ϫ1 appeared in the photolysis and disappeared in the hydrolysis reaction, indicating a protonation of a carboxyl group from an Asp or Glu (56) in the GTP state (Fig. 5). To our knowledge this is the first time a protonation change has been observed in GTPases. For a clear cut assignment further studies with sitedirected mutations have to be performed.
Phosphate vibrations were assigned using isotopically labeled nucleotides, namely ␣-18 O 2 -pHPcgGTP, -18 O 3 -pHPcgGTP, and ␥-18 O 4 -NPEcgGTP. Double difference spectra of FTIR measurements using unlabeled and labeled nucleotides showed exclusively band shifts caused by the isotopes and allow band assignments of the phosphate region. In the photolysis spectrum the bands at 1240, 1224, and 1155 cm Ϫ1 were assigned to the asymmetric stretching vibrations of ␣-, -, and ␥-GTP (Fig. 6, A and B). The vibrations for and ␥-GTP appear as clear bands, the ␣-band appears as a shoulder only in the photolysis spectrum but is more distinct in the hydrolysis spectrum. Band assignments of the hydrolysis reaction confirmed ␣-, -, and ␥-GTP vibrations at 1243, 1224, and 1156 cm Ϫ1 . The vibrations of the product state were assigned to 1214 cm Ϫ1 for ␣-GDP, 1134 and 1103 cm Ϫ1 for -GDP, and 1078 and 991 cm Ϫ1 for the cleaved free phosphate (Fig. 6, C and D). The cleaved phosphate is not protein bound, as the vibrations at 1078 and 991 cm Ϫ1 are typical for free phosphate. Proteinbound phosphate intermediates are blue-shifted, e.g. in case of Ras⅐GAP an intermediate band appears at 1192 cm Ϫ1 (57). Because a protein-bound phosphate intermediate was not observed as in case of the Ras⅐GAP catalyzed reaction, bond breakage is the rate-limiting step in the hydrolysis reaction of G␣ i1 (Fig. 4). Summarizing, the hydrolysis reaction of G␣ i1bound GTP to GDP and P i was monitored label free at atomic resolution and in the millisecond time scale. Individual asymmetric stretching modes of GTP and GDP bound to G␣ i1 and P i were assigned clear cut.
In addition, we performed the same experiments with the G␣ i1 ⅐RGS4 1:1 complex at 5°C. Addition of RGS4 further catalyzed the hydrolysis reaction by almost 2 additional orders of magnitude (Fig. 7). As for the intrinsic measurements, global fit analysis resulted in one exponential rate, which demonstrates that again bond breakage is rate-limiting. No protein-bound FIGURE 3. Three-dimensional spectrum (global fit) of G␣ i1 -WT. The first spectrum represents the photolysis spectrum, subsequently hydrolysis takes place and was monitored. Time dependence of the bands at 1155 cm Ϫ1 (gray) and 1078 cm Ϫ1 (blue) is indicated. The absorbance change of these bands is shown in Fig. 4. SCHEME 1. Reaction scheme observed in FTIR measurements. (Fig. 6)). Solid lines represent the monoexponential global fit, dots represent data points. FIGURE 5. Photolysis and hydrolysis spectrum of G␣ i1 . Bands facing downward in the photolysis spectrum represent the pHPcgGTP state of G␣ i1 , bands facing upward depict the GTP bound state. Bands facing downward and upward in the hydrolysis spectrum represent the educt and product state of the hydrolysis reaction that takes place in G␣ i1 , respectively. The spectral region between 1680 and 1620 cm Ϫ1 is superimposed by water absorptions and not further regarded.
phosphate intermediate was observed and the absorptions of protein-bound GTP disappeared with the same rate as the absorptions of free P i developed. RGS4 contributed numerous protein bands and altered the GTP/GDP binding modes. Assignments of these bands by isotopic labeling and site-specific mutagenesis will be part of future work.
In the following, various G␣ i1 residues were investigated to determine their role in nucleotide exchange and hydrolysis using site-directed mutagenesis. Investigations included the intrinsic arginine finger mutant G␣ i1 -R178S that is known to have a slowed down hydrolysis rate (58), as well as mutations affecting the interface of the Ras-like and the all-␣ domain (G␣ i1 -D229N/G␣ i1 -D231N), and a residue that is participating in nucleotide binding (G␣ i1 -D272N) (Fig. 8). Multiple turnover measurements via reversed phase HPLC, nucleotide exchange experiments via fluorescence spectroscopy, and single turnover measurements via time-resolved FTIR spectroscopy were used to investigate these mutants.
Steady-state Measurements-The multiple turnover GTPase reaction of wild type and mutant G␣ i1 , consisting of nucleotide exchange (k off,GDP and k on,GTP ) and the hydrolysis rate k hyd , was investigated by reversed phase HPLC at 30°C according to Equation 1. Results can be grouped into four classes. One turnover is a cycle consisting of GDP release, GTP binding, and GTP hydrolysis that took 12.7 Ϯ 0.2 min for G␣ i1 -WT. The arginine finger mutant G␣ i1 -R178S was slowed down to 29.6 Ϯ 1.6 min per turnover. G␣ i1 -D229N and -D231N shared an accelerated turnover time of 3.9 Ϯ 0.03 min and G␣ i1 -D272N was accelerated even more to 0.5 Ϯ 0.02 min per turnover (Fig. 9A). It is generally accepted that the GDP release step is rate-limiting in multiple turnover measurements of G␣-proteins (59). To further investigate the underlying rate constants we additionally performed nucleotide exchange and single turnover hydrolysis experiments.
Nucleotide Exchange Experiments-In contrast to multiple turnover experiments, tryptophan fluorescence spectroscopy can monitor solely the nucleotide exchange reaction from GDP to GTP␥S of G␣ i1 as Trp 211 is sensitive for binding of the third phosphate group (Fig. 10). Hydrolysis cannot proceed as GTP␥S is a non-hydrolyzable GTP analogue. The results of nucleotide exchange can be grouped into three classes. In contrast to multiple turnover measurements, the half-life value for nucleotide exchange to GTP␥S of G␣ i1 -R178S was similar to G␣ i1 -WT (12.8 Ϯ 0.9 and 10.7 Ϯ 0.2 min, respectively). Nucleotide exchange was accelerated by a factor of about 3 in G␣ i1 -D229N and -D231N to 3.3 Ϯ 0.2 and 3.7 Ϯ 0.1 min and even more accelerated in G␣ i1 -D272N (0.8 Ϯ 0.03 min) (Fig. 9B). Hence it can be concluded that the acceleration in multiple turnover measurements of G␣ i1 -D229N, -D231N, and -D272N can be explained by accelerated dissociation times for GDP and/or association times for GTP. On the other hand the decelerated multiple turnover time for G␣ i1 -R178S is not caused by nucleotide exchange, indicating that nucleotide exchange is not the rate-limiting step for this mutant.
Single Turnover Hydrolysis Measurements Using FTIR-In contrast to multiple turnover measurements, time-resolved FTIR spectroscopy can determine the hydrolysis reaction label free under actual single turnover conditions with high spatiotemporal resolution. The half-life value was obtained from the results of the global fit procedure (44). The measured kinetics can again be grouped into three classes. G␣ i1 -WT and G␣ i1 -D231N had similar half-life values of 32.7 Ϯ 2.5 and 27.8 Ϯ 2.6 s, respectively. The single turnover hydrolysis reaction of G␣ i1 -D229N and -D272N was slightly slowed down to 50.2 Ϯ 4.5 or 49.8 Ϯ 4.1 s. The hydrolysis reaction of G␣ i1 -R178S was noticeably slowed down by 2 orders of magnitude to 3400 Ϯ 400 s (Fig. 9C). Thereby the deceleration of G␣ i1 -R178S in multiple turnover measurements can be explained solely by the slowed down single turnover hydrolysis reaction. Due to the change of the rate-limiting step, the slowdown in the multiple turnover assay appears to be only about 2-fold, whereas the single turnover FTIR assay yield the true slowdown by 2 orders of magnitude.
Molecular Dynamics Simulations-To further examine the molecular interactions taking place in the interface between the Ras-like and all-␣ domain of G␣ i1 , molecular dynamics simulations were performed to elucidate the role of Asp 229 and Asp 231 at atomic detail. Simulations of wild type G␣ i1 and mutants G␣ i1 -D229N and -D231N were performed for 100 ns each. Subsequently contact matrix analysis was carried out for every simulation. Contacts were sampled in time windows of 1 ns and the interaction partners of Asp/Asn 229 and Asp/Asn 231 were depicted in Fig. 11. Polar contacts of the side chain groups are indicated by black bars.
Asp 229 formed a stable interdomain contact to the all-␣ domain through Arg 242 that bound Gln 147 in wild type G␣ i1 (Fig. 11A). When mutated to Asn 229 , this interdomain contact triad was interrupted after the first 30 ns. Thereby the contact loss between Asn 229 and Arg 242 happened simultaneously to the contact loss of Arg 242 to Glu 43 and Gln 147 . Hence Asp 229 seems to position Arg 242 allosterically, so that Arg 242 forms an interdomain contact that tightly binds and stabilizes the Raslike and the all-␣ domain.
Similar to Asp 229 , Asp 231 formed an interdomain contact to Arg 144 within a 100-ns MD simulation (Fig. 11B). It is notable, that the contact Asp 231 -Arg 144 does neither exist in the starting structure generated from PDB code 1GIA, nor in the original crystal structure, but formed de novo in the simulation. The initial contact to Lys 277 persisted through the simulation time. When mutated to Asn 231 the initial contact to Lys 277 was weakened, but the contact to Arg 144 was completely lost.
Summarizing the results from multiple and single turnover measurements, nucleotide exchange experiments and simulation data, we understand the effects of the point mutations in G␣ i1 . G␣ i1 -R178S was slowed down in multiple turnover measurements, but even slower in single turnover measurements that depict only the hydrolysis reaction itself. Its nucleotide exchange ability appeared unaltered. Slowdown of multiple turnover is solely from hydrolysis in this case. Thus Arg 178 only participates in the hydrolysis reaction as described elsewhere (5,58).
The interface mutations D229N and D231N were both accelerated in multiple turnover measurements. Investigations of the nucleotide exchange reaction showed that the exchange time for both mutants was accelerated. Simulation data suggest an allosteric (Asp 229 ) and direct (Asp 231 ) interdomain binding mode of both amino acids. Mutations affecting the guanosine binding moiety Asp 272 resulted in accelerated half-life values in multiple turnover measurements that can be originated to an accelerated nucleotide exchange behavior as shown via fluorescence spectroscopy.
Discussion
Molecular mechanisms that take place in G␣ i1 have been investigated by numerous studies including structural (5, 34, 60), computational (61), and biochemical (13,14,33) assays. In particular, multiple turnover GTPase assays like malachite green or radiometric phosphate tests using [␥-32 P]GTP are widely used even though it is commonly known that GDP/GTP exchange is the rate-limiting step in intrinsic multiple turnover measurements (9 -12) and thereby determines k obs . Pre-steadystate measurements using GTP or [␥-32 P]GTP pre-loaded G␣ subunits that are triggered via Mg 2ϩ addition are also able to depict single turnover conditions, but the percentage of nucleotide loading (G␣⅐GTP versus G␣⅐GDP) and the altered GTP binding affinity due to Mg 2ϩ -binding (62) may cause systematic errors such as side reactions like nucleotide exchange. However, in FTIR measurements the percentage of loaded cgGTP versus GDP does not influence the kinetics due to the method of phototriggered difference spectroscopy. Additionally we checked the loading rate via HPLC (always Ͼ95% cgGTP) and removed non protein-bound nucleotides. The determined single turnover rate for wild type G␣ i1 measured via FTIR spectroscopy (0.02 s Ϫ1 at 15°C) is in good agreement to the literature (0.03 s Ϫ1 at 30°C (58) and 0.03 s Ϫ1 to 0.04 s Ϫ1 at 20°C (13,64)). In addition the ensemble of methods enables a classification of effects caused by point mutations in high detail. Effects caused by the intrinsic arginine finger mutant G␣ i1 -R178S were quantified correctly in single turnover measurements (2 orders of magnitude) but not in multiple turnover measurements (factor of 2). The unaltered nucleotide exchange rate of G␣ i1 -R178X mutants has already been described elsewhere (5).
Single turnover FTIR spectroscopy unravels for the first time the rate-limiting step of the intrinsic GTP hydrolysis in G␣ i1 . Analogue experiments with small GTPases revealed that in some cases the bond breakage and in others the P i release is rate-limiting (57). For G␣ i1 no protein-bound cleaved phosphate intermediate could be observed, thus bond breakage is the rate-limiting step in this reaction. This is surprising due to the tight coordination of the nucleotide by G␣ i1 . The narrow protein environment is still able to release free phosphate to the periphery, probably through a small channel located near the ␥-phosphate. The measured IR bands for GTP and GDP are very sensitive to changes in the protein environment and depict for the first time the coordination of the natural nucleotides GDP and GTP in G␣ i1 in contrast to GTP analogues, which were described to have poor affinities for G␣ i1 (65). After we have successfully assigned the ␣-, -, ␥-, and the free phosphate vibrations it will be possible to assess the effect of point mutations in the binding pocket of G␣ i1 in the future supported by theoretical IR spectra calculation from QM/MM simulations as performed for the small GTPase Ras (66) to further decode the experimental spectra. In addition to the phosphate bands, various bands caused by the protein itself were nicely resolved, which will enable investigations of the hydrolysis mechanism taking place in G␣ proteins with improved spatio-temporal resolution. The observed band at 1784 cm Ϫ1 is the first protonation change observed in GTPases to our knowledge. In fact, heterotrimeric G proteins have been speculated to function as pH sensors (67) and a protonation change close to the surface of G␣ i1 could function as a key player in this reaction.
In addition to the intrinsic GTPase reaction of G␣ i1 we were also able to measure the hydrolysis reaction catalyzed by RGS4 via FTIR spectroscopy. Hydrolysis was thereby accelerated by almost 2 orders of magnitude (Fig. 7). As for intrinsic G␣ i1 , bond breakage is the rate-limiting step.
We were able to show with our orchestration of different methods that two point mutations in the G␣ i1 Ras-like/all-␣ interface (G␣ i1 -D229N/G␣ i1 -D231N) are able to weaken the coordination in the protein domain interface. Our measurements together with MD simulations demonstrate the importance of the amino acid triad Asp 229 -Arg 242 -Gln 147 for the interface coordination in G␣ i1 . Asp 229 holds Arg 242 in a position to bridge the interface to Gln 147 . Investigations on the mutant G␣ i1 -R242A confirmed its role (nucleotide exchange: 3.09 Ϯ 0.21 min/single turnover hydrolysis: 32.5 Ϯ 3.5 s) and resulted in similar values as G␣ i1 -D229N. In agreement, accelerated nucleotide exchange for G␣ i1 -R242A has recently been described for the analogue R243H in G␣ o (68). Our findings on the other amino acid in the domain interface, Asp 231 , suggest a direct binding mode of the side chain of Asp 231 across the interface to Arg 144 . This contact is not observable in any of the deposited structures of G␣ i1 in the Protein Data Bank, except structure 4PAQ where the side chain of Arg 144 is slightly tilted toward Asp 231 with occupancy of 0.46 (69). In contrast to tightly packed crystal structures, the dynamics of G␣ i1 in our experiments and in our simulations are much more comparable with physiological conditions, so we hereby demonstrate the importance of the salt bridge Asp 231 -Arg 144 , which is not observable in the crystal structures. Our findings are summarized in an advanced interface binding model of G␣ i1 as shown in Fig. 12.
In summary, we were able to measure the isolated rates of nucleotide exchange and GTP hydrolysis, which both contribute to the signaling state of G␣ i1 . In addition we identified the individual phosphate vibrations of GTP, GDP, and P i during the hydrolysis reaction of G␣ i1 . We demonstrated the importance of the intrinsic arginine finger for the hydrolysis reaction and the relevance of Asp 272 for nucleotide binding. Furthermore, we identified novel key players in the coordination of the Raslike/all-␣ interface. Asp 229 stabilizes the interface allosterically via Arg 242 and Asp 231 forms a direct H-bond to Arg 144 . Orchestration of our methods will further elucidate the molecular mechanisms taking place in G␣ i1 in the future. | v3-fos-license |
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} | pes2o/s2orc | Validation of a UV Spectrometric Method for the Assay of Tolfenamic Acid in Organic Solvents
The present study has been carried out to validate a UV spectrometric method for the assay of tolfenamic acid (TA) in organic solvents. TA is insoluble in water; therefore, a total of thirteen commonly used organic solvents have been selected in which the drug is soluble. Fresh stock solutions of TA in each solvent in a concentration of 1 × 10−4 M (2.62 mg%) were prepared for the assay. The method has been validated according to the guideline of International Conference on Harmonization and parameters like linearity, range, accuracy, precision, sensitivity, and robustness have been studied. Although the method was found to be efficient for the determination of TA in all solvents on the basis of statistical data 1-octanol, followed by ethanol and methanol, was found to be comparatively better than the other studied solvents. No change in the stock solution stability of TA has been observed in each solvent for 24 hours stored either at room (25 ± 1°C) or at refrigerated temperature (2–8°C). A shift in the absorption maxima has been observed for TA in various solvents indicating drug-solvent interactions. The studied method is simple, rapid, economical, accurate, and precise for the assay of TA in different organic solvents.
TA occurs as a white or slightly yellow crystalline powder and is practically insoluble in water [29,30]. The official pharmacopoeial assay method for TA involves direct titration against sodium hydroxide solution [29]. Previously, validated methods for the quantitative analysis of TA, both as a pure compound and in tablet dosage form, have been reported using FTIR and UV spectrometry [31]. Both methods showed good accuracy and precision for the assay of TA with the UV method showing comparatively better results. Both techniques were found to be statistically comparable with the official titrimetric method [31].
The present study has been designed to validate the UV spectrometric assay procedure for the analysis of pure TA in different organic solvents according to the guidelines of International Conference on Harmonization (ICH) [32]. TA is a water insoluble drug and such study would provide useful data which would help in its determination with high accuracy and precision in various pharmaceutical systems incorporating organic solvents. this study were of analytical grade having the highest degree of purity. The details of the solvents used in this study are reported in Table 1.
Thin Layer Chromatography (TLC). TLC was performed
to check the purity of TA used in this study according to the method reported in British Pharmacopoeia [29]. The substance (25 mg) was dissolved in a mixture of methanol and methylene chloride (1 : 3, v/v) and diluted to 10 mL with the same mixture and 10 L of the solution was applied to 250 m silica gel GF 254 plates. It was developed with the mobile phase up to 2/3 distance of the plate. The plate was dried and viewed under 254 nm UV lamp (Uvitec, Cambridge, UK).
Ultraviolet Spectrometry.
All absorbance measurements and spectral determinations were carried out on a Shimadzu UV-visible spectrophotometer (model UV-1601) using quartz cell of 10 mm path length. The cells were employed always in the same orientation using appropriate control solutions in the reference beam. The baseline correction was made by the built-in baseline memory at the initializing period while auto-zero adjustment was made by one-touch operation. The wavelength scale was also calibrated automatically by the instrument. The instrument was calibrated for the absorbance scale according to the method described in British Pharmacopoeia [29], by using 0.057-0.063 g/lit of potassium dichromate in 0.005 M sulfuric acid. The absorbencies of the corresponding series of solutions in each solvent were measured against a reference of the same solvent in the region of 250-400 nm. Quartz cells were closed with a cap to prevent evaporation of the organic solvent during absorbance measurements.
Preparation of Stock and Test Solutions for Validation
Studies. The stock solutions of TA for validation studies were prepared in a concentration of 1.0 × 10 −4 M (2.62 mg%) in the individual solvent ( Table 1). The stock solutions were thoroughly stirred each time by the aid of a magnetic stirrer for 30 min. During stirring the solutions were kept in a tightly closed container to avoid evaporation of the organic solvent. The test solutions in each solvent were prepared from the stock by making appropriate dilutions in the concentration range of 1.0-8.0 × 10 −5 M. The stock solutions and the respective dilutions were found to be completely transparent in appearance. Each time fresh solutions were prepared. The solutions were protected from light and the absorbance was then measured immediately. All experiments were performed in triplicate.
Validation of the Analytical Method.
The UV method for the assay of TA was validated according to the guidelines of ICH [32]. Different parameters of validation for TA were studied which are described as follows.
2.5.1. Linearity and Range. The linearity of the method was determined by preparing calibration curves of absorbance versus the concentration of TA of the test solutions in the concentration range of 1.0-8.0 × 10 −5 M for each solvent. The linearity was statistically determined by regression analysis of five concentrations used in triplicate. The linearity range was selected on the basis of absorbance values in the region of around 0.2-0.8. This range of absorbance is known to provide values with the highest precision [34]. The molar absorptivity and A (1%, 1 cm) values were also determined from the calibration curve.
Accuracy.
The accuracy of the proposed method was determined by adding known concentrations of the drug in the solutions followed by their analysis by the UV spectrometric method. Three different concentrations in triplicate from the studied range were selected and analyzed for the recovery.
Precision.
The precision of the developed method was calculated by performing nine determinations at three concentrations covering the specified range. The precision Journal of Pharmaceutics 3 was determined by calculating relative standard deviation (%RSD) of the mean recoveries. (LOQ). LOD and LOQ of the developed method were calculated from the standard deviation of the -intercept and slope of the calibration curve using the following formulae:
Limit of Detection (LOD) and Limit of Quantitation
where is the standard deviation of the intercept and is the slope of the calibration curve.
2.5.5.
Robustness. The robustness of the method was determined by studying small changes in the assay wavelength (±2 nm). This parameter was studied thrice in the similar range used for the determination of TA (i.e., 1.0-8.0 × 10 −5 M). The accuracy and precision of the method were determined.
Solution Stability.
The stability of stock solutions of TA was studied at room (25 ± 1 ∘ C) and refrigerated temperature (2-8 ∘ C). The stock solutions of TA were prepared in pure solvents at a concentration of 1 × 10 −4 M (2.62 mg%). The samples were stored in tightly sealed glass containers protected from light. A 5 mL aliquot of the sample was taken each time and the absorbencies were measured at 0-, 1-, 2-, 3-, and 24hour time interval.
Confirmation of Purity of Tolfenamic Acid.
In order to study spectrometric characteristics of a compound, it is necessary to confirm the purity of the material to avoid any effect on the position and intensity of the absorption maxima as well as on the validation of the assay method. In the case of TA a thin layer chromatography (TLC) examination was conducted to detect any spots other than that of TA on TLC plates. The TLC test for TA has been carried out according to the method described in British Pharmacopoeia [29]. TA appeared as a single spot confirming the purity of the material.
Nature of Solvents and Spectral
Characteristics of Tolfenamic Acid. The use of solvents in UV-visible spectrometric measurements depends on the nature of the compound to be characterized or analyzed. The solvent must be transparent in the region in which the compound exhibits absorption spectrum. The compound should have enough solubility to obtain a reasonably clear absorption spectrum. It is also important to consider any possible interaction of the solvent with the absorbing molecule to impart a shift in the absorption maxima. It has been reported that polar solvents such as water, alcohols, esters, and ketones (containing lone pair of electrons) tend to obscure vibrational spectra. The nonpolar solvents such as cyclohexane, chloroform, and benzene give spectra somewhat similar to that of a gas (better band resolution) [35]. The maximum absorption wavelength of the absorption band depends on the degree of solutesolvent interaction and the nature of solvent [36][37][38][39]. The solvent dependent spectral shifts arise from either nonspecific (dielectric enrichment) or specific (e.g., hydrogen bonding) solute-solvent interactions. Considering the interactions between the solute and solvent molecule and the intensity of these interactions, a change in the absorption spectrum of the molecule (e.g., max and max ) can be expected. Such a change has been described as solvatochromism [40]. The organic solvents have a different polar character as indicated by the dielectric constant of the medium. It has been observed that an electronic transition of a compound may lead to a modification of the charge distribution by the solvent used. This would result in some change in the position and intensity of the absorption maxima depending on the nature of the solvent. The extent of solute-solvent interaction would give an indication of the type of electronic transition undergone by the molecule [41].
The lower wavelength limit of common solvents in the UV and visible spectra strongly depends on the purity of the solvent (Table 1). For example, ethanol and the hydrocarbon solvents are frequently contaminated with benzene which absorbs below 280 nm [35]. Therefore, the highest/spectroscopic grade solvents should always be used for the measurement of the absorption spectra of organic compounds; otherwise the true spectral characteristics of a compound may not be obtained due to the presence of interfering impurities.
The spectral characteristics of TA including the value of absorption maxima, respective molar absorptivities ( ), and specific absorbance [ (1%, 1 cm)] in various organic solvents are reported in Table 2. A consideration of the values of absorption maxima of TA in various organic solvents shows that their 1max range from 286 to 294 nm and 2max from 332 to 354 nm ( Figure 1, Table 2) with regression values ( 2 ) of 0.99905-0.99988 showing very small scatter of the points around the calibration curves (Table 3).
Similarly, a variation in the values of max in these solvents is also observed ( Table 2). This is probably due to the degree of interaction between the solute and the solvent to cause a shift in the absorption maxima with accompanying change in the intensity of absorption as indicated by the values of max . The high values of max indicate - * electronic transition in the molecule. The values of 1 range from 7930 to 10960 M −1 cm −1 and those of 2 from 5310 to 8967 M −1 cm −1 .
Validation of the Assay Method.
The UV spectrometric assay of TA in various solvents has been validated according to the guidelines of ICH [32], including the following parameters.
3.3.1. Linearity. Linearity determines the ability of the method to obtain the results that are directly proportional to the concentration of the analyte within a given range by plotting a calibration curve. TA is 2-[(3-chloro-2methylphenyl)amino]benzoic acid and gives two peaks in the region of 280-360 nm (Figure 1). The short wavelength peak in the region below 300 nm is more prominent with a greater intensity than the one present above 300 nm. Therefore, calibration curves of TA in each solvent have been prepared with respect to the short wavelength peak in the majority of solvents (Table 2). On the contrary, in the solvent that showed some interference or has a cutoff point near or above the prominent peak of TA such as acetone (330 nm), benzyl alcohol (282 nm), and toluene (286 nm), it has been assayed and validated with respect to the long wavelength peak (Figure 1). Although the calibration curves in benzyl alcohol and toluene have been prepared with respect to the short wavelength peak due to their interfering cutoff points they have further been validated for TA assay using the long wavelength peak. A linear relationship has been found for TA in each solvent and the statistical data are reported in Table 3.
The intercept values are significantly close to zero in each case thus confirming the peak purity of TA. The overlay spectra of TA in acetonitrile are shown in Figure 2.
Range.
It is defined as the interval between the upper and lower concentrations of the analyte that have been demonstrated to be determined with acceptable precision, accuracy, and linearity. The absorbance values in the range of 0.2-0.8 are known to offer the highest precision [34]. Therefore, similar pattern has also been followed in this study in determining the range of TA in each solvent. The ranges for the assay of TA in each solvent are reported in Table 3 which corresponds well to the points in calibration curves.
Accuracy.
The accuracy of an analytical method is defined as the degree to which the determined value of Journal of Pharmaceutics an analyte in a sample corresponds to the true value. The results for the percent recovery of TA in different organic solvents are reported in Table 3. Although the results show good accuracy for TA in each solvent comparatively the mean recovery in 1-octanol followed by ethanol and methanol is better than that of the others due to minimum standard deviations. The standard deviations are small in all cases indicating that the method can be used with high accuracy for the determination of TA in the studied organic solvents.
Precision.
Precision of an analytical method is the closeness of agreement between a series of measurements obtained from multiple samples of the studied drug under prescribed conditions. The results for the precision of the method for the assay of TA in various solvents are reported in Table 3. These indicate that the %RSD in the majority of cases is less than 2% and is minimum in the case of 1-octanol with nearly the same values in ethanol and methanol. Thus the studied method is highly reliable for the assay of TA in different solvents.
LOD.
It is the lowest concentration of an analyte in a sample that can be detected but not necessarily quantified. It is considered as limit test that indicates that the analyte is above or below a certain value which is usually expressed as percentage of the analyte in the sample. The LOD of TA in each solvent is reported in Table 3. The minimum detection limit of 1.97 × 10 −6 M (0.05 mg%) has been found in 1-octanol while the highest of 6.47 × 10 −6 M (0.17 mg%) has been found in 1-butanol. This indicates that the UV spectrometric technique is highly sensitive for the detection of TA in various organic solvents.
3.3.6. LOQ. The LOQ determines the lowest concentration of an analyte in a sample that can be quantified with acceptable precision and accuracy under the documented operational conditions of the drug being assayed. The minimum quantification limit of 5.98 × 10 −6 M (0.16 mg%) has been found in 1-octanol while the highest of 1.96 × 10 −5 M (0.51 mg%) has been found in 1-butanol. The values of LOQ of TA in each solvent are reported in Table 3. All solvents have been found to correspond well with the quantification of TA by UV spectrometric technique indicating that the method is accurate and precise for its assay.
3.3.7.
Robustness. The robustness of an analytical method is a measure of its capacity to obtain acceptable results when perturbed by small but deliberate variations. It is basically an indicator of method suitability and reliability during normal use. Absorbance of a solution is dependent upon wavelength, solvent, pH, and temperature. Therefore, these parameters should remain constant throughout the course of the analysis; otherwise significant errors may arise in the quantitative analysis of the samples [34]. In the present study, the reliability of the method has been tested by determining the absorption maxima in each solvent and by changing the assay wavelengths at room temperature (25±1 ∘ C). The results showed that small changes in the wavelength of absorption maxima do not affect the accuracy and precision of the assay of TA (Table 4). This indicates that the method is robust under the studied conditions in the majority of the solvents. The highest robustness has been found in acetonitrile whereas the lowest has been found in chloroform followed by acetone (Table 4).
Solution Stability.
The solution stability is a measure of the extent to which the studied drug is stable in a solvent being used for the assay over a particular period of time under specified conditions. It is an essential requirement that the analyte should not undergo any chemical change and should remain stable in the particular solvent [34]. The study of TA was carried out at room temperature (25± 1 ∘ C) and refrigerated temperature (2-8 ∘ C). The consistency in absorbance indicated the stability of TA solutions. In all solvents no significant change has been observed in the absorbance of TA after 24 hours of storage either at room temperature or in a refrigerator. However, in spite of the stability of TA in the organic solvents for at least 24 hours, fresh solutions were used for the validation study.
Conclusion
The present study has employed thirteen commonly used solvents for the validation of a UV spectrometric method for the determination of TA. The results indicated that the method is accurate, precise, robust, economical, and rapid for the assay of TA with a stock solution stability of 24 hours in each solvent. TA exhibits two peaks in the UV region of 280-360 nm. The major short wavelength peak is in the region of 285-295 nm that showed good results for the assay of TA. Those solvents that have a cutoff point in this region or interfere with the major peak can also be used for the determination of TA with respect to the minor long wavelength peak in the region of 335-355 nm.
The results of this study highlight the effect of different solvents on the spectral characteristics of organic molecules of pharmaceutical importance. Some shifts in the absorption maxima of TA have been noted probably due to drugsolvent interaction while absorptivity constants of TA in each solvent have also been determined. These shifts can affect the wavelengths used for the assay of a compound and, therefore, it is necessary to use a particular solvent for assay purpose. It is also necessary to confirm the purity of the solvent used for assay and its interference in the spectral region of the compound to be studied. A detailed investigation of the effect of solvent parameters on the spectral characteristics of a compound is required to develop an understanding of the changes observed. Such study would help the pharmaceutical formulators and analysts to determine TA in pharmaceutical systems incorporating organic solvents.
Conflict of Interests
There is no conflict of interests regarding the publication of this paper. | v3-fos-license |
2016-04-15T09:12:14.267Z | 2014-07-01T00:00:00.000 | 18702740 | {
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} | pes2o/s2orc | Preparation of Polyphosphazene Hydrogels for Enzyme Immobilization
We report on the synthesis and application of a new hydrogel based on a methacrylate substituted polyphosphazene. Through ring-opening polymerization and nucleophilic substitution, poly[bis(methacrylate)phosphazene] (PBMAP) was successfully synthesized from hexachlorocyclotriphosphazene. By adding PBMAP to methacrylic acid solution and then treating with UV light, we could obtain a cross-linked polyphosphazene network, which showed an ultra-high absorbency for distilled water. Lipase from Candida rugosa was used as the model lipase for entrapment immobilization in the hydrogel. The influence of methacrylic acid concentration on immobilization efficiency was studied. Results showed that enzyme loading reached a maximum of 24.02 mg/g with an activity retention of 67.25% when the methacrylic acid concentration was 20% (w/w).
Synthesis and Characterization of PBMAP
PBMAP was synthesized by the nucleophilic substitution of sodium methacrylate (Figure 1a). Generally, the substitution of chlorine atoms by methacrylate groups is more difficult than traditional alkoxy substitutions because of the lower reactivity of carboxylates as nucleophiles compared to alkoxides [22]. To optimize the macromolecular substitution, the temperature was raised to ~ 35 to 40 °C during the reaction. The structure of the resultant polymer was confirmed by FT-IR and 1 H-NMR spectra. In the FT-IR spectrum of PBMAP (Figure 2a), the bands corresponding to C=O (1716 cm −1 ), C=C (1635 cm −1 ) and P−O−C (976 cm −1 ) stretching demonstrated that alkyne side groups were successfully incorporated into the polyphosphazene. In addition, bands at 1238 cm −1 and 1190 cm −1 confirmed the presence of P−N and N=P groups, respectively. The 1 H-NMR confirmed that the chlorine atoms were substituted by sodium methacrylate (Figure 2b). The signals at δ = 5.25 and 5.58 correspond to CH 2 =C groups while the signal at δ = 1.83 corresponds to −CH 3 groups. Moreover, because of the presence of the reactive vinyl group, the polymer underwent some extent of c ross-linking during the purification process, which was suggested by the chemical shift at δ = 2.16. A well-known method of protecting the vinyl group from radical reactions is the addition of stabilizers such as 2,2-diphenyl-1-picrylhydrazyl. However, such stabilizers were avoided in this work where we aimed to use a greener chemistry procedure to develop a more purified material for biological purposes.
Preparation of PBMAP Hydrogel
Hydrogels were prepared by adding PBMAP to a methacrylic acid solution and exposing the hydrogel to UV light ( Figure 1a). This cross-linking process was carried out without the use of cross-linker substance. The inter-chain cross-links were responsible for keeping the material as a gel. The PBMAP solution was radiated by UV light for 1-15 min in the presence of a 20% (w/w) methacrylic acid solution. Macroscopically, no obvious hydrogel forming was observed within 3 min. The turbid solution appeared to be stratified at 5 min. After 15 min, the cross-linking reaction was complete and a light yellow hydrogel was obtained.
Physical Properties of PBMAP Hydrogel
Thermal characterization of the hydrogel was performed with DSC, as shown in Figure 3. Two heating rounds were conducted. The hydrogel easily absorbed water when exposed to air because of the presence of carboxyl groups. During the first heating round, water in the hydrogel absorbed heat and was vaporized. In the second round, no obvious endothermic phenomenon was observed and the hydrogel showed good thermal stability; no degradation or cleavage appeared when the hydrogel was heated even up to a temperature of 150 °C. Moreover, the hydrogel maintained a similar thermal performance at temperatures above 100 °C, verifying that vinyl groups in PBMAP were completely cross-linked. The hydrogel was weighed before and after immersing in ultra pure water with excess water carefully removed. The swelling degree was calculated by the equation: where W t and W 0 are the weights of the swollen and dry hydrogels, respectively.
In Figure 4a, the water uptake of the hydrogel indicated remarkable absorbing ability. The absorption occurred for approximately 72 h, after which, the hydrogel had gained weight to 35 times that of the dry material. Figure 4b,c show PBMAP hydrogel in the dry and swollen states, respectively. According to the literature, the most absorbing polymer gels are derived from backbones containing ionic groups such as methacrylic acid. In fact, the structure of PBMAP hydrogel presented a synergetic absorption behavior by the poly(methacrylic acid) branches and the highly flexible backbone of the polyphosphazene. Moreover, the cross-linked network allowed for hydrogel expansion, which facilitated the diffusion of water to the hydrogel. We also studied the effect of methacrylic acid concentration on the hydrogel swelling ability. When the methacrylic acid concentration was lower than 20% (w/w), the hydrogel was weak and could be easily broken; the concentration was thus kept above 20%. All the samples tested here were placed in ultra pure water until saturated. The results in Figure 5 show that maximum water uptake was achieved at a methacrylic acid concentration of 20% (w/w). At this concentration, the lengths of branches and the cross-linking density were optimized. Further increasing the concentration increased the cross-linking density and decreased the absorption ability. Figure 5 also shows that the compressive modules increase with the increase of the methacrylic acid concentration.
Effect of Initial Lipase Concentration on Enzyme Loading
In the entrapment method, lipase entered the network of the hydrogel through diffusion. In order to verify that the proteins were entrapped within the hydrogel, we used the FITC-labeled bovine serum albumin (BSA) as a model protein for the same immobilization study. Then the BSA-immobilized hydrogel was cut and the cross-section was observed under an emission wavelength of 488 nm with a fluorescence microscope (Nikon Ti-U). The result was shown in Figure 6. According to this figure, the proteins were successfully entrapped into the hydrogel. Enzyme loading reflects the interactions between enzymes and substrates. Figure 7 shows the amount of lipase entrapped under different lipase concentrations as indicated by the enzyme loading. The hydrogel used here was prepared in the presence of a 20% (w/w) methacrylic acid solution. According to this figure, the enzyme loading increased with increasing lipase concentration and reached a maximum of 24.02 mg/g, indicating that the hydrogel owned a high capacity for biomolecules.
Effect of Methacrylic Acid Concentration on Enzyme Loading and Activity
The lipase-immobilized hydrogel system has its advantages. The immobilized lipases retain the ability to catalyze a wide range of reactions such as alcoholysis, hydrolysis, trans-esterifications, aminolysis and enantiomer resolution. The immobilization technique also offers better catalytic stability, feasible catalyst recycling, significant operational cost reduction and simplified product purification in practical applications. The network structure of the hydrogel is vital for enzyme immobilization, and from above, we have found that the methacrylic acid concentration can affect the cross-linking degree of the hydrogel. Consequently, the lipase entrapment efficiency at a range of methacrylic acid concentrations was tested. The results are shown in Figure 8. Enzyme loading decreased with the increase of methacrylic acid concentration. This is due to an increase in the cross-linking density in the hydrogel; in this situation, diffusion of lipases into the hydrogel is difficult. The activity retention of the immobilized lipases decreased with an increase in methacrylic acid concentration, which was also due to the greater cross-linking degree: the denser the network structure, the greater the mass transfer limitation. According to this figure, when the concentration of methacrylic acid solution was 20% (w/w) and the enzyme loading reached a maximum of 24.02 mg/g, the activity retention was 67.25%.
Reuse Stability of Immobilized Lipase
Reuse stability of the immobilized lipase is important for ensuring the economical use of the enzyme in repeated batch or continuous reactions. If the immobilized lipase has a relatively long lifetime, the cost will be remarkably decreased and its industrial implementation will be accelerated. In the reusability study, the activity of immobilized lipases in subsequent batch cycle reactions was compared with the activity in the first cycle. A slight decrease in activity appeared after the second usage. After four cycles of batch operation, less than 50% of the original activity remained (Figure 9). The activity loss was partially related to the inactivation of the lipase by continuous use. However, it was mostly due to the loss of entrapped lipases, which was due to the relative loose physical interaction of lipases with the hydrogel. Because this polyphosphazene hydrogel presented high absorbing ability, it can be dried and immersed in fresh lipase solution for a new round of immobilization. Moreover, the enzyme leakage can be overcome by different chemical immobilization procedures. For example, we can covalently immobilize the lipase into the hydrogel via an N-(3dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide activation process [6]. The benefit of the polyphosphazene matrix lies in its synthetic flexibility, and in this way the design of many variations of the polymer is possible; as a result, we can introduce glyoxyl, epoxyl and glutaraldehyde groups to the polymer for chemical binding with the lysine residues of the lipases [39][40][41]. Also, the lipases can be cross-linked to decrease enzyme leakage [42]. Apart from producing a covalent linkage, other methods such as chemical or physical modification of the enzymes with large polymers and usage of pre-immobilized enzymes are also possible to improve the enzyme stabilities [43][44][45][46].
Materials
Hexachlorocyclotriphosphazene (HCCP, Boyuan New Material & Technology Co. Ltd., Ningbo, China) was purified by recrystallization from heptane at a temperature of 60 °C, followed by two cycles of vacuum sublimation. Poly(dichlorophosphazene) (PDCP) was synthesized via thermally initiated ring-opening polymerization of HCCP in a sealed Pyrex tube at 250 °C [38]. Tetrahydrofuran (THF) was dried by refluxing over a Na/K alloy and distilling under nitrogen. Sodium methacrylate and methacrylic acid were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China) and used without purification. Heptane was also purchased from Sinopharm Chemical Reagent Co. Ltd. and molecular sieves (4 Ǻ) were added to the heptane 24 h before use. Lipase (from Candida rugosa) powder (1150 units/mg solid), Bradford reagent and bovine serum albumin (BSA, molecular mass 67,000 Da) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and used as received. All other chemicals were of analytical grade and used without further purification.
Synthesis and Analysis of PBMAP
The synthesized PDCP (2.00 g, 17.3 mmol) was rinsed with petroleum ether to remove any remaining monomers and oligomers, and then dissolved in dry THF under nitrogen protection. In a rounded flask, 7.52 g (69.6 mmol) of sodium methacrylate was dissolved in 50 mL of dry THF. PDCP solution was added dropwise to the stirring sodium methacrylate suspension. The mixture was protected from light and stirred at ~35 to 40 °C for 48 h. A 50% excess of sodium methacrylate was used to assure total chlorine substitution. The reacted solution was then added dropwise to an excess amount of ethyl acetate-water (1:1 volume ratio) to precipitate the polymer product. The product was purified by washing with heptane-water (1:1 volume ratio) followed by ethyl acetate-water. After evaporation of the solvent, the material collected was a white and brittle solid. This solid could be partially dissolved in water and the insoluble part was composed of gel.
The chemical structure of PBMAP was characterized by FT-IR and 1 H-NMR. The FT-IR spectrum of the polymer in KBr was obtained on a Brucker Vector 22 FT-IR spectrometer, while the 1 H-NMR spectra of the sample solution in D 2 O were recorded on a Brucker Advance DMX500 nuclear magnetic resonance spectrometer.
PBMAP Hydrogel Formation
The hydrogel was visually detected during the purification steps. Its formation was due to the spontaneous cross-linking of the material during the reaction and when exposed to the atmosphere. This spontaneously cross-linked hydrogel was very soft and easy to break. In order to obtain hydrogels with improved mechanical properties, methacrylic acid was used for a further cross-linking process. PBMAP was added to a methacrylic acid-water solution and 2,2-dimethoxy-2-phenylacetophenone (0.05 equiv. with respect to the double bonds) was added as the photoinitiator. The system was irradiated with UV radiation (λ max = 365 nm, 0.6 mW/cm 2 ) for 15 min for hydrogel formation.
For the differential scanning calorimetry (DSC) test, the sample (~8 mg) was placed in an aluminum pan under a nitrogen atmosphere and heated in the first scan to 96 °C at a heating rate of 10 °C/min followed by cooling to −40 °C. A second scan was carried out by heating to 150 °C at the same heating rate used previously. The second heating run was used to observe the thermal events.
Swelling Behavior and Mechanical Property Measurements
Before measurements, samples were dried in a vacuum at room temperature for 24 h. The dry samples were then weighed and placed in tubes containing ultra pure water. After the swelling process, samples were collected using a spatula and transferred to a Petri dish where excess water was carefully removed using strips of filter paper. After this process, hydrogels were again weighed. The compressive stress-strain measurements were performed on hydrogels by using a CHT 4000 electronic universal testing machine (SANS, Shenzhen, China) at a velocity of 0.1 mm/min. The cylindrical gel samples were 15 mm in diameter and 10 mm in thickness.
Lipase Entrapment
The entrapment of lipase was performed by immersing hydrogels in lipase solution and allowing them to swell until equilibrium. Lipase solution was prepared by dissolving lipase powder in phosphate buffer solution (PBS, 0.05 M, pH 7.0). The immobilization was performed at ambient temperature. After reaching equilibrium, the samples were withdrawn and washed thoroughly with plenty of PBS (0.05 M, pH 7.0) until no free lipase was detected in the washing solution. The enzyme loading was determined using Coomassie brilliant blue reagent, following Bradford's method [47]. BSA was used as a standard to construct a calibration curve. The entrapment capacity of lipase in the hydrogel was calculated from the protein mass balance among the initial and final lipase solutions and washings. The enzyme loading was defined as the amount of protein (milligram) per gram of hydrogel. Each reported value was the mean of at least three experiments, and the standard deviation was within ca. ± 5%.
Assay of Lipase Activity
The activity of the immobilized lipase in an aqueous medium was determined using a previously reported method [48]. Briefly, the reaction was initiated by immersing an immobilized lipase preparation in a reaction mixture composed of 1.0 mL ethanol solution containing 14.4 mM p-nitrophenyl palmitate and 1.0 mL PBS (0.05 M, pH 7.0). The mixture was then incubated at 25 °C under reciprocal agitation. After 5 min, the reaction was terminated by adding 2.0 mL of 0.5 M Na 2 CO 3 , followed by centrifugation for 10 min (10,000 rpm). A 0.50 mL aliquot of the supernatant was diluted 10-fold with deionized water and measured at 410 nm in a UV-Vis spectrophotometer (UV-2450, Shimadzu, Japan) against a blank without enzyme that had been treated in parallel.
One enzyme unit is defined as the amount of biocatalyst liberating 1.0 µmol p-nitrophenol min −1 for the above conditions. The activity retention value is the ratio of the specific activities of immobilized and free lipase. Each data value shown was the average of at least three parallel experiments, and the standard deviation was within ca. ± 5%.
Conclusions
In this work, PBMAP hydrogels were successfully prepared and lipase from Candida rugosa was immobilized in the hydrogel by entrapment. The hydrogel showed high absorbing ability and withstood temperatures up to 150 °C. The influence of cross-linking degree on immobilization efficiency was studied. Results showed that enzyme loading was as high as 24.02 mg/g with an activity retention of 67.25% when the methacrylic acid concentration was 20% (w/w). This work broadens the biological applications of polyphosphazenes, and based on this pathway, an efficient enzyme immobilization system can be conveniently fabricated. | v3-fos-license |
2019-06-27T10:16:51.069Z | 2019-06-17T00:00:00.000 | 195729664 | {
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} | pes2o/s2orc | Oscillatory Shear Stress Induces Oxidative Stress via TLR4 Activation in Endothelial Cells
Background Oscillatory shear stress (OSS) disrupts endothelial homeostasis and promotes oxidative stress, which can lead to atherosclerosis. In atherosclerotic lesions, Toll-like receptor 4 (TLR4) is highly expressed. However, the molecular mechanism by which TLR4 modulates oxidative changes and the cell signaling transudation upon OSS is yet to be determined. Methods and Results Carotid artery constriction (CAC) surgery and a parallel-plate flow chamber were used to modulate shear stress. The results showed that OSS significantly increased the oxidative burden, and this was partly due to TLR4 activation. OSS activated NOX2 and had no significant influence to NOX1 or NOX4 in endothelial cells (ECs). OSS phosphorylated caveolin-1, promoted its binding with endothelial nitric oxide synthase (eNOS), and resulted in deactivation of eNOS. TLR4 inhibition restored levels of nitric oxide (NO) and superoxide dismutase (SOD) in OSS-exposed cells. Conclusion TLR4 modulates OSS-induced oxidative stress by activating NOX2 and suppressing eNOS.
Introduction
Arterial endothelium homeostasis is associated with the distribution of shear stress, a dragging force generated by blood flow which has a profound effect on endothelial function [1]. Endothelial cells (ECs), which comprise the inner surface of the vessels, are exposed to various flow patterns such as laminar shear stress and oscillatory shear stress (OSS) directly [1,2]. OSS prefers to appear at the curvatures, bifurcations, and branches in the artery, where the fluid mechanical environment is distinct from the straight sections of the vessel wall [3]. Cells in regions undergoing OSS are characterized by accumulated reactive oxygen species (ROS), decreased nitric oxide (NO) bioavailability, and prevailed inflammation [4]. Endothelial dysfunction is the initial factor leading to atherosclerosis lesions, where an accumulation of Toll-like receptors (TLRs) has been found [5,6].
TLRs, pattern recognition receptors, are part of the innate immune system and respond to pathogenic factors or cellular damage to elicit an effective defense [5,7]. Recently, a growing body of evidence has elucidated their role in regulating the inflammatory response and maintaining endothelial homeostasis [5]. TLR4, the first identified TLR, has also been implicated in the development and progression of cardiovascular disease [8,9]. Several studies have reported a role of TLR4 in promoting ECs proliferation and neointima formation [10]. Lu et al. identified a high expression of TLR4 in endothelial cells and macrophages in atherosclerotic plaques [11]. Our previous genome analysis indicated that TLR4 was the most differentially expressed mRNA in sheared cells compared with static cultured cells [12]. We have also shown that acute exposure to shear stress results in extensively oxidative damage in ECs [13,14]. However, the effect of TLR4 activation in sheared cells and the related mechanism remain unclear. In the present study, we demonstrate that OSS activates TLR4, causing downstream effects on NOX2 and eNOS that result in oxidative damage.
Material and Methods
2.1. Cell Culture. Human umbilical vein endothelial cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in Dulbecco's Modified Eagle's Medium culture medium supplemented with 10% fetal bovine serum and cultured at 37°C in a humidified incubator with 5% CO 2 . When grown to confluence, cells were trypsinized, harvested, resuspended, and seeded to a 0.1% gelatin-coated glass. After adherence, cells were used for shear stress study.
2.2. Parallel-Plate Flow Chamber Study. A parallel-plate flow chamber that exerts continuous flow was made by sandwiching a silicon gasket between two stainless steel plates with a cover slip sink in the base plate. The chamber and all parts of the circuit were sterilized by steam autoclaving, before a glass plate containing monolayer cells was placed into the flow chamber. Shear stress is calculated as τ = 6 * Q * μ/ w * h 2 , in which τ is the target shear stress acting tangentially on the cells, Q is the flow rate, μ is the viscosity of the perfusate, and w and h refer to the width and height of the flow chamber. In this experiment, the wall shear stress used was 4 dynes/cm 2 , the viscosity of the medium was 0 009 g/cm * s, and the width and height of the chamber were 28 mm and 440 μm, respectively.
Carotid Artery Constriction.
Carotid artery constriction (CAC) was conducted to modify flow pattern. Briefly, 6-8 weeks old rats were anesthetized with pentobarbital (20 mg/kg, ip) and subjected to CAC surgery using a modified cast, which was cone-shaped and made with silicone ( Figure 1(a)). In the sham group, the right carotid was exposed but was left unconstricted. Blood velocity was measured using a Doppler ultrasound at 7 days postsurgery, the rats were subsequently sacrificed, and their carotids were removed for further study. The experimental and animal surgery procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the animal ethics board of Nanjing Medical University.
2.4. Tissue Section of Human Coronary Artery. Human coronary arteries were collected from patients undergoing heart transplant surgery that has been approved by the Institutional Review Board of Nanjing First Hospital. We separated the left main coronary artery, left descending artery, and left circumflex artery carefully. Arteries were divided into bifurcation and nonbifurcation groups. After that, tissues were embedded with OCT and cut into slices which were then stained with primary antibodies followed with another incubation of fluorescence-conjugated secondary antibodies. Images were obtained using a confocal microscope (ZEISS, German).
2.6. Coimmunoprecipitation. Following flow treatment, ECs were lysed and incubated with caveolin-1 overnight at 4°C with gentle shaking. Following further incubation with protein A+G for another 4 h, the precipitate was washed five times with RIPA at 1,000 g for 5 min and then resuspended in SDS-PAGE loading buffer. After being heated at 99°C for 5 min, samples were separated by SDS-PAGE and transferred to a PVDF membrane to detect total eNOS. Primary antibodies against NOX2 and caveolin-1 were from Abcam. (Cambridge, UK). Antibodies against p47 and eNOS were purchased from Cell Signaling Technology (Massachusetts, USA) and R&D Systems Inc. (Minneapolis, USA), respectively. Protein A+G was purchased from Millipore (Massachusetts, USA).
Western Blot Analysis.
Monolayer cells grown on a glass plate were subjected to OSS for different lengths of time (0, 20, 40, 80, and 120 min); cells were then lysed in a cocktail of RIPA, proteinase inhibitor, and phosphatase inhibitor. After that, the lysates were centrifuged at 12,000 g for 15 min at 4°C, and the supernatant was collected for concentration determination and protein detection. Protein concentration was quantified using a bicinchoninic acid protein assay according to the manufacturer's instructions (KeyGen Biotech, Nanjing, China). In total, 60 μg protein was separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane, which was then incubated overnight at 4°C with primary antibody. Following this, the membrane was washed and incubated with horseradish peroxidaseconjugated secondary antibody for 2 h at room temperature and developed using an enhanced chemiluminescence kit. Bands were analyzed using ImageJ software. Primary antibodies against GAPDH, caveolin-1, and p47 were obtained from Cell Signaling Technology (Massachusetts, USA). Primary antibodies against NOX1, NOX2, and NOX4 were from Abcam (Cambridge, UK). Primary antibody against eNOS was from R&D Systems Inc. (Minneapolis, USA). Antibody against TLR4 was purchased from Santa Cruz (Dallas, USA). Horseradish peroxidase-coupled secondary antibody was purchased from Santa Cruz (Dallas, USA).
Confocal
Microscopy. Cells or tissues mounted on glass slides were gently washed twice with PBS, followed by fixation with 4% paraformaldehyde for 20 min. After permeabilization with 0.1% TritonX-100 for 5 min, they were incubated with 3% BSA for 1 hour at room temperature and then with primary antibodies overnight at 4°C. After that, they were incubated with Fluor 488-or Fluor 555conjugated secondary antibodies for 2 hours at room temperature, and DAPI was used to counter stain nuclei. Finally, images were obtained using a confocal microscope (ZEISS, German). Primary antibodies against NOX2, caveolin-1, and CD31 were from Abcam (Cambridge, UK). Antibody 2.9. Measurement of ROS Accumulation. ROS level was detected through either chemiluminescence or flow cytometry. For chemiluminescence assay, cells following treatment (static culture, OSS 30 min, LPS, and TAK-242+OSS 30 min) were gently washed twice with ice-cold PBS, incubated with ROS Fluorescent Probe-DHE (5 μmol/L, Beyotime) at 37°C for 20 min under light-protected conditions and stained with DAPI. Images were obtained with a confocal microscope (ZEISS, German). For flow cytometry analysis, cells that are treated with apocynin, allopurinol, tempol, and rotenone were applied with OSS; after that, cells were trypsinized, resuspended, and incubated with ROS Fluorescent Probe-DHE (5 μmol/L, Beyotime) for analysis. At least 10,000 events were analyzed, and the intensity of fluorescence was determined using the PE-A channel. Data were analyzed using FlowJo V10.3.0 software. LPS was purchased from Sigma-Aldrich (St. Louis, USA). TAK-242 was from MedChemExpress (Shanghai, China). Apocynin, allopurinol, tempol, and rotenone were purchased from Selleck (Houston, USA).
2.10. Assessment of NO Bioavailability. NO was analyzed to confirm the protection of TLR4 inhibition on OSS-treated cells. Cells subjected to OSS for 30 min with or without TLR4 inhibition were lysed, scraped, and centrifuged. Subsequently, the supernatant was incubated with Griess Reagents, and the mixture was examined at 540 nm according to the manufacturer's instructions (KeyGen Biotech Co. Ltd, Nanjing, China).
2.11. Determinant of SOD Activity. SOD was tested to confirm the antioxidant effect of TLR4 inhibition on OSStreated cells. In brief, sheared cells with TLR4 inhibited or not were lysed, and the lysates were centrifuged. After that, supernatant was collected and was incubated with WST-8/enzyme working solution and SOD detected buffer solution for 30 min at 37°C. At last, the mixture was assayed at 450 nm. This detection was conducted according to the manufacturer's instructions (Beyotime, Nanjing, China).
Data Analysis and Statistics.
Quantitative data were presented as means ± SEM. Student's t-test was used to analyze data between two groups, and one-way analysis was utilized to compare data from more than two groups. Statistical significance was assumed as p < 0 05, and all analyses were performed with SPSS version 19.0 (Chicago, USA).
CAC Induces Flow Change in Rat Carotid Arteries.
A silicone-made, cone-shaped cast was used to modify shear stress in the left common carotid artery (LCCA) of rats (Figure 1(a)). After placement of the constricted cast, the downstream region will be exposed to oscillations in shear stress. The tapering region, where the vessel diameter decreases from 1 mm to 0.5 mm, induces a gradual increase in shear stress. In the upstream region of the cast, blood velocity reduced and shear stress decreased (Figure 1(b)). Blood flow was analyzed by a Doppler ultrasound at 7 days postsurgery. Compared to sham, the constricted arteries exhibited reverse flow at the proximal part of the cast, which suggested an oscillatory flow pattern. In addition, the modified vessels showed narrowing and reduced maximal blood velocity (Figures 1(c)-1(e)).
OSS Activates TLR4 Both
In Vivo and In Vitro. We have previously conducted GeneChip analysis to generate an mRNA expression profile in shear stress-treated cells and found that TLR4 was one of the most differentially expressed genes. In this study, we assayed the TLR4 expression in carotid arteries and found increased levels of TLR4 in the OSS exposure regions (Figures 2(a) and 2(b)). In vitro study, a parallel-plate flow chamber was used to mimic flow changes. In response to OSS, TLR4 was activated at 30 min and sustained until 120 min (Figures 2(c) and 2(d)). Oxidative changes appeared immediately after the application of shear stress; hence, (Figures 2(e) and 2(f)). Taken together, the above results suggested that the oxidative burden trigged by OSS was at least in part due to TLR4 activation.
OSS Activates NADPH Oxidase.
To explore the mechanisms in mediating OSS-induced oxidative stress, apocynin (10 μmmol/L), allopurinol (1 mmol/L), tempol (1 mmol/L), and rotenone (100 μmmol/L) were used prior to stimulation by OSS, followed by the examination of oxidative state. The flow cytometry results showed that the four inhibitors restored ROS accumulation, while apocynin, an NADPH oxidase inhibitor, being the most effective (Figures 3(a) and
3(b)
). In ECs, activity of NADPH oxidase requires the assembly of NOX regulatory isoforms with its catalytic isoforms. The major NOX subunits in ECs are NOX1, NOX2, and NOX4. As shown by immunoblots, OSS significantly activated NOX2 and had no distinctive effect on NOX1 or NOX4 (Figures 3(c) and 3(d)). Moreover, OSS promoted translocation of p47 from cytoplasm to membrane and combination with NOX2 (Figures 3(e)-3(h)). To confirm NOX2 activation by OSS, we assayed its expression in human coronary artery. Compared to the nonbifurcation site, the bifurcation site had an increased expression of NOX2 ( Figure 4(e)). The above results suggested that NOX2 plays an important role in OSS-induced redox reactions.
OSS Accelerates eNOS Membrane
Localization. NO derived from eNOS plays an important role in scavenging oxidant. In ECs, eNOS is functionally inhibited through the binding of caveolae-scaffolding domain. The phosphorylation of tyrosin-14, which is required for caveolae-mediated trafficking, showed a marked increase in OSS-treated cells (Figure 4(a)). In addition, OSS increased eNOS accumulation on membrane and promoted its colocalization with caveolin-1 (Figures 4(b)-4(d)). To verify their combination in vivo, we assayed the expression of eNOS and caveolin-1 in the aortic arch, where the outer curve is the normal stress region and the inner curve is the OSS region. Compared to the outer curve, an increased association of eNOS and caveolin-1 can be detected in the inner curve (Figures 5(a)-5(f)). The data identified that with OSS exposure, eNOS accumulated at membrane where it formed a complex with caveolin-1 and maintained inactive.
TLR4 Inhibition Deactivates NOX2 and Restores eNOS
Activity. Since the TLR4 expression paralleled ROS generation, we posited that there might be bidirectional feedback between TLR4 activation and ROS production. Hence, we treated cells with siTLR4 or siNOX2 prior to OSS stimulation. We found that siTLR4 decreased the NOX2 expression and restored eNOS activity, whereas siNOX2 had no significant effect on TLR4 in sheared cells (Figures 6(a)-6(h)). Disrupting caveolae membrane domains with methyl-βcyclodextrin (10 mmol/L) slightly increased TLR4 and NOX2 regardless of OSS treated or not (Figures 6(i) and 6(j)). Moreover, TLR4 inhibition restored NO production as well as SOD levels ( Figure 6(k)). These results indicated that TLR4 acts as an upstream regulator of NOX2 and eNOS in OSS-induced oxidative damage (Figure 7).
Discussion
In the current study, we explored the mechanism of TLR4 activation in OSS-induced oxidative stress. The major findings were (1) OSS increased the TLR4 expression both in vivo and in vitro experiments, (2) TLR4 modulated OSSinduced oxidation by activating NOX2 and suppressing eNOS, and (3) TLR4 inhibition alleviated OSS-induced oxidative stress. Atherosclerotic lesions are preferentially located at branch points and curved regions of the arterial tree, where blood flow is disturbed, suggesting that shear force contributes to the distribution of atherosclerotic lesions [3]. The application of shear stress to ECs can activate a number of mechanosensors that are associated with adaptor proteins and lead to modulation of signaling pathways [2,4]. Emerging studies reveal that shear stress is converted into biochemical signals that are mediated by a variety of microdomains and membrane molecules, including caveolae, the glycocalyx, the cytoskeleton, ion channels, and G-protein-coupled receptors, followed by the almost simultaneous activation of multiple downstream signaling pathways [4]. TLRs, membranespanning proteins, have been implicated in the progression and development of many chronic diseases [15]. TLR4 has also been implicated in the development and progression of cardiovascular disease by inducing endothelial dysfunction [8,10]. Study showed that TLR4 knockout led to marked reduction of aortic plaque area, decreased inflammation, and reduced oxidative damage [16]. In addition, TLR4 antagonist inhibited vascular inflammation and atherogenesis in ApoE -/mice [11]. Qu et al. reported that fibronectin containing the extra domain A (FN-EDA) was the activator of TLR4 under disturbed shear stress [17]. Together with our previous findings, we speculated that TLR4 could be a regulator of OSS-induced oxidative damage. In this study, we employed the CAC model and a parallel-plate flow chamber to mimic oscillatory flow change and assayed the TLR4 expression. Consistent with our hypothesis, cells in OSS regions showed a marked increase of the TLR4 expression both in vivo and in vitro study.
Oxidative stress is evoked immediately after imposing shear force [18]. In order to explore oxidative changes in response to TLR4 activation, we examined ROS accumulation in LPS-treated cells. LPS induced ROS production comparable with OSS exposure. Excess ROS can result in impairment of redox signaling which leads to cellular damage and dysfunction. An imbalance in the production of ROS and its breakdown can be both a cause and a consequence of oxidative stress [4,19]. In the vasculature, several enzyme systems contribute to ROS formation, including NADPH oxidase, xanthine oxidase, cytochrome P-450 monooxygenases, mitochondrial oxidase, and uncoupled eNOS [20]. Although all of these enzymes contribute to oxidative burden, evidence is accumulating that NADPH oxidase acts as a major source of ROS [21,22]. To verify this hypothesis, inhibitors of NADPH oxidase, xanthine oxidase, cytochrome P-450 monooxygenases, and mitochondrial oxidase were used prior to OSS exposure. As shown in our results, apocynin was the most efficient, suggesting that NADPH oxidase contributed to OSS-induced oxidative damage. NADPH oxidase consists of 7 catalytic subunits (NOX1-5, Duox1, and Duox2) and 5 regulatory subunits (p22, p47, p67, p40, and Rac). Of these, NOX1, NOX2, and NOX4 are expressed in cardiovascular ECs and participate in regulating endothelial function. In resting state, NOX2 binds with p22 and forms a membrane complex, while p67 links with p47 or p40 and thus forms a trimer in the cytosol. Upon stimulation, p47 transfers to membrane and binds with NOX2 to form an active enzyme [22]. Hence, we tested NOX1, 2, and 4 in the present study. OSS activated NOX2 and had no significant influence on NOX1 or NOX4. There are some controversy in shear stress-regulated NOXs. Hwang et al. reported activated NOX2 and NOX4 in ECs exposed to shear stress for 4 h [20]. Siu et al. have also reported that NOX2 is downregulated by LSS and NOX1 is actually affected by OSS [23]. The difference of flow pattern and stimulating time might contribute to these contradictory results. We think that the translocation of p47 from the cytoplasm to the membrane and its association with NOX2 may be the major source of redox activation under the acute stimulus of OSS. Chen et al. reported that deactivated eNOS was accompanied by endothelial dysfunction in disturbed flow regions. eNOS serves as a critical enzyme in maintaining endothelial homeostasis, and the activation correlates with its intracellular localization [24]. In brief, eNOS is functionally inhibited through the binding of caveolae, a 50-100 nm diameter cell surface plasma membrane invagination, that plays a role in transcytosis, endocytosis, lipid homeostasis, and mechanotransduction [25,26]. Caveolin-1 is the main functional protein of caveolae, and a loss of caveolin-1 results in the loss of caveolae. Direct binding of eNOS to caveolin-1 is a wellaccepted mechanism for inactivating eNOS, while the absence of caveolin-1 is thought to promote eNOS dysfunction [27]. In this study, we assayed the eNOS membrane expression and its association with caveolin-1 to determine its activation state. Our study showed that OSS accelerated eNOS accumulation at the membrane, where it colocalized with caveolin-1 and resulted in deactivated eNOS. While disrupting caveolae with methyl-β-cyclodextrin excited TLR4 and NOX2 for the defection of caveolae usually leads to an extensively influence on cellular function, the regulation of caveolin-1 to TLR4 and NOX2 is controversial and needs to be explored further.
Several studies have reported the interrelation between TLR4 and NOXs. Wang et al. elaborated that TLR4 blocking reduced salt-induced prehypertension response via NADPH oxidases [28], while a study from Kim et al. also suggested a regulation effect of NOX4 on TLR4 [29]. We wondered the directional regulation; hence, siRNA against TLR4 or NOX2 were used prior to OSS exposure in ECs. TLR4 inhibition blocked NOX2 activation, while siNOX2 did not affect the TLR4 expression in sheared cells. In static-cultured cells, siNOX2 slightly decreased the TLR4 expression. Our data suggested that TLR4 acted as an upstream regulator of NOX2 in OSS-induced oxidative stress. Additionally, a bidirectional regulation existed between TLR4 and NOX2 in static-cultured cells which need further exploration.
There are several limitations to our study. In vivo study, a constriction cast was used to modify shear stress in carotid; however, we cannot exclude the injury caused by the constricted surgery; hence, a nonconstricting cast will be better used as sham. In the physiological state, the vessel wall is exposed to multiple stimuli in addition to shear stress; hence, potential contribution of other stimuli such as stretch force or chemical irritants is difficult to study. In addition, endothelial dysfunction is a consequence of chronic and complex stimuli and cannot be fully explained by acute stimulus; thus, a prolonged time course study should be conducted in the future. Moreover, classical TLR activation contributes to immune regulation through macrophages instate of mediated oxidative stress in ECs. Therefore, dual directional regulation of macrophages and ECs in a coculture system should be further explored.
In conclusion, our study identifies the specific roles that TLR4 plays in the response of endothelial cells to OSS. Under OSS condition, TLR4 promotes the translocation of p47 from cytoplasm to the membrane and combination with NOX2 which results in the production of O2-. On the other hand, OSS triggers the colocalization of eNOS with caveolin-1 which in turn deactivates eNOS and inhibits the production of NO. These results not only elucidate the key details of OSS regulation of endothelial redox status but also identify possible therapeutic targets that can be exploited in the treatment of endothelial injury that are high prevalent in vasculature exposed to oscillatory flow.
Data Availability
The data including ultrasound testing data, immunochemistry data, western blot data and biochemistry data used to support the findings of the study are available from the corresponding author upon request.
Conflicts of Interest
No competing interests exist.
Authors' Contributions
Zhimei Wang and Feng Wang contributed equally to this article. | v3-fos-license |
2021-08-08T13:16:57.115Z | 2021-08-03T00:00:00.000 | 238810022 | {
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} | pes2o/s2orc | Free amino acids quantification in cloud water at the puy de Dôme station (France)
Abstract. Eighteen free amino acids (FAAs) were quantified in cloud water sampled at the puy de Dôme station (PUY – France) during 13 cloud events. This quantification has been performed without concentration neither derivatization, using LC-MS and the standard addition method to avoid matrix effects. Total concentrations of FAAs (TCAAs) vary from 1.2 µM to 7.7 µM, Ser (Serine) being the most abundant AA (23.7 % in average) but with elevated standard deviation, followed by Glycine (Gly) (20.5 %), Alanine (Ala) (11.9 %), Asparagine (Asn) (8.7 %), and Leucine/Isoleucine (Leu/I) (6.4 %). The distribution of AAs among the cloud events reveals high variability. TCAA constitutes between 0.5 and 4.4 % of the dissolved organic carbon measured in the cloud samples. AAs quantification in cloud water is scarce but the results agree with the few studies that investigated AAs in this aqueous medium. The environmental variability is assessed through a statistical analysis. This work shows that AAs are correlated with the time spent by the air masses in the boundary layer, especially over the sea surface before reaching the PUY. The cloud microphysical properties fluctuation does not explain the AAs variability in our samples confirming previous studies at PUY. We finally assessed the sources and the atmospheric processes that potentially explain the prevailing presence of certain AAs in the cloud samples. The initial relative distribution of AAs in biological matrices (proteins extracted from bacterial cells or mammalian cells, for example) could explain the dominance of Ala, Gly and Leu/I. AA composition of aquatic organisms (i.e., diatoms species) could also explain the high concentrations of Ser in our samples. The analysis of the AAs hydropathy also indicates a higher contribution of AAs (80 % in average) that are hydrophilic or neutral revealing the fact that other AAs (hydrophobic) are less favorably incorporated into cloud droplets. Finally, the atmospheric aging of AAs has been evaluated by calculating atmospheric lifetimes considering their potential transformation in the cloud medium by biotic or abiotic (mainly oxidation) processes. The most concentrated AAs encountered in our samples present the longest atmospheric lifetimes and the less dominant are clearly efficiently transformed in the atmosphere, potentially explaining their low concentrations. However, this cannot fully explain the relative contribution of several AAs in the cloud samples. This reveals the high complexity of the bio-physico-chemical processes occurring in the multiphasic atmospheric environment.
ND: Not determined LOQ: Limit of Quantification (≈ standard deviation, see Figure S3, Table S3 and Section 3.1) Unlike Table S3, negative values are considered as below the LOQ. ND: Not determined LOQ: Limit of Quantification (≈ standard deviation, see Figure S3, Table S3 and Section 3.1) Unlike Table S3, negative values are considered as below the LOQ. Figure S3, Table S3 and Section 3.1) Unlike Table S3, negative values are considered as below the LOQ. Figure S3, Table S3 and Section 3.1) Unlike Table S3, negative values are considered as below the LOQ. Table S3. Concentration (µg L -1 with dilution 9:1, detailed in Figure S3), calibration curve and R² data for the 18 amino acids (AA) analyzed in the 13 clouds sampled at PUY. The calculation method (detailed in Figure S3) might mathematically provide negative values for the concentration. However, if the concentration (Conc) may turn out to be positive due to a higher STD (STD > |Conc|), the values are left as is (e.g., Asn -1 ± 4). Otherwise (STD < |Conc|), we assume to be below the limit of quantification (< LOQ). ND: Not Determined. Gly > Ala = Pro Zhu et al. (2021) Estimated lifetimes of AAs : Description of the calculations performed in Table 4. Table 4)
1-Calculations of the lifetimes considering theoretical HO • , O3 and 1 O2 * concentrations (column (A) in
Aqueous concentrations of HO • , O3 and 1 O2 * are respectively equal to 10 -14 , 5.0 10 -10 and 1.0 10 -12 M. The concentration of HO • derives from the study of Arakaki et al. (2013); the concentration of O3 is calculated considering a 50 ppb concentration of gaseous O3 and its Henry's law constant (H(O3) = 10 -3 M atm -1 ). 1 O2 * concentration is estimated to be 2 orders of magnitude more concentrated than HO • . All the kinetic constants derive from the Jaber et al. (2021) study (considering T and pHdependency when necessary and available). The lifetimes for individual AA are calculated as following: Table 4) Table 2 in Jaber et al., 2021). For Arg, Asn, Asp, Gln, Gly, Lys and Pro, lifetimes cannot be calculated since a production is observed during the experiment.
2-Calculations of the lifetimes using irradiation experiments in artificial cloud medium (column (D) in
The lifetimes for individual AA are calculated as follows:
Quantification and uncertainty (Figure S3)
In standard addition, known quantities of analyte (AA) are added to the unknown quantity in the sample. From the increase in signal, we deduce how much analyte was originally in the sample. This method requires a linear response to analyte (Broekaert, 2015).
The magnitude of the intercept on the x-axis is the original concentration of Gly. The equation of the trendline is y = a x + b. The x_intercept is obtained by setting y = 0: x = -b / a, with a = slope of the curve, b = y_intercept, x = the concentration of the AA, y= the mass spectral area: Gly: a = 410.49; b = 13607 → |x_intercept| = [Gly] = 33.1 µg L -1 (negative value) The obtained values are then corrected by the dilution factor of 10 % (due to the ratio 9:1 volume cloud: volume added standard). Final value is: [Gly]= 33.1 × %: ; = 36.8 µg L -1 .
The uncertainty in the x_intercept is sx: where a is the absolute value of the slope of the trendline, n is the number of data points, ? @ is the mean value of y for the points, D = are the individual values of D, D̅ is the mean value of y for the points, and : < is the standard deviation for y: | v3-fos-license |
2018-10-05T01:42:48.984Z | 2018-09-26T00:00:00.000 | 207370170 | {
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"Medicine",
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} | pes2o/s2orc | Characterization of novel glycosyl hydrolases discovered by cell wall glycan directed monoclonal antibody screening and metagenome analysis of maize aerial root mucilage
An indigenous maize landrace from the Sierra Mixe region of Oaxaca, Mexico exhibits extensive formation of aerial roots which exude large volumes of a polysaccharide-rich gel matrix or “mucilage” that harbors diazotrophic microbiota. We hypothesize that the mucilage associated microbial community carries out multiple functions, including disassembly of the mucilage polysaccharide. In situ, hydrolytic assay of the mucilage revealed endogenous arabinofuranosidase, galactosidase, fucosidase, mannosidase and xylanase activities. Screening the mucilage against plant cell wall glycan-specific monoclonal antibodies recognized the presence of carbohydrate epitopes of hemicellulosic polysaccharides like xyloglucan (both non-fucosylated and fucosylated), xylan (both substituted and unsubstituted xylan domains) and pectic-arabinogalactans, all of which are potential carbon sources for mucilage microbial residents. Mucilage metagenome annotation using MG-RAST identified the members forming the microbial community, and gene fragments with predicted functions associated with carbohydrate disassembly. Data from the in situ hydrolytic activity and monoclonal antibody screening assays were used to guide the selection of five full length genes with predicted glycosyl hydrolase function from the GenBank database that were similar to gene fragments of high relative abundance in the mucilage metagenomes. These five genes were then synthesized for recombinant production in Escherichia coli. Here we report the characterization of an α-N-arabinofuranosidase (GH51) and an oligosaccharide reducing-end xylanase (GH8) from Flavobacterium johnsoniae; an α-L-fucosidase (GH29) and a xylan β-1,4 xylosidase (GH39) from Spirosoma linguale, and a β-mannosidase (GH2) from Agrobacterium fabrum. Biochemical characterization of these enzymes revealed a β-Mannosidase that also exhibits a secondary activity towards the cleavage of galactosyl residues. We also describe two xylanases (GH8 and GH39) from underexplored glycosyl hydrolase families, one thermostable α-L-Fucosidase (GH29) and a thermostable α-N-Arabinofuranosidase (GH51).
Introduction
The large increase in shotgun metagenomic sequence data from environmental samples collected around the world provides extensive information regarding the taxonomic distribution of microbial communities. The sequence information contained within these metagenomes also serves as a potential resource for the discovery of novel enzymatic machinery, which can be achieved by establishing links between a given environmental sequencing data set and the metabolic processes that confer functions of interest within the targeted community [1][2][3]. Additionally, environmental sequencing studies also enable researchers to streamline the development of biocatalyst pipelines in a more efficient manner. Conducting stand-alone enzyme screening assays in high throughput, automated formats for a desired functionality is likely to be inefficient when compared to more targeted approaches that utilize environmental sequence analysis at the community-level to extract specific protein coding sequences from a well-characterized environment [4][5][6].
The application of functional metagenomics targeting the investigation of bacterial carbohydrate active enzymes (CAZymes) has recently emerged, where ecological changes that affect global carbon cycling in natural environments can now be monitored. This approach has provided unique opportunities to rapidly scan the microbial functionality of any ecosystem for new pools of glycosyl hydrolase (GH) biodiversity that can be used to create biocatalysts for the improvement of biotechnological processes [7]. However, the proliferation and establishment of each species within the microbial community of a given environment will largely depend on a number of factors, including the presence of specific glyconutrients with high bioavailability [3,8].
Discovery of GHs with new substrate specificities from metagenome environments that are rich in non-cellulosic sugar linkages, or with unique linkages, are extremely valuable for sustainable technologies that utilize biomass conversion of non-cellulosic polymers contained within plant-based feedstocks such as tailored prebiotic fibers from bio-refineries [9,10]. In this study, a unique variety of maize from the Sierra Mixe region of Oaxaca, Mexico was selected as the candidate source for enzyme discovery based on its observed development of an elaborate aerial root network that extensively secretes a carbohydrate-rich gel matrix or "mucilage" [11]. Preliminary in-situ assays for endogenous GH activities within the aerial root exudate suggested that the mucilage environment harbored CAZymes that act upon arabinosyl, galactosyl, fucosyl, mannosyl and xylosyl sugar residues derived from mucilage glycans. Furthermore, using an enzyme-linked immunosorbent assay (ELISA) to monitor plant-cell wall glycan epitopes present in the secreted mucilage provided structural insights and corroborated the in-situ enzyme activities detected. Metagenomes from aerial root mucilage were found to harbor a microbiome with a high relative abundance of GH sequences, and the integration of all three data types guided our selection of five gene sequences from the mucilage metagenomes that exhibited high sequence similarity to GH sequences within the GenBank and Carbohydrate-Active enZYmes (CAZy) databases [12,13]. Here we report the gene synthesis, recombinant production and biochemical characterization of five GHs, and collectively, the results validate the strategy of combining glycome profiling, environmental sequencing, genome analysis, and synthetic biology to elucidate the functional characteristics of novel subgroups of enzymes (GHs, other CAZymes, or any other enzymes) of specific relevance to ecosystems of interest (Fig 1). 4-Nitrophenyl (4NP), 4-Nitrophenyl (4NP)-β-D-glucopyranoside (4NP-β-Glc), 4NP-β-Dxylopyranoside (4NP-β-Xyl), 4NP-β-D-galactopyranoside (4NP-β-Gal), 4NP-β-D-manopyranoside (4NP-β-Man), 4NP-α-L-fucopyranoside (4NP-α-Fuc), 4NP-β-D-xylopyranoside (4NP-β-Xyl), 4NP-α-D-mannopyranoside (4NP-α-Man) and 4NP-α-L-arabinopyranoside (4NP-α-Ara) were purchased from Sigma-Aldrich (Steinheim, Germany). 4NP-βxylotrioside (4NP-β-(Xyl) 3 ) was purchased from Megazyme (Wicklow, Ireland).
In situ glycosyl hydrolase activities endogenous to the aerial root mucilage
To assay mucilage for endogenous GH activities, a high throughput colorimetric assay utilizing 4-Nitrophenyl (4NP)-conjugated glycosides was implemented in 96 well format similar to previously reported methods for determining extracellular enzyme activity in environmental samples [14]. Carrying out this assay required large volumes of fresh mucilage samples, which were generated by collecting mucilage from greenhouse-grown Sierra Mixe maize plants at U. C. Davis. The mechanism of detection for this assay relied on quantification of 4NP molecules that were liberated from their respective 4NP-conjugated glycosides following incubation with freshly collected mucilage at 28˚C. This assay was conducted as a time course study over 48 hours of continuous monitoring with 30-minute intervals between absorbance measurements at a wavelength of 400 nm using the BioTek Synergy™ Mx microplate reader. Quantification of 4NP levels within each mucilage-incubated sample was achieved by interpretation from a linear standard curve that was generated by measuring the absorbance values of 4NP dissolved in fresh mucilage over a range of concentrations (0.02-1 mM). To prepare the standard curve wells, stock solutions were prepared by dissolving the respective amounts of 4NP with Molecular Biology Grade water. Next, 100 μL of each stock solution was combined with 150 μL of freshly collected mucilage such that the final concentration of 4NP in each well reflected a value within the range of the standard. For each 4NP substrate (4NP-β-Glc, 4NP-β-Xyl, 4NPβ-Gal, 4NP-β-Man, 4NPα-Fuc, 4NP-α-Xyl, 4NP-α-Man, 4NP-α-Ara), 100 μL of a stock solution was combined with 150 μL of mucilage to achieve a final assay 4NP concentration of 4 mM. Each 4NP standard and sample was analyzed over 8 replications.
Plant cell wall glycan directed monoclonal antibody screening of mucilage
Fresh aerial root mucilage samples collected off Sierra Mixe maize plants grown in greenhouses at U.C. Davis were utilized to generate aerial root mucilage samples that lacked low Pipeline for the discovery of novel glycosidic enzymes using aerial root mucilage. Aerial root mucilage samples were analyzed using three different approaches: an in-situ assay for endogenous glycosyl hydrolase (GH) activity; an enzyme linked immuno sorbent assay (ELISA) to detect non-cellulosic glycan epitopes using monoclonal antibodies (mAbs) and shotgun metagenomics. The metagenomes from five maize aerial root mucilage samples were analyzed using MG-RAST tools by querying all of the metagenome sequences against the SEED database. This enabled the identification of partial sequences from the mucilage metagenome samples that had high relative abundance and high sequence similarity to full length GenBank coding sequences for genes encoding GH activities. Results from the in-situ GH activity and ELISA assays of Sierra Mixe mucilage revealed GH activities that were likely to be relevant in mucilage polysaccharide disassembly. These data were then used to guide the selection of five full length GH encoding gene sequences with the targeted functional predictions, which were then subjected to artificial gene synthesis, recombinant production and biochemical characterization. molecular weight sugar molecules. To achieve this, ethanol-insoluble mucilage samples (EIMS) were generated by precipitating the high molecular weight polysaccharide present in the fresh mucilage samples with high concentrations of absolute ethanol at a volumetric ratio of 3:1 (ethanol:mucilage) [15,16]. The ethanol-soluble fraction of the solution was decanted, and the precipitate was re-constituted in Milli-Q water to generate the EIMS. These EIMS were then subjected to hydrolysis using trifluoroacetic acid (TFA) at different final concentrations (0.05 M, 0.2 M and 0.5 M) for 1 hour at 100˚C. Each hydrolysis reaction began with 3 mg of lyophilized mucilage that was re-constituted in 1 mL of sterile Milli-Q water. The hydrolysis reactions were initiated by adding appropriate volumes of 1 M TFA to achieve the desired final concentration and were subsequently terminated and neutralized through the dropwise addition of 1 M sodium hydroxide. The neutralized samples containing the long-chain oligosaccharides were then transferred to a 96-well plate and allowed to dry overnight at 37˚C. Next, the enzyme-linked immunosorbent assay (ELISA) for cell wall glycan-directed monoclonal antibody based screening was performed on each TFA-hydrolyzed mucilage sample [17]. ELISA screening of the mucilage preps was conducted using a comprehensive suite of plant cell wall glycan-directed monoclonal antibodies (mAbs) directed against diverse glycan epitopes dispersed among all major non-cellulosic glycans of plant cell walls except rhamnogalacturonan II [18]. These mAbs were obtained from laboratory stocks (CCRC, JIM and MAC series) at the Complex Carbohydrate Research Center (available through CarboSource Services; http://www.carbosource.net) or from BioSupplies (Australia) (BG1, LAMP). Additional information about the antibody library used for these experiments can be found at Wall-MabDB (www.wallmabdb.net).
Aerial root mucilage metagenome analysis with MG-RAST
The five sequenced metagenomic libraries used in this study were generated as part of a related project examining the microbiomes associated with maize plants from the same geographic origin [11]. All five metagenomes were derived from mucilage samples that were collected from maize plants grown in the Sierra Mixe region of Mexico. Four of the samples were collected on August 1, 2008 and the fifth was collected on August 27 th , 2013. Each collected sample was shipped to U.C. Davis, where total DNA was then extracted using a DNA isolation kit (Mo Bio Laboratories, Inc, USA). Illumina sequencing libraries were prepared using total DNA extracts from each mucilage sample with a procedure modified from the Nextera transposase-based library construction method and multiplex barcoding. All mucilage metagenome libraries were sequenced using the Illumina MiSeq and HiSeq 2000 instruments. Sequences were de-multiplexed and trimmed using Trimmomatic (version 0.33) with the following parameters: Illuminaclip 2:30:10, Headcrop:15, Leading:20, Trailing:20, Sliding window:4:20, and Minlen:100. PhiX and maize sequences (genomic, chloroplast and mitochondrial) within the mucilage metagenomes were screened for using Bowtie2 [19] against the PhiX genome (Genbank acc# NC_001422.1) and Zea mays cultivar B73 draft genome (RefSeq assembly acc# GCF_000005005.2). These sequences were then filtered out of the mucilage metagenomes that were uploaded to MG-RAST.
The trimmed and filtered sequence data for all five mucilage metagenomes were analyzed using MG-RAST web servers and exist as public records that were assigned the following IDs: samples from 2008 (mgm4504365.3, mgm4504364.3, mgm4504362.3 and mgm4504361. 3) were uploaded to the server as fastq files containing the reads that survived the read processing described above, and the sample from 2013 (mgm4550815.3) was uploaded to the server as a list of contigs in fasta format that were assembled from the surviving fastq reads using the de Bruijn graph assembly program IDBA-UD v1.1.0 [20]. In terms of the total number of uploaded sequences for the five mucilage metagenomes on MG-RAST, mgm4504365. 3 bp, 149 ± 23 bp, and 1,475 ± 345 bp respectively. The reads from each aerial root mucilage metagenome library were analyzed collectively to evaluate the relative abundance of functional gene categories within the subsystems database using MG-RAST version 4.0.3. S1 Fig shows the workflow of the analysis made using MG-RAST. The overall microbial diversity within the aerial root mucilage samples was then assessed by querying the reads of all five metagenomes against the Refseq database using the analysis tool with the default settings, also using MG-RAST version 4.0.3 [21]. Results from the query of all reads against the Refseq database were then filtered based on phylum (S2 Fig, S1 Table) and class (S2 Table).
Using MG-RAST version 3.3.6, subsytems annotation of all aerial root mucilage metagenome samples was carried out using the default settings (maximum e-value cut off value of 1e -5 , minimum percent identity cutoff value of 60, minimum alignment length cutoff value of 15 (S3 Fig. and S3 Table). The subsystem designated as "Carbohydrate" was evaluated in detail to identify bacterial genes that were predicted to encode CAZymes involved in mucilage polysaccharide catabolism. This process enabled the identification of partial DNA sequences from the mucilage metagenomes that were similar to known GenBank sequence annotations of previously reported bacterial genomes. The identified reference genome sequences were then utilized for artificial gene synthesis rather than using a PCR based approach to extract the actual enzyme coding sequences from the environmental samples because the original DNA extractions that were used to make the sequencing libraries were not available. These sequences of interest for CAZyme production are presented in Table 1, which provides relevant information regarding how the sequences were selected based on having a relatively high number of abundance hits across all five mucilage metagenome libraries, their alignment scores to genomic sequences in the GenBank database, the length of the reported sequence alignment, and the sequence alignment e-value scores. Table 1. Glycosyl hydrolases selected for gene synthesis and metagenomic sequence query results. Analysis of the five mucilage metagenomes using the strategies depicted in Fig 1 and S1 Fig led to the selection of the following full-length coding sequences that were annotated in genomes that had been previously deposited to the GenBank database. "GH Family" corresponds to the selected enzyme coding sequence's designation within the CAZy database. "Abundance Hits" refers to the number of partial sequences across all five mucilage metagenome samples that had sequence alignment hits to the selected full-length GH coding sequence. "Sequence Similarity" indicates the percentage of sequence similarity produced by alignment between the mucilage metagenome partial sequences and the full-length coding sequence from Gen-Bank that was selected for gene synthesis.
Gene synthesis, recombinant protein production and purification
The five genes for which the gene products were predicted to have activities similar to those involved in mucilage polysaccharide catabolism that were selected for further characterization are listed in Table 1 with their corresponding Uniprot IDs [22]. The species origin and predicted function of each are as follows: α-L-fucosidase (D2QK56) and a xylan β-1,4 xylosidase (D2QHX5) from Spirosoma linguale, an α-N-arabinofuranosidase (A5FF88) and an oligosaccharide reducing-end xylanase (A5FD37) from Flavobacterium johnsoniae, and a β-mannosidase (Q7CZ23) from Agrobacterium fabrum str. C58. DNA sequences for these genes were codon optimized for recombinant production in Escherichia coli (E. coli) (S4 Table). Transformation constructs were generated by GenScript (Piscataway, NJ, USA) using chemically synthesized genes. The synthetic genes were cloned into the pET-28a (+) vector (Novagen) that contains a C-terminal, 6x poly-histidine tag. All constructs were transformed into E. coli BL21 (DE3) pLysS competent cells (Novagen) by heat shock for protein production. Small batch protein production and purification of each enzyme was carried out as described previously [23]. Briefly, the isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration was optimized over a range from 0.1 to 0.5 mM for 50 mL cultures using Terrific Broth (TB). The incubation temperature was set to either 16 or 37˚C to improve the production of each soluble protein (if the production at 37˚C was low, the incubation temperature was lowered to 16˚C following the addition of IPTG) and the total protein from each was purified using the His-Spin Protein Miniprep kit from Zymo Research (Irvine, U.S.A). Confirmation of successful small-scale production was achieved by carrying out protein production in 200 mL of TB that was supplemented with 50 μg/mL kanamycin using a 1 L shake flask. Initial incubations were at 37˚C with shaking at 200 rpm. Induction of enzyme production was performed using the optimized amount of IPTG depending on the construct administered once the optical density of the liquid culture at λ = 600 nm reached a value of 0.6. Following chemical induction of protein production, cultivation times continued up to 16 hours with shaking at 200 rpm. The cell cultures were centrifuged (13,000 ×g, 10 min, 4˚C), and cell pellets were re-suspended in lysis buffer (20 mM imidazole, 20 mM Tris-HCl, 0.75 M NaCl, pH 7.5). Cell suspensions were lysed enzymatically as described by the European Molecular Biology Laboratory Protocol (www.embl.de), followed by treatment with Benzonase1 Nuclease (EMD Millipore) for 30 minutes. Cell lysates were then immediately subjected to an additional round of centrifugation (30 min, 15,000 ×g, 4˚C). The His-tagged proteins were purified from the resulting supernatant using 1 mL HisTrap affinity columns (GE health care, Germany) that utilize a nickel sulfate solution to carry out immobilized metal ion affinity chromatography (IMAC). Fractions containing the purified enzyme were pooled and dialyzed against 50 mM citrate phosphate buffer over a range from pH 3 to pH 5 and phosphate buffer from pH 6 to pH8. The successful purification of each enzyme was confirmed by SDS-PAGE using a
Biochemical characterization of glycosyl hydrolases
Substrate specificity for each enzyme was determined by carrying out individual reactions with each of the following 4NP substrates: 4NP-β-Glc, 4NP-β-Xyl, 4NP-β-Gal, 4NP-β-Man, 4NP-α-Fuc, 4NP-α-Xyl, 4NP-α-Man, or 4NP-α-Ara. The optimal reaction conditions for hydrolysis of the 4NP-conjugated glycosides that corresponded to the specificity of each GH were determined by continuously monitoring sample absorbance at λ = 400 nm for a total duration of 60 minutes with 30 second intervals using different pH buffered solutions. Citrate-Phosphate buffer was used for reactions occurring over a pH range from 3.0 to 5.0 and Phosphate buffer was used for reactions occurring over a pH range from 6.0 to 8.0. Once the optimal pH was determined for each GH, reactions were carried out at different temperatures ranging from 30˚C to 60˚C to identify the optimal temperature for enzymatic hydrolysis. Standard curves were generated using 4NP for each combination of pH and temperature. Next, each 4NP substrate listed above was screened for hydrolytic activity against all five recombinant GH enzymes by adding the substrate to a final concentration of 1 mM, and the reactions were carried out under the optimal hydrolytic conditions for each enzyme over 50 minutes with 50 mM final buffer concentrations. If a secondary activity was detected for a recombinant GH protein was detected during the 4NP substrate screen, further characterization for the secondary activity was then carried out at the enzyme kinetic level. The kinetic parameters were obtained using the optimum pH at two temperatures using the software Graph Pad Prism6 (Graph Pad Software Inc., San Diego, CA) by fitting the rate of the reaction and substrate concentration to the Michaelis-Menten equation. All enzymatic reactions were run in triplicate.
Phylogenetic analysis of glycosyl hydrolase family sequences
Multiple sequence alignment and gene tree analyses were carried out for each GH family that corresponded to the enzymes characterized in the present study. Amino acid sequences of bacterial enzymes from GH families 2, 8, 29, 39 and 51 were identified from the respective subsets of "Characterized" sequences within the CAZy database, where the subset of "Characterized" sequences refers to those that had been assigned enzyme class (EC) numbers based on experimental data according to rules set by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). These "Characterized" sequences from each GH family of interest were subsequently downloaded in FASTA format from the NCBI GenBank database (see S5 Table for sequence accessions used in the analysis). These sequences were then subjected to multiple sequence alignment and phylogenetic tree building using Geneious version 11.5. Multiple sequence alignments were carried out using ClustalW v 2.1 using the default settings of the Geneious module, and the trees were created using the UPGMA method with bootstrap resampling over 100 replications.
Results and discussion
As an integral component of their development, plants generate root exudates that are primarily comprised of high molecular weight polysaccharide (mucilage) and specialized metabolites that serve as a means to facilitate information exchange with the root microbiome (rhizobiome) in order to meet demands imposed by their surrounding environment [24]. Because mucilage polysaccharides from the aerial roots of Sierra Mixe maize have the potential to be utilized as a carbon source by the resident microbial community, we sought to gather insight regarding the enzymatic machinery that is likely to be associated with polysaccharide disassembly within the aerial root mucilage environment.
Detection of in situ glycosyl hydrolase activities endogenous to mucilage
The production of mucilage by the aerial roots is likely to be a dynamic process, where the polysaccharide structures present within the mucilage at any time represents a balance of newly secreted mucilage and the evolving structures of mucilage polysaccharides that are being metabolized by the diversity of mucilage-localized microbes. In an attempt to confirm the presence of enzymatic activity within the mucilage, 4NP-conjugated glycosidic substrates were spiked into freshly collected mucilage samples in order to monitor the GH activity of the mucilage in situ. While this colorimetric method allowed us to observe and quantify the presence of GH activity endogenous to the aerial root mucilage of Sierra Mixe maize, it also provided a direct line of evidence to support the presence of GH activities that target a diverse range of monosaccharide types within the mucilage (Fig 2).
The highest enzymatic activity observed within the mucilage was determined to be from enzymes acting on the 4NP-α-arabinofuranoside substrate, where these incubations exhibited the release of 4NP residues at a rate of 3.37 μmol•min -1 •mL -1 . Independent incubations of mucilage with 4NP-β-xylopyranoside, 4NP-α-mannopyranoside and 4NP-β-galactopyranoside substrates each demonstrated similar levels of 4NP liberation at 2.02, 1.78 and 1.76 μmol•min -1 •mL -1 respectively. Endogenous enzymes that facilitated the liberation of 4NP residues from 4NP-α-fucopyranoside substrates showed only the fifth highest glycosidase activity rate (1.08 μmol•min -1 •mL -1 ) in these assays. Enzymatic activities within the mucilage on 4NP-αxylopyranoside, 4NP-β-glucopyranoside and 4NP-β-mannopyranoside substrates were determined to be the lowest among the eight substrates used in this set of assays (rates of 0.77, 0.59 and 0.37 μmol•min -1 •mL -1 ). While the endogenous CAZyme activities detected within the mucilage cannot be concluded to be fully attributed to microbial CAZyme activities because of the possibility that plant-derived CAZymes may also populate the aerial root mucilage, this experiment was crucial for motivating further investigation because it revealed the presence of active enzymes likely to be involved in the catabolism of mucilage polysaccharide. These differences in substrate specificities indicated that the mucilage possesses a diverse range of GH activities and we can presume that relative levels of GH enzymes may be dynamic and vary over time, with our assays providing a snapshot in time.
Mucilage polysaccharide structural insights
A screening assay to detect non-cellulosic plant cell wall glycan epitopes using a suite of monoclonal antibodies (mAb) was conducted to characterize the structural features of the complex mucilage glycans. This screening assay confirmed that the glycan content within the mucilage is highly heterogeneous (Fig 3). The ethanol-insoluble mucilage samples (EIMS1 and EIMS2) exhibited binding to the following cell wall glycan-directed mAbs that recognize epitopes in hemicellulosic polysaccharides (xyloglucans, xylans): non-fucosylated xyloglucan-1 and -2 antibody clades, which bind to the L side-chain of xyloglucans (Tuomivaara et al., submitted for publication), a single mAb from the fucosylated xyloglucan clade (CCRC-M84) that is specific for difucosylated xyloglucan (nFFG) (Tuomivaara et al., submitted for publication); one antibody in xylan-4 (CCRC-M154) that is specific for arabino-substituted xylans [25], several antibodies specific for 4-O-methyl glucuronic acid side-chains on xylans (xylan-5 clade) [25], and a couple of antibodies from the xylan-7 clade, which bind unsubstituted xylan epitopes [25]. In addition, a number of antibodies in the RG-I/AG clade, which mostly recognize β-1,6 galactan epitopes with varying degrees of substitution [25] that are present on rhamnogalacturonan I (RG-I) (13) and arabinogalactan glycoproteins (AGPs) bind moderately.
Performing the same assay with mucilage samples that had been treated by mild acid hydrolysis (0.05M TFA, 100˚C) resulted in a different pattern of mAb binding. Most of the hemicellulosic (xyloglucan and xylan) epitopes were lost after the mild acid hydrolysis. Only weak binding by a couple xyloglucan-directed antibodies that bind to L-side-chain or the nFFG epitope remained. The binding of the xylan-directed antibodies was completely eliminated by the mild acid treatment, indicating that these epitopes were attached to the mucilage polymer by acid-labile linkages, perhaps in a configuration similar to that described for APAP1 [26]. In contrast, binding of antibodies in the RG-I/AG clade was increased in the 0.05M TFA hydrolyzed mucilage sample, probably because acid-labile substituents on the galactans (e.g., arabinosyl residues) were removed by the acid treatment thereby uncovering the underlying galactan epitopes recognized by these antibodies. Stronger (0.5 M) TFA hydrolysis treatment resulted in the almost complete removal of all antibody binding to the mucilage except for a couple of RG-I/AG antibodies, probably due to extensive hydrolysis and fragmentation of the mucilage polymer and hence loss of epitopes in the stronger acid environment (Fig 3). Collectively, the top five most abundant GH activities detected by the in situ GH activity assay of fresh mucilage, the decrease in binding intensity by mAb that recognize fucosylated and nonfucosylated xyloglucan carbohydrate epitopes following mild acid hydrolysis of the EIMS, and the increase in binding intensity by mAb that recognize RGI/AG epitopes following mild acid hydrolysis of the EIMS suggested that GH enzymes conferring arabinofuranosidase, fucosidase, galactosidase, mannosidase and xylosidase activities are likely to be important in disassembly of the maize aerial root mucilage polysaccharide.
Mucilage metagenome analysis using MG-RAST
The microbiota associated with the aerial root mucilage of the Sierra Mixe maize landrace was investigated by analyzing five shotgun metagenome libraries of DNA extracted from mucilage sampled in Mexico. As depicted in the workflow shown in S1 Fig, DNA sequencing reads from all mucilage metagenomes were queried against sequences from the Refseq database using MG-RAST in order to quantify the abundance of ribosomal RNA (rRNA) genes that were present in each mucilage sample [21]. This analysis revealed that the most abundant rRNA genes were those from bacteria (S2 Fig, S1 and S2 Tables). Among the bacterial phyla that were identified within the mucilage metagenomes, the Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were found to have the first, second, third and fourth highest relative abundances, respectively. Considering each of the five mucilage metagenomes together, the Proteobacteria classes with the highest relative abundance were Betaproteobacteria and Gammaproteobacteria, which were observed to have similar relative abundance levels. Looking at the Bacteroidetes, the classes Flavobacteria and Sphingobacteria were found to have the highest relative abundance.
Data from the subsystems analysis tool of MG-RAST [13,27] indicated that the subsystem with the highest relative abundance in each metagenome was determined to be "Carbohydrates" (S3 Fig and S3 Table). This result prompted the application of a subsystem filtering strategy to the metagenome reads that were annotated using MG-RAST, which has been diagramed in S1 Fig. To begin filtering the annotated metagenome reads, a "Carbohydrates" level 1 subsystem filter was applied to the annotated mucilage metagenome sequence reads. Next, the diversity of GH encoding genes within the mucilage metagenomes was achieved by selecting the "Monosaccharides" subsystem. From this point, tertiary subsystem filters were then applied to identify annotated metagenome reads that corresponded to monosaccharides of interest (arabinose, fucose, mannose and xylose) based on the in-situ GH activity and mAb cell wall glycan screening assays of aerial root mucilage.
Following the iterative application of tertiary subsystem filters to target protein coding sequences related to the utilization of monosaccharides that were determined to be of interest by the previous mucilage assays, the surviving mucilage metagenome reads were investigated to identify GH encoding genes within the samples. Specifically, reads with predicted GH function that were assigned to each monosaccharide subsystem were evaluated based on the parameters of their sequence alignment to reference sequences from the SEED database. GH sequences of interest for enzyme production and characterization were chosen by selecting those with alignments to mucilage metagenome reads that had the highest relative abundance within the samples, the lowest relative e-value among the candidate metagenome sequences, the highest sequence alignment percent identity, and highest relative bit score. However, because full-length protein coding sequences were required for gene synthesis and the metagenome reads that were annotated by MG-RAST only represented partial gene fragments, the full-length gene sequences from the SEED database of MG-RAST were utilized for gene synthesis instead.
The observed epitope binding events and endogenous glycosyl hydrolase activities of aerial root mucilage from Sierra Mixe maize samples served as guides during the process of mining the previously generated mucilage metagenome sequence libraries to identify GH-encoding genes that were likely to confer functional activities associated with mucilage polysaccharide catabolism. A summary of the reference database matches to the mucilage metagenome queries that were selected from the MG-RAST analysis to acquire complete reference sequences for artificial gene synthesis is presented in Table 1. The five GHs that were selected from this analysis were found to belong to bacterial species in the Proteobacteria or Bacteriodetes phyla. The annotation of these in Uniprot are as follows: α-N-arabinofuranosidase (FjArf51), α-Lfucosidase (SlFuc29), β-mannosidase (AfMan2), a xylan β-1,4 xylosidase (SlXyn39) and an oligosaccharide reducing end xylanase (FjXyn8) from Flavobacterium johnsoniae UW101, Spirosoma linguale DSM 74, Agrobacterium fabrum C58, Spirosoma linguale DSM 74, and Flavobaterium johnsoniae UW101 respectively [22].
These five enzymes proved to be interesting based on the taxonomy of the organisms from which they were found to derive, where previous reports in the literature described taxonomically related bacteria as being associated with grass plants. Specifically, using a 16S ribosomal RNA probing method of different soil samples, Bouffaud et al. reported positive correlations for associations between poaceous plant species and targeted microbial taxa from Agrobacterium and Spirosoma [28]. In addition, the recent report from Kolton et al. described that Flavobacteria are well suited to survive in environments that are rich in plant-synthesized glycans, which reinforced our decision to pursue the characterization of GHs from the Flavobacterium johnsoniae UW101 genome [29]. Results from these studies corroborate the recent comprehensive analysis of genomes derived from plant associated bacteria by Levy et al., which revealed the trend for plant associated bacteria to exhibit diverse enzymatic machinery related to carbohydrate metabolism, thus making them an ideal source for enzyme discovery [30]. While the five predicted gene sequences with putative glycosyl hydrolase function were selected from the aerial root mucilage metagenomes based on their whole genome sequence annotations from the UniProt database, the next step was to validate the predicted function of each enzyme by using a combination of comparative sequence analysis to those with similar annotations and biochemical characterization.
Phylogenetic analysis of targeted glycosyl hydrolase families
The CAZy database is a comprehensive repository of information for well characterized bacterial enzymes that includes the classification of GH enzyme sequences under different GH family numbers. Enzymes that are listed under each GH family are then directly linked to the accession pages for the nucleotide and amino acid sequences stored within the NCBI GenBank database. Amino acid sequences for enzymes that corresponded to the same GH family and monosaccharide utilization annotations as those of the enzymes investigated in this study were acquired in FASTA format from the NCBI GenBank database by using the CAZy database to guide the selection of "characterized" (experimental data has been generated resulting in the assignment of an enzyme class number according to IUBMB rules) bacterial enzymes from GH families 2, 8, 29, 39 and 51. The selected "characterized" sequences corresponding to each GH family in this study, were then used to generate multiple sequence alignments and the subsequent phylogenetic trees that are presented in Fig 4. While the CAZy database is a continuously growing database, the number of "characterized" bacterial enzymes present within the database for each GH family was found to be variable during the time of our GH family sequence analysis.
The GH families containing the highest number of "characterized" bacterial enzyme sequences with similar substrate specificities to the targeted enzymes of interest were GH51 and GH29. GH51 had 45 enzyme sequences with proven arabinofuranosidase activity that were incorporated into the tree shown in Fig 4A, including that of the FjArf51 enzyme from Flavobacterium johnsoniae UW101. The FjArf51 sequence formed a clade with only one other sequence among the 44 GH51 sequences that were included in the group (an arabinofuranosidase sequence from Streptomyces chartreusis) [31]. This result suggested that FjArf51 does not exhibit high sequence similarity to the majority of "characterized" enzymes from GH51, as this pair of sequences (FjArf51 and S. chartreusis_AFase) were not found to be deeply nested within either of the major clades on the tree. However, the observed clustering of the FjArf51 sequence with other GH51 sequences from the CAZy database provides one level of confirmation for the sequence annotation-based assignment of putative arabinofuranosidase function.
GH family 29 had the second highest number of bacterial enzyme sequences with biochemically proven fucosidase activity incorporated into its phylogenetic analysis with 24 (SlFuc29 and 23 other sequences) members comprising the tree depicted in Fig 4B. Phylogenetic analysis of these sequences revealed that the sequences clustered as a sister clade to SlFuc29 were assigned "Mfuc" as an enzyme prefix and derived from uncultured bacteria that were previously reported in a soil metagenome study by Lezyk et al. in 2016 [32]. Further investigation into these "Mfuc" sequences revealed that SlFuc29 shared the conserved nucleophile sequence TPEQ with all five "Mfuc" members included in the phylogenetic analysis, while Mfuc1, Mfuc2, Mfuc4 and Mfuc5 all had similar kM values to that of SlFuc29 (0.18) when hydrolyzing 4NP-α-Fuc.
The number of "characterized" bacterial enzyme sequences incorporated into the phylogenetic trees corresponding to GH families 2, 8 and 39 were found to be lower with 10, 14 and 13 sequences included into each group respectively. Fig 4C shows that well-characterized mannosidase sequences from GH2 had the lowest representation among the GH family groups we explored in the CAZy database, where the AfMan2 sequence from Agrobacterium fabrum str. C58 formed a clade with two other GH2 mannosidase sequences: one sequence from Bacteroides thetaiotaomicron and another from Paenibacillus polymyxa [33,34]. The SlXyn39 enzyme sequence from Spirosoma linguale DSM74 was analyzed phylogenetically along with 13 other bacterial enzymes from GH39 with specificities for xylosyl-residues in Fig 4D, which revealed that SlXyn39 was the sister taxon to 12 other sequences with which it formed a monophyletic group. Finally, the sequence for the FjXyn8 enzyme from Flavobacterium johnsoniae UW101 was analyzed alongside 12 other well-characterized bacterial enzymes from GH8 with reported activity on xylose-containing sugar molecules. Fig 4E shows that FjXyn8 formed a two-member clade with the Rex8A enzyme from Bacteroides intestinalis DSM17393.
Biochemical characterization of glycosyl hydrolases
The selected GH genes encoding putative GH enzymes from Table 1 were codon optimized for recombinant production in E. coli, synthetized, and subsequently cloned into the pET-28a The selected full-length protein sequences forming each phylogenetic tree derived from the CAZy database that were listed as "characterized" enzymes with bacteria origin (S4 Table). A) members of GH family 51 with α-N-arabinofuranosidase activity (FjArf51); B) members of GH family 29 with α-L-fucosidase activity (SlFuc29); C) members of GH family 2 with βmannosidase activity (AfMan2); D) members of GH family 39 with xylan β-1,4-xylosidase activity (SlXyn39); E) members of GH family 8 with oligosaccharide reducing end xylanase activity (FjXyn8).
https://doi.org/10.1371/journal.pone.0204525.g004 (+) vector from Novagen. Protein was then produced by induction using the appropriate final concentration of IPTG (0.1 to 0.4 mM). Cell pellets were enzymatically lysed, and the soluble protein was purified for all five of the recombinant GHs. The amount of soluble protein obtained from 200 mL Terrific Broth cultures differed for each recombinant protein. SlFuc29 had the highest expression level with 25 mg of soluble, purified protein recovered. For the remaining four recombinant proteins, purified recovery totals were as follows: FjArf51 (20 mg), AfMan2 (12.5 mg), SlXyn39 (12.5 mg) and FjXyn8 (10 mg). The purified, soluble protein samples, before dialysis, have been presented in the SDS-PAGE gel image of S4 Fig. Given the gel concentration (12%) and the estimated molecular weight for each of the five purified GH proteins, the total migration distance of each sample was found to be appropriate in relation to the bands of the molecular weight protein standard (S4 Fig, Table 1). Enzyme characterization was carried out for each of the five CAZymes using the corresponding commercial substrates (4NP-substrates). The kinetic constants were calculated at the optimum pH over two temperatures, 30˚C and 50˚C and the data have been summarized in Table 2.
The FjArf51 protein showed kinetic parameters that included substrate accommodation with a lower K M value when operating at 30˚C, and higher turnover at 30˚C (31090 min -1 mM -1 ) when compared to that at 50˚C (22873 min -1 mM -1 ). The more relevant enzyme characteristic of FjArf51 was its demonstrated thermostable hydrolytic activity at different temperatures (30˚C,40˚C, 50˚C and 60˚C) over an incubation period of 40 minutes (S5 Fig). The kinetic parameters for SlFuc29 indicated that the enzyme activity was enhanced at the higher temperature of 50˚C (52605 min -1 mM -1 ), where it exhibited a higher turnover rate, and a lower K M . At first glance, the primary difference between SlFuc29 and the Mfuc enzymes appears to be that SlFuc29 exhibited optimal activity at pH5 and was determined to be thermostable with activity still present as high as 60˚C (S5 Fig), while the Mfuc enzymes reported by Lezyk et al. were shown to display heightened activity at higher pH values that ranged from 7 to 9 but were not found to be thermostable.
The AfMan2 enzyme presented a stable activity at different temperatures with similar turnovers at both 30˚C (119018 min -1 mM -1 ) and 50˚C (140453 min -1 mM -1 ). Interestingly, this β-mannosidase showed another substrate specificity towards the 4NP-β-Gal, and this secondary activity exhibited a kinetic turnover that was equal to more than half of the kinetic turnover observed with the 4NP-β-Man substrate at both 30˚C (65014 min -1 mM -1 ) and 50˚C (55178 min -1 mM -1 ). This sequence is classified under GH family 2, which is a family that currently holds 9 "characterized" bacterial members with reported activity on mannose. These sequences were used alongside AfMan2 to construct the phylogenic tree in Fig 4C that depicts AfMan2 within a clade of three taxa on its own branch as a sister taxon to two other sequences included in the analysis. Biochemical assay of AfMan2 revealed that it was active on both the 4NP-β-Man and 4NP-β-Gal substrates (Table 2), which could potentially be explained by the LacZ (COG3250), ebgA (PRK10340), and Glyco_hydro_2 (pfam00703) conserved domains that are present within its protein sequence. This feature makes AfMan2 the first reported β-mannosidase with dual-activity. While the observed activity may potentially be due to the presence of a galactose-binding-like domain, further investigation into the structure and functional promiscuity of this enzyme is required in order to fully elucidate the mechanism by which it is capable of accommodating different hexose isomers as substrates for hydrolysis.
The SlXyn39 demonstrated a higher substrate affinity for 4NP-β-(Xyl) 3 at 30˚C (K M 0.08 and k cat / K M 1615 min -1 mM -1 ), but the higher temperature (50˚C) made the substrate interaction less specific. On the other hand, FjXyn8, with an inverting mechanism, had greater substrate accommodation at higher temperature with the 4NP-β-(Xyl) 3 substrate at 50˚C (K M 0.150 and k cat / K M 3629 min -1 mM -1 ). The use of xylanases from well described GH families such as GH10 and GH11 have been reported for numerous applications, whereas the lack of abundance in well-characterized xylanases from GH families 8 and 39 at both the biochemical and sequence levels may be account for their lack of incorporation into biotechnological processes [10]. Interestingly, the comparative genomic analysis of Kolton et al. described the presence of CAZy genes from GH51 within the genomes of terrestrial Flavobacterium species but does not mention their possession of genes that could encode xylose-acting CAZymes from GH 8 [29]. This development provides further validation of the described strategy to use metagenome sequence analysis in combination with whole genome sequence database mining, where this result has served the purpose of complementing previous reports through the presentation of new information related to GHs from terrestrial species of Flavobacterium.
Conclusion
This work demonstrates how metagenomic approaches to mine specific environmental samples is a useful strategy for guiding the discovery of gene sequences within large genome sequence databases that may then be utilized for the generation of new enzymatic tools and applications in biotechnology. Furthermore, utilizing the in-situ GH activity and non-cellulosic plant cell wall glycan mAb screening assays of aerial root mucilage from Sierra Mixe maize informed the shotgun metagenomic analysis, which expanded the repertoire of knowledge for GH families that derive from plant associated bacteria of terrestrial environments that are rich in complex carbohydrate and provided insight into the enzymatic strategies that are potentially employed by the microbiota of Sierra Mixe maize for the catabolism of mucilage polysaccharide. While a strong indication of taxonomic differences among the key players Table 2. Determination of kinetic parameters for mucilage derived glycosyl hydrolases. Experiments were run in triplicate under the optimal pH and temperature for each enzyme. Both the enzyme concentration and 4NP substrates concentration varied over ranges of 8-20 μM and 0.2 to 1mM respectively. All reactions were carried out in the corresponding buffer (either citrate-phosphate or phosphate) with a final buffer concentration of 50 mM.
α-N-Arabinofuranosidase
4NP-α-Ara 30˚C, pH 7 affiliated with the aerial root mucilage was observed, the findings of the present work also suggest that distinguishable enzymes from within the same microbial ecosystem may work synergistically to hydrolyze this compositionally diverse mucilage polysaccharide. The results from biochemical and phylogenetic analyses of GHs reported in this study serve the scientific community by expanding both the characterization of enzymes derived from microbes associated with plants and the reported biodiversity of the corresponding GH families within the CAZy database. Overall, we highlight the utility of combining metagenomics and synthetic biology to discover and validate enzymatic activities that derive from microbes of geographically isolated ecosystems.
Supporting information S1 Fig. Diagram of workflow for mucilage metagenome analysis using MG-RAST. Metagenome datasets for the five Sierra Juarez mucilage samples were selected for the MG-RAST analysis. Microbial community analysis was carried out by selecting the Refseq database, which then allowed for the classification of ribosomal sequences and relative abundance estimations of taxa within the samples at different taxonomic levels. The annotation of reads based on function was achieved by selecting the SEED database for subsystems analysis. Level 3 filters that corresponded to monosaccharides related to the aerial root mucilage sugars were applied separately to the sequence reads from all five samples. The filtered reads surviving for each monosaccharide category were then compared based on the relative sample abundance, percent similarity of the alignment to database reference sequences, the identity of positions in the alignments, and the e-value. Reference sequences that aligned to metagenome reads with the lowest e-value, highest sequence similarity, highest identity value and highest relative abundance were then selected for artificial gene synthesis. Table. DNA sequences of the codon optimized synthetic genes. Nucleotide sequences were acquired from NCBI Genbank and were codon optimized (red colored nucleotides) for artificial gene synthesis and cloning into the pET-28a(+) vector (Novagen) by Genscript Inc.
The following sequences were downloaded from NCBI GenBank after browsing the CAZy database and were incorporated into the phylogenetic analysis used to generate the trees shown in Fig 4. (DOCX) | v3-fos-license |
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} | pes2o/s2orc | Metabolomics study of dried ginger extract on serum and urine in blood stasis rats based on UPLC‐Q‐TOF/MS
Abstract Blood stasis syndrome (BSS) is the pathological basis of many cardiovascular diseases. Ginger is often used as herbal medicine, condiment, and health food in China and Southeast Asia to improve some symptoms of cardiovascular disease, but its mechanism of efficacy and metabolic processes is not clear enough. In this study, a rat model of BSS was successfully established and treated with different doses of dried ginger extract. After the end of the administration period, the blood and urine of 5 groups of rats were collected for metabonomic analysis. Multivariate statistical analysis was used to explore metabolites and metabolic pathways, and the correlation between metabolites and pharmacodynamic indicators was further explored. The experimental results show that the pharmacodynamic indicators of dried ginger group (DG) extracts of different doses have different degrees of changes than model group (MG), and the high dose of dried ginger group (GJH) changes is the most significant (p < .05 or p < .01). Besides, 22 different metabolites were identified in the experiment. These metabolites mainly involve seven metabolism pathways in different impact value. DG has therapeutic effects on BSS rats by regulating multiple metabolic pathways. This study provides an effective method for understanding the metabolic mechanism of DG extracts on BSS.
| INTRODUC TI ON
Cardiovascular disease is one of the leading causes of death worldwide, affecting not only high-income but also low-and middle-income countries. Nearly 80 percent of all estimated cardiovascular disease-related deaths worldwide now occur in low-and middle-income countries, where nearly 30 percent of all deaths are attributable to cardiovascular disease (Kelly et al., 2012). BSS is a state of poor blood circulation due to increased blood viscosity and concentration, which is closely related to many diseases, such as coronary heart disease, infertility, and cancer (Xiao-yan et al., 2018). It is a complex pathological system, usually accompanied by vascular endothelial injury, inflammation, liver and kidney injury, and other symptoms, which further promote the occurrence of blood stasis (Liu et al., 2017;Ma et al., 2009). In modern medicine, the cognition of BSS mainly refers to insufficient blood circulation, decreased blood flow to the body, or impurities in the blood (Jian et al., 2012). The pathological indexes of BSS include hemorheology, coagulation function (Cao et al., 2018). The perceptions of BSS are changing, as well as its management. Lifestyle factors involving diet now feature prominently as the more important contributors to the pathogenesis of BSS than genetics, as a result, TCM and healthy food have received extensive attention in the treatment of BSS due to their natural nature.
Ginger (Zingiber officinale Rosc.) is taxonomically characterized as perennial, aromatic, tuberous, and nontuberous rhizomes (Jatoi et al., 2007). Mainly produced in China, India, and other places and used as food seasoning ingredients and medicinal resources, it is rich in phytochemicals, such as gingerols, shogaols, and zingerone (Brahe et al., 2016). In recent years, ginger has attracted increasing attention due to its pharmacological properties, such as its anti-inflammatory, anticarcinogenic, and antioxidant activities (Ezzat et al., 2018;Lee et al., 2008Lee et al., , 2015, especially for the treatment of diseases related to inflammation, oxidative stress, and cardiovascular diseases (Gunathilake & Rupasinghe, 2015;Peng et al., 2012;HASANAIN et al., 2016;Singh et al., 2008;Tohma et al., 2017). In China, Ginger is a commonly used medicinal and food dual-use traditional Chinese medicine, it has a long history of medicinal use and significant clinical efficacy, and it is first published in Shennong Bencao Jing and has a history of more than 2,000 years (Fang et al., 2017). The main function of ginger is to treat colds, dispel cold, warm the stomach, and stop vomiting. The results of pharmacological studies have confirmed that ginger has the effects of vomiting, strengthening the heart, and anti-inflammatory. It has a good therapeutic effect on cold and coughing, and has a certain preventive effect on the occurrence of cardiovascular, cerebrovascular diseases, and tumors. Generally, dried ginger is obtained by drying fresh ginger at low temperature.
Due to the heating and drying process, dried ginger and fresh ginger have some changes in the content and composition of chemical components. The efficacy of dried ginger is mainly warming blood and promoting blood circulation, warming the lungs, relieving cough and vomiting, and treating abdominal pain, etc. Pharmacological research shows that DG has effects such as improving cardiovascular function, protecting liver and choleretic anti-ulcer, anti-inflammatory, anti-tumor and anti-pathogen, etc. Moreover, the studies have shown that ginger can improve hemorheology events, inhibit platelet aggregation, prevent thrombosis, and play an anticoagulant effect (He et al., 2012;Jia et al., 2014). However, the understanding of its treatment of BSS is limited, and this is a problem worthy of our attention.
Metabolomic is a scientific technology for quantitative measurement of the time-related multiparametric metabolic response of multicellular systems to pathophysiological stimuli or genetic modification (Han et al., 2016), which provides a unique chemical fingerprint of a specific organism and reveals the essence of the syndrome and the therapeutic effect of Chinese medicine; it involves "holistic-dynamic-comprehensive analysis" (You et al., 2009). High-throughput metabolomics analysis has been used to reveal metabolic profiles.
Owing to the massive amount of accurate chemical data, high-speed data acquisition, and high resolution, UPLC-Q-TOF/MS-based metabolomics has been widely used in dynamic analyses of biochemical changes during drug intervention, which is useful for elucidating intervention mechanisms (Chen et al., 2017;Okada & Morihito, 2012).
Combined with multivariate data analysis, the metabolic profiles of each intervention group or NG can be visually displayed, and endogenous metabolites with significant differences between groups can be identified as biomarkers (Huang et al., 2020).
In this study, we established a rat model of BSS and administered different doses of dried ginger extract to the model rats. After the treatment period expired, the sample of the animal's urine, blood, and aortic vessels was collected. Pharmacodynamic indicators such as blood viscosity, aortic vascular pathology, ESR, PCV, deviation index (DI), and red blood cell accumulation index (EAI) are tested.
In addition, UPLC-Q-TOF/MS combined with multivariate statistical analysis of serum and urine was used to conduct multi-dimensional analysis on the metabolic spectrum and to explore the changes of its metabolites. We hope that this study can provide a new explanation for the mechanism of dried ginger in treating BSS.
| Extraction and content identification of DG
In brief, 1.0 kg of DG was decocted for 40 min with 10 times distilled water repeatedly for two times. The decoctions were collected, mixed, filtered, concentrated under reduced pressure, and dried by vacuum at 75°C. The w/w yields of DG were 11.74%. The DG extract was re-dissolved in distilled water to a final concentration of 2 g/ ml (equivalent to the dry weight of crude drugs) before being used and further analyzed with a Waters Acquity H-Class UPLC equipped with an Acquity BEH C 18 column (100 mm × 2.1 mm with the particle size of 1.7 μm), which consisted of a photodiode array detector, and the mobile phase was comprised of acetonitrile (A) and water (B).
The gradient mode was as follows: initial 3% A linear gradient to 85%
| Animals and treatments
Male SD rats weighing 200-220 g were purchased from the Animal Center of Anhui Medical University. They were kept in plastic cages at 25 ± 2°C with free access to pellet food and water and on a 12 hr light/dark cycles. Animal welfare and experimental procedures were carried out following the guidelines for the care and use of laboratory animals (National Research Council of USA, 996). The experimental protocol was approved by the Committee on the Ethics of Animal Experiments of Anhui University of Chinese Medicine. All surgical procedures were performed under anesthesia with pentobarbital (50 mg/kg body weight), and all efforts were made to minimize suffering.
| Experimental model and drug administration
A total of 60 SD rats were randomly divided into five groups equally, including normal group (NG), model group (MG), high dose of dried ginger group (GJH), middle dose of dried ginger group (GJM), and low dose of dried ginger group (GJL), (2.10, 1.05, 0.53 g/kg, respectively).
TCM intervention groups were orally administered different doses of DG extract, respectively, and the control group was orally administered an equivalent volume of distilled water. BSS was induced by placing the rats of the model and TCM intervention groups in ice water (0-2°C) for 5 min daily for 14 consecutive days. After that, except for the NG group, other groups were injected with 0.1% adrenaline hydrochloride twice, 0.8 ml kg −1 each time, with an interval of 4 hr. After the first injection, rats were immersed in ice water (0-2°C) for swimming for 5 min. Rats fasted overnight, and the administration of DG extract was continued after performing the model. Blood samples were collected on the following day at 40 min after the last administration.
| Sample collection
Urine samples were taken from six rats in each group between 18-24 hr after the second injection of epinephrine, and the rats were sodium pentobarbital anesthesia (2 ml/kg) at 24 hr after the last injection of Adr, stored at −80°C before analysis. Serum was isolated by centrifugation at 3,500 rpm for 10 min at 4°C and then frozen −80°C before metabolomics detection. Then, the other rats were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg body weight) 1 hr after the administration on the second day. The blood collection was carried out by carotid artery intubation, and the anticoagulation was carried out at the ratio of 1:9 with 3.8% sodium citrate. The whole blood viscosity, plasma viscosity, and clotting time were measured using a fully automatic hemorheometer. The blood samples of the remaining six rats in each group were drawn from the abdominal aortic to determine hemorheological variables. Blood was collected into plastic tubes with 3.8% sodium citrate for plasma anticoagulation and detected for whole blood viscosity (WBV), erythrocyte sedimentation rate blood (ESR), and packed cell volume (PCV).
Then, plasma was separated from blood by centrifugation at 3,500 g for 10 min and detected for plasma viscosity (PV) and plasma anticoagulation. All experiments were completed within 4 hr after blood collection.
| Viscosity determination
Six samples of 800 μl whole blood were taken from each group was used to determine the viscosity with a one-plate viscometer (Model LG-R-80B, Steellex Co., China) at different shear rates maintained at 37°C. WBV was measured with shear rates varying from 1 to 200/S. PV was measured at the high shear rate (200/S) and low shear rate (50/S). ESR and PCV measurements are a total of 1,000 μl blood that was put into an upright Westergren tube. The rate of red blood cells falling to the bottom of the tube (mm per hour) was observed and reported. The volume of packed red blood cells was immediately measured in the tube after centrifugation (3,000 g for 30 min). Thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen content (FIB) were examined with commercial kits following the manufacturer's instructions by a coagulometer (Model LG-PABER-I, Steellex Co., China). TT was determined by incubating 50 μl plasma solutions for 3 min at 37 °C, followed by the addition of 100 μl thrombin agent. PT was determined by incubating 50 μl plasma solutions for 3 min at 37°C, followed by the addition of 100 μl thromboplastin agents. APTT was determined by incubating 50 μl plasma with 50 μl APTT-activating agent for 3 min at 37°C, followed by addition of 50 μl CaCl 2 . FIB was determined by incubating 10 μl plasma with 90 μl imidazole buffers for 3 min at 37°C, followed by addition of 50°C FIB agent. The anticoagulation activity was assessed by assaying the prolongation of the plasma clotting time of TT, APTT, increase INR of PT, and reduction of FIB content (Sysmex CA7000, Japan). Besides, a random sample is split into six parts and processed in the same way. These six samples were continuously analyzed injected to validate the repeatability of the sample preparation method.
| UPLC-Q-TOF/MS conditions
Perform serum or urine metabolic profiling on UPLC-Q-TOF/MS system coupled to a Waters Q-Tof Premier Mass Spectrometer. Perform urine and serum chromatography on a Waters Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) with the temperature of the column set at 30°C. The flow rate was 0.3 ml/min-1, and the mobile phase was ultrapure water with 0.1% formic acid (A) and acetonitrile (B). The gradient elution procedure is as follows: 0-1 min, 1% → 10% B; 1-2 min, 10% → 30% B; 2-4 min, 30% → 75% B; 4-7 min, 75% → 75% B; 7-9 min, 75% → 100% B; 9-11.5 min, 100% → 100% B; 11.5-12 min, 100% → 1% B; 12-13.5 min, 1% → 1% B. Sample analysis time was 13.5 min, and the sample injection volume was 2 μl. The autosampler maintained at 4°C. The ESI source has two working modes: positive and negative patterns. The quality test parameters are set as follows: The N 2 flow rate is set to 650 L/h, and the positive and negative ion mode is 600 L/h, respectively. The gas temperature was 350°C. The source temperature was set to 110°C, with a cone gas flow of 100 L/hr. The capillary voltage was set at 1.5 kV in positive ion mode, and 1.8 kV in negative ion mode and the sample cone voltage was set at 100 V. All sample detections were acquired by using the lock-spray to ensure accuracy and reproducibility. A lock-mass at a concentration of 200 pg/ml was employed via a lock-spray interface. The MS/MS analyses of the ions were performed at different collision energy parameters that ranged from 5 and 50 eV for plasma samples and from 10 and 50 eV for the urine samples. The ESI interface was used, and the profile data were collected in full scan mode from m/z 50-1,000. Leucine-enkephalin was used as the lock-mass reference compound (m/z 556.2771 in positive mode, m/z 554.2615 in negative mode) and the flow rate was 20 μl/min.
| Data processing and analysis
The mass data acquired were imported to Markerlynx XS (Waters Corporation, MA, USA) within the Masslynx software for peak detection and alignment. The original data were processed using the following parameters: The retention time range was 0-13 min, the mass range was 50-1,000 Da, the retention time tolerance was 0.01 min, and the mass tolerance was 0.1 Da. Multivariate statistical analysis in the form of PLS-DA and OPLS-DA was performed using Pareto scale data. Extract potential biomarkers from S-maps were constructed by OPLS-DA analysis, and VIP is also used to select potential biomarkers. Variable importance in projection (VIP) score > 1 and t test p-value < .05 were prerequisite conditions for biomarkers.
PCA, OPLS-DA, clustering heatmap analysis, correlation analysis, relative intensity analysis, and pathway analysis with MetaboAnalyst 4.0 (http://www.metab oanal yst.ca/). Other statistical analyses were performed using SPSS 22.0, and the experimental data were expressed as the mean ± SD. Comparisons between groups were performed using one-way analysis of variance (ANOVA). Bilateral P values less than 0.05 were considered statistically significant.
Through the identification of biomarkers and the construction of metabolic pathways, research was conducted on essential ions found from statistical analysis to determine whether they provided potential biomarkers. These ions were tentatively identified based on their m/z value and mass spectrum using in-house data.
More specifically, the following web-based search engines were also used to provide potential identities for these ions: Human
| Content determination results of DG
The components were identified and quantified by comparison of retention time and calculation of peak areas from the chromatograms with those of known standards. The contents of 6-gingerol, 8-gingerol, 6-shogaol and 10-gingerol in DG extract were 6.95, 0.99, 0.42 and 1.32mg/g, respectively (Figure 1).
| Pathological observation of vascular
The microstructures of the abdominal aorta in rats were observed.
Vascular obstruction and a small amount of microthrombosis were observed in the MG. Some endothelial cells fell off from the vascular wall, endothelial cells swelled, and intima thickened. Also, inflammatory cell infiltration was observed. The above pathological symptoms were alleviated in the administration group, especially in the GJH (C). The results are shown in Figure S1. Histological results indicate that the vascular function of the dried ginger treatment group is protected. Concerns over the side effects and other adverse effects resulting from synthetic compounds in the treatment of BSS have been factors leading to medicinal plants as alternative choices. In view of the ability of DG extract can relax blood vessels by releasing nitric oxide and prostacyclin, activating cGMP-KATP channels, muscarinic receptor stimulation, and transmembrane calcium channels or Ca 2+ release from intracellular storage, it is a treatment for BSS Good drug candidate (Razali et al., 2020).
| Effect of DG on blood viscosity
The impact of WBV is shown in Figure 2 and Table S1. In the MG of BSS, WBV increased significantly at all shear rates. After administration, the WBV of each group was significantly reduced at high shear rates (p < .01 or .05). And the table also shows the effects of DG on plasma viscosity. The model rats had a significantly higher plasma viscosity than the controls. The plasma viscosity in the GJH, GJM, GJL was significantly decreased compared to the MG (p < .01 or .05).
| Effect of DG on ESR, PCV, deviation index (DI), and erythrocyte accumulation index (EAI)
The results of ESR, PCV, EAI, and DI for each group are shown in Table S2. All four indexes were significantly higher in the MG than in the NG. GJH and GJM reduced ESR and PCV (p < .05). All DG dose groups decreased DI and EAI (p < .05 or p < .01). It can be seen from the above that DG extract can improve blood rheology indicators.
| Effect of DG on plasma coagulation parameters
The impacts of DG on blood coagulation were evaluated by assays of APTT, PT, TT, and FIB in the plasma. PT was decreased, FIB was increased; APTT and TT were significantly shortened in the model rats compared with the NG levels, as showed in Table S3. After administration, compared with the MG, the PT of GJH and GJM was significantly increased, and the FIB was significantly decreased (p < .05). In terms of TT and APTT, the DG group was significantly longer (p < .05). The results show that the DG extract has an effect on the coagulation system of blood stasis syndrome.
| Metabolic profiling analysis
To obtain the maximum possible information for each metabolite, the experiments were performed in both positive and negative F I G U R E 1 Content determination of 6-gingerol, 8-gingerol, and 10-gingerol in the DG extract ionization modes and analyzed serum and urine samples under the same chromatographic conditions. The typical total ion chromatograms (TICs) of the serum and urine samples from the NG, MG, and GJH collected in the experiment are presented in Figure S2. To further analyze changes between complex sets of data, the multivariate data analysis techniques, including PCA-X, PLS-DA, and OPLS-DA, were used to analyze the data.
PCA analysis was used to assess the difference in metabolite profiles between serum and urine samples of NG and MG. The apparent separation between them was obtained in the PCA scores plot ( Figure 3a,b), which indicated that the two groups had utterly different metabolic profiling. Then, the PLS-DA method was used to systematically evaluate the metabolomics of BSS rats (permutation number: 200). In PLS-DA, the NG was more distinct from the MG (Figure 3c,d).
The PLS-DA model parameters were as follows: R 2 = 0.846 and Q 2 = −0.0669 in serum, and R 2 = 0.831 and Q 2 = −0.00977 in urine, which showed an excellent predictive power (Figure 3e,f). To screen differential metabolites and maximize the discriminatory ability of serum and urine metabolites between the groups, orthogonal partial least squares discriminant analysis (OPLS-DA) was used. As showed in the score plot (Figure 3g,h), the serum and urine samples in the MG were significantly different from those in the NG. The S-plots ( Figure 3i,j) showed differential metabolites between the two groups, and VIP was obtained based on OPLS-DA with a threshold than 1 would be viewed as potential biomarkers. Combined with the results of the S-plot and VIP-value plot together, the UPLC-Q-TOF/MS analysis platform provided the retention time, precise molecular mass, and MS/MS data for the structural identification of biomarkers. The same procedures were utilized to analyze the plasma samples derived from the NG, GJL, GJM, and GJH.
Besides, we investigated the differences in metabolic profiles between the MG and the GJL, GJM, and GJH, using OPLS-DA analysis. Score 3D plots ( Figure S3a,b) from OPLS-DA were used to maximize the discrimination of metabolite differences among the five groups. The figure shows that the metabolites of serum and urine in GJL, GJM, and GJH gradually approach the NG. Meanwhile, the GJH was the closest to the NG, the GJL was the closest to the MG, and the GJM was between the GJH and the GJL. Therefore, the intervention of DG on endogenous metabolites in rats with BSS shows a significant dose-effect relationship.
| Biomarker identification
To select potential biomarkers related to BSS, the first principal com-
| Metabolic pathway analysis
Possible ways to further explore the effects of BSS, using online Glycolysis is a common way for all organisms to extract energy from glucose, and gluconeogenesis provides a source for new glucose molecules. Glucose is produced from small carbohydrate substrates such as pyruvate, lactic acid, glycerin, and glucose amino acids, and to synthesize glucose from simple starting materials to meet the needs of various tissues (Ramirez et al., 2014;Tang et al., 2018). And in our study, when BSS occurred, the model group showed significant upregulation of pyruvate, suggesting that glucose metabolism disorders may occur, and glucose metabolism disorders indicate there appeared abnormal coagulation mechanism. After DG intervention, the pyruvate metabolism of the DG group was different from that in the model group, which indicated that the effect of DG on rats with BSS may involve the regulation of glucose metabolism. dysfunction. This effect is mainly caused by the PI3K/Akt signaling pathway after PI3K activates Akt. When PI3K is activated and Akt is inhibited, signal transduction cannot continue, and the above effect disappears (Siragusa et al., 2010). These studies indicated the crucial role of inositol phosphate metabolism in angiogenesis.
In our study, decreased myoinositol and pathway analysis suggested disruption of inositol phosphate metabolism was a characteristic of BSS, and DG extract was normalized myoinositol levels.
However, the specific mechanisms of these effects have not been characterized.
Pyruvate can stimulate the transcription of fibroblast growth factor receptors and vascular endothelial growth factor m RNAs and promote the aggregation of new blood vessels in tissues. Studies have shown that pyruvate can reduce intestinal ischemia-reperfusion injury (IRI) and effectively protect internal organs by shortening hypoxemia time, removing oxygen free radicals, inhibiting inflammatory response, increasing pH value, and increasing energy production (Zhang et al., 2020). In this study, compared with the NG, the metabolic pathway of pyruvate in the MG was destroyed, and pyruvate was abnormally increased, indicating that pyruvate level
| Correlation analysis between biomarkers and pharmacology Indicators
A correlation map of rat serum and urine metabolites of biomarkers and pharmacological indicators of BSS was conducted based on Pearson's correlation coefficients. The correlation heatmap in Figure 5 shows that the metabolites of Sm1 (serum, Pyruvate),
| Correlation analysis of biomarkers in each group
The heat map ( Figure 6) Due to the different contents of its components, each group had a different impact on promoting blood circulation and removing blood stasis. It is dose-dependent. Gingerol has been reported to inhibit inflammatory factors, angiotensin II activity, platelet cyclooxygenase activity, thromboxane synthesis, and advanced glycation end products, promote myocardial sarcoplasmic reticulum Ca 2+ ATPase activity, regulate the expression of related enzymes and proteins in the process of blood lipid metabolism, and exert cardiovascular pharmacological effects, such as cardiotonic, antiplatelet, hypolipidemic, anti-atherosclerosis (Ying-Zhi et al., 2017). This study can provide a scientific basis for further understanding of the mechanism of activating blood circulation and removing blood stasis by DG.
| CON CLUS ION
In this study, we found that DG extract can improve the pharmacodynamic indicators of blood stasis model rats, such as WBV, ESR, PCV. UPLC-Q-TOF/MS metabolomics and multivariate statistical analysis were used to compare the efficacy of different doses of DG extract on BSS. Through the research of metabolites in rats with BSS, it was found that 22 metabolites in rats with BSS had significant metabolic differences, which may be potential biomarkers or therapeutic targets in the development of BSS. The effect of DG on BSS may be related to the regulation of glycolysis/ gluconeogenesis, phosphatidylinositol signaling system, and pyruvate metabolism. In addition, we found that DG has a dose-dependent increase or decrease in the metabolism of BSS; these findings reveal the possible mechanism of action of DG in the treatment of BSS. Therefore, this study also provides a basis for exploring DG as an alternative drug for treating blood stasis syndrome; however, the specific mechanism of pharmacodynamics is not fully clear, and future studies are needed to investigate the potential roles of DG in the regulation of the selected endogenous metabolites associated with BSS.
CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.
AUTH O R CO NTR I B UTI O N S
Min Su carried out the main experiments and drafted this manuscript. Gang Cao and Daniel Raftery participated in metabonomics research and data analysis. Xiaoli Wang completed the determination F I G U R E 6 Heat map analysis of different groups of metabolites (with the deepening of red, the expression level of endogenous substances gradually increased, and with the deepening of blue, the expression level of endogenous substances gradually decreased) and analysis of UPLC. Yan Hong designed part of the efficacy experiment and revised the manuscript. Yanquan Han directed the experimental research and manuscript writing. All authors read and approved the final manuscript.
E TH I C A L A PPROVA L
The experimental protocol was approved by the Committee on the Ethics of Animal Experiments of Anhui University of Chinese Medicine (Anhui, China; permit number: 2019AH-038-04).
DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article. | v3-fos-license |
2019-04-09T13:01:44.766Z | 2006-01-02T00:00:00.000 | 103993160 | {
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} | pes2o/s2orc | Fiber Optic Detection of Ammonia Gas
Rapid detection and location of ammonia gas leaks poses a serious problem at all facilities utilising almost pure ammonia gas, such as large-scale refrigerating systems (breweries, dairies, abattoirs, logistic centres, ice-rinks, etc.), chemical plants producing ammonia by the Haber-Bosh reaction, and fertilizer plants. Every year, several accidents caused by ammonia are reported, resulting in severe damage to health and general distress. The concentration limit of human ammonia perception is about 50 ppm, but even lower concentrations are harmful for the human respiratory system. The long-term allowed concentration is defined as 20 ppm. Higher concentrations (500–1000 ppm) lead to a serious attack on the respiratory system. The lethal concentration limit is estimated as 5000–10000 ppm [1]. Such a level can be readily reached in the event of a highly concentrated ammonia gas leak. The standard architecture of an ammonia detection system used at present is based on a network of individual conductometric sensing heads equipped with a metal-oxide film [1]. Clearly, both the cost and the electric power consumption of such a sensing network rise rapidly with the number of sensors installed. Low gas selectivity and insufficient long-term stability of the sensing elements are also serious problems [1]. Numerous sensing schemes and optical systems have been tested to overcome these drawbacks (e.g. [1, 3–8]). One approach employs a suitably sensitized optical fibre. The two most extensively tested principles of this type are based on absorptionand fluorescence-based intrinsic sensing fibres. Selected reagent is embedded in the fibre cladding; ammonia gas then induces spectral variations of the fibre optical absorbance or fluorescence through its chemical reaction with the reagent. Short sections of such a fibre can be employed in fabricating the individual sensing heads, whereas various modifications of the optical reflectometry method (optical time domain reflectometry, OTDR, optical frequency domain reflectometry, OFDR, optical low coherence reflectometry, OLCR, optical time-of-flight chemical detection, OTOF-CD) provide a basis for constructing distributed sensing systems [9–11]. The crucial part of any intrinsic fibre optic sensor design is the selection of a reaction mechanism transforming an analyte exposure event into variations of the optical properties of the fibre. For instance, a pH-sensitive dye can be embedded into the fibre cladding. When exposed to alkaline ammonia gas, the optical absorption band of the dye is shifted; the change in cladding absorption modifies the light spectrum (guided in the fibre core as individual waveguide modes) through evanescent electromagnetic field components. Unfortunately, such utilization of an acid base reaction suffers from its strong dependence on the humidity level (the presence of hydronium ions is necessary for the creation of NH4 ions) and the obvious low sensing selectivity. The latter drawback can be partly compensated by using a proper gas selective membrane [1]. The ammonia detection approach employed here uses the ligand exchange reaction proposed in [12]. It is in principle capable reducing the humidity dependence and enhancing the selectivity in comparison with the acid base reaction (see the Experimental section for more details). In the research stage described here, the following objectives were adopted: (i) to choose reagent(s) suitable for further studies on optical fibres by evaluating the optical absorption spectra of two suitable organic dyes and their metallic complexes in solution, (ii) to prepare sensing fibre samples by diffusion of the reagent into a plastic clad silica (PCS) fibre cladding using the frequently applied procedure elaborated by our research group in frame of the CEC Copernicus programme [13, 9, 14], (iii) to test the concentration and temporal response to ammonia gas exposure by VIS-NIR absorption spectroscopy measurements on short fibre sections, and (iv) to pre-evaluate the qualitative features of an OTDR signal by measurements on a longer fibre, only partially sensitized with the selected reagent. Preparation, optimization, and quantitative characterization of a real distributed fibre optic ammonia sensor will be carried out in the next step, and the results will be published in a forthcoming paper.
Introduction
Rapid detection and location of ammonia gas leaks poses a serious problem at all facilities utilising almost pure ammonia gas, such as large-scale refrigerating systems (breweries, dairies, abattoirs, logistic centres, ice-rinks, etc.), chemical plants producing ammonia by the Haber-Bosh reaction, and fertilizer plants. Every year, several accidents caused by ammonia are reported, resulting in severe damage to health and general distress. The concentration limit of human ammonia perception is about 50 ppm, but even lower concentrations are harmful for the human respiratory system. The long-term allowed concentration is defined as 20 ppm. Higher concentrations (500-1000 ppm) lead to a serious attack on the respiratory system. The lethal concentration limit is estimated as 5000-10000 ppm [1]. Such a level can be readily reached in the event of a highly concentrated ammonia gas leak.
The standard architecture of an ammonia detection system used at present is based on a network of individual conductometric sensing heads equipped with a metal-oxide film [1]. Clearly, both the cost and the electric power consumption of such a sensing network rise rapidly with the number of sensors installed. Low gas selectivity and insufficient long-term stability of the sensing elements are also serious problems [1].
Numerous sensing schemes and optical systems have been tested to overcome these drawbacks (e.g. [1,[3][4][5][6][7][8]). One approach employs a suitably sensitized optical fibre. The two most extensively tested principles of this type are based on absorption-and fluorescence-based intrinsic sensing fibres. Selected reagent is embedded in the fibre cladding; ammonia gas then induces spectral variations of the fibre optical absorbance or fluorescence through its chemical reaction with the reagent. Short sections of such a fibre can be employed in fabricating the individual sensing heads, whereas various modifications of the optical reflectometry method (optical time domain reflectometry, OTDR, optical frequency domain reflectometry, OFDR, optical low coherence reflectometry, OLCR, optical time-of-flight chemical detection, OTOF-CD) provide a basis for constructing distributed sensing systems [9][10][11].
The crucial part of any intrinsic fibre optic sensor design is the selection of a reaction mechanism transforming an analyte exposure event into variations of the optical properties of the fibre. For instance, a pH-sensitive dye can be embedded into the fibre cladding. When exposed to alkaline ammonia gas, the optical absorption band of the dye is shifted; the change in cladding absorption modifies the light spectrum (guided in the fibre core as individual waveguide modes) through evanescent electromagnetic field components. Unfortunately, such utilization of an acid base reaction suffers from its strong dependence on the humidity level (the presence of hydronium ions is necessary for the creation of NH 4 + ions) and the obvious low sensing selectivity. The latter drawback can be partly compensated by using a proper gas selective membrane [1]. The ammonia detection approach employed here uses the ligand exchange reaction proposed in [12]. It is in principle capable reducing the humidity dependence and enhancing the selectivity in comparison with the acid base reaction (see the Experimental section for more details). In the research stage described here, the following objectives were adopted: (i) to choose reagent(s) suitable for further studies on optical fibres by evaluating the optical absorption spectra of two suitable organic dyes and their metallic complexes in solution, (ii) to prepare sensing fibre samples by diffusion of the reagent into a plastic clad silica (PCS) fibre cladding using the frequently applied procedure elaborated by our research group in frame of the CEC Copernicus programme [13,9,14], (iii) to test the concentration and temporal response to ammonia gas exposure by VIS-NIR absorption spectroscopy measurements on short fibre sections, and (iv) to pre-evaluate the qualitative features of an OTDR signal by measurements on a longer fibre, only partially sensitized with the selected reagent. Preparation, optimization, and quantitative characterization of a real distributed fibre optic ammonia sensor will be carried out in the next step, and the results will be published in a forthcoming paper.
Preparation of tested fibres
A custom-made PCS fibre (n c = 1.458, 200/260 mm core/cladding diameter, Dow Corning Optigard siloxane cladding, produced at IRE ASCR, Prague, CR) was utilized throughout the experiments. The fibre sections were washed in acetone for at least 12 h prior to further sensitization. The reagent was soaked into the fibre cladding from its ethanol/chloroform solution (1:1 wt.), washed in ethanol, and left to dry for 12 h in room conditions. Two types of fibres were prepared: short sections (12 cm) intended for VIS-NIR absorption spectroscopy measurements and a long fibre (120 m, sensitized within the interval 104-110 m) tested by OTDR. The whole preparation and subsequent characterization of the fibres was performed at room temperature (RT).
Ligand exchange reaction
The ammonia sensing fibres prepared in this work employ the ligand exchange reaction where L is an organic chromatic ligand, Me is a bivalent positive metal ion forming a complex ion with L (see also Table 1), A is a suitable counter-anion, and n is the integer number, which depends on the type of Me co-ordination. The superscripts indicate the degree of ionization of the particular fragment.
Experimental characterization
The VIS-NIR absorption spectra of the solutions and short fibre sections were measured using a setup containing a white light source (halogen lamp, 25 W), a cuvette holder with focusing optics (solution spectroscopy measurements) or a sealed testing chamber attached to a gas mixing system (spectroscopy measurements of fibre sections), an optical pigtail collecting light from the sample under test and transferring it into an Ocean Optics S1000 array spectrometer (200-1200 nm wavelength range, 1 nm resolution, single channel operation) controlled by a PC. In the case of fibre testing, the light beam was focused into the tested fibre through a microscopic objective and collected by an integration sphere (1 mm in diameter) at the end of the pigtail. The light source, the testing chamber, and the collecting pigtail were all placed on a common optical rail.
The OTDR setup consisted of a Photodyne 5500 XFA OTDR unit (laser diode wavelength l = 850 nm, pulse width 20 ns, average pulse power 30 mW, repeating frequency 3.1 kHz), HP 54615B digital oscilloscope (maximum sampling speed 1 GSa/s, signal bandwidth 500 MHz), a sealed testing chamber (4 dm 3 ) attached to the cylinder with 10000 ppm ammonia/nitrogen gas, and a PC controller. The OTDR unit launched laser pulses into the tested fibre, registered a back-scattered light intensity using an internal PIN Si diode, and logarithmically amplified the detected signal. The temporal signal course was then recorded by the digital oscilloscope and transferred to the PC. The final pulse width limited the spatial resolution along the fibre length to~4 m (cf Eq. 3). The OTDR curves showing a full temporal course were recorded with the oscilloscope set to collect data within the time range Dt = 2000 ns. The curve details were examined with Dt = 200 ns. A numeric data averaging procedure (128 single-shot spectra were accumulated) was employed to reduce the signal-to-noise ratio of the resulting OTDR curves.
Variable concentrations of ammonia gas in nitrogen were prepared by volumetrically dosing the concentrated ammonia/nitrogen mixture (10000 ppm) into the gas circuit (including the measuring chamber) filled with nitrogen. A membrane circulation pump included in the circuit provided fast homogenization of the prepared gas mixture.
Basic theory
The VIS-NIR absorption spectra were obtained by a standard absorption spectroscopy method [15].
The OTDR technique relies on interrogation of an attached optical fibre by short monochromatic laser pulses, and a subsequent temporal analysis of the light intensity P(x) returning to the OTDR unit from the fibre distance x, x = D t c/n c (c -light velocity in a vacuum, n c -refractive index of the fibre core, Dt -time interval between the pulse onset and the measuring time point). Two types of processes have to be considered as the dominant signal sources in the case of an intrinsic absorption-based sensing fibre: Rayleigh scattering along the fibre length, and Fresnel reflection from the fibre splices and the free end [16].
The recorded OTDR curves (log(P(x) versus x) provided information about the immediate optical properties of the tested fibre along its length. The total Fresnel reflection intensity P F (x) is proportional to the forward pulse power P + (x) at position x, multiplied by the square of the corresponding reflection coefficients R i averaged over all reflected light modes M guided within the multimode fibre of the core diameter D and numeric aperture NA [15,17] The integration runs over pulse spatial width w c n c = t ; t is the pulse duration. The intensity of the Rayleigh back-scattered light can be expressed as [10,16,18] where C is a constant characterizing the coupling efficiency of the optics, I L is the laser intensity injected into the fibre, S B and S R are respectively a back-scattering factor and the Rayleigh scattering coefficient at position x. The latter relates on a microscopic level to the local polarizability a(x) of the fibre as [15] S The total fibre attenuation a(x) caused by scattering and absorption effects is obtained as [9] a x a l l where ¢ a l ( ) is a local attenuation coefficient and the integration runs over the fibre length 0-x.
For example, in the case of a uniform fibre (S R (x), S B (x), NA(x), and a(x) independent of x), it follows from (3) that the Rayleigh contribution to the OTDR logarithmic output, log(P R (x)), should be a linear function of x. Contingent variations of ¢ a x ( ) and/or S R (x), S B (x) and NA(x) along the fibre length lead respectively to the corresponding slope changes and/or local extremes appearing on the resulting OTDR curve.
Results and discussion
As expected, dyes L1 and L2 showed strong absorption bands in the spectral range 500 nm-800 nm with maxima at 626 nm and 690 nm, respectively (Fig. 1). Creation of metallic complexes of the dyes resulted in bathochromic shifts (Dl) of the absorption bands due to widening of the corresponding electronic resonant systems. The complex ions containing ligand L1 showed remarkably larger shifts than those containing ligand L2; the greatest Dl-value was observed for complex dye [L1-Cu-L1] SO 4 (Table 1, Fig. 2). The impact of the counter-anion (SO 4 2and (Cl -) 2 were tested) on the Dl value was small. The [L1-Cu-L1] SO 4 complex dye (hereafter referred to as reagent R) was selected for the subsequent sensing tests performed on the sensitized fibre samples. Studies employing other interesting complexes, especially those containing Co 2+ , are now in progress and will be published in a separate paper. As expected (cf Eq. 1), the VIS-NIR absorption spectra showed that ion [L1-Cu-L1] 2+ decomposes in contact with ammonia liquor and the ligand absorption spectrum is restored (Fig. 2). A similar reaction was also observed on short fibre sections sensitized with reagent R and exposed to 10000 ppm ammonia gas (Fig. 3). The forward reaction time t 90 (the time necessary to reach 90 % of the total light intensity drop) was~30 sec, much faster than the fibre recovery time; full spectral relaxation was obtained after~5 minutes of nitrogen blow.
Ammonia, as a stronger electron donor than the organic ligand, substitutes the latter in the metallic complex, giving rise to a new [(NH 3 ) n Me] 2+ complex ion (cf Eq. 1). The decomposition of the original chromatic complex leads to the observed colour changes. The equilibrium constant of reaction (1) depends not only on the ammonia concentration, but also on the actual degree of dissociation of the individual ionic species. The degree of dissociation varies with the type of ions (metallic ions as well as anions) and with the permittivity of the solvating medium. If a polymer matrix acts as the solvent, the possible diffusion of water into the polymer bulk can also modify the actual degree of dissociation, thus contributing to a remarkable dependence of the sensor signal on the ambient humidity. Such behaviour was indeed experimentally observed [12,14]. The presence of hydroxyl and hydronium ions in the polymer matrix can also lead to an alternative chemical process -the creation of an ammonium salt competing with the reaction (1), thus disturbing the sensor function. Careful optimization of the cladding polymer and counter-anion type is therefore necessary to reduce undesired effects. We are currently researching in this direction, and our work will be presented in a forthcoming paper. For this study, only dry gas was used throughout the experiments; thereby reaction scheme (1) could be adopted.
Spectroscopic measurements also confirmed the crucial importance of the initial fibre wash for the long-term spectral stability of the sensitized fibres. The rapid decay of the optical absorption of the reagent R observed for an unwashed sample (Fig. 4) resulted very likely from a chemical decomposition of the reagent fractions due to a reaction with remains of a UV-initiator/catalyser in the siloxane cladding. The optical properties of fibres properly treated with acetone remained stable for several months of storage in laboratory conditions; the stability and reversibility in field conditions are currently being tested.
The optical absorption of a short sensing fibre sample integrated within the spectral interval (840-860) nm decayed with increasing ammonia gas concentration (Fig. 5); the corresponding concentration sensitivity descends with growing analyte concentration and approaches a saturated level at ammonia concentration~4000 ppm. The saturated state likely corresponds to a complete reagent decomposition (cf reaction (1) involving all reagent molecules embedded in the cladding). Thus, increasing the reagent concentration in the fibre cladding could potentially increase the ammonia sensitivity 44 Acta Polytechnica Vol. 46 No. 2/2006 Czech Technical University in Prague threshold, but it would also enhance the total fibre attenuation (undesirable for longer fibres), and likely decrease the resolution at low concentrations. This is because the diffusing analyte molecules will mostly react with the reagent molecules located in the outer shell of the cladding, which interacts only weakly with the evanescent field of the fibre core [17]. The cladding thickness and reagent concentration profile therefore also have to be carefully optimized. The low concentration resolution r within the interval 0-1000 ppm can be roughly estimated from the course in Fig. 5. Assessing the standard deviation (STD) value as s @ 0002 . and taking in account the signal change c. 0.092 we get for r @ @ 1000 2 0092 50 s .
ppm. As already mentioned, the practical reagent concentration which can be achieved in a sensing fibre cladding is generally limited. Therefore, the detection resolution can be further improved mainly by reducing the noise level. In our case, we estimate that the primary noise source was the poor light coupling between the tested fibre and the measuring system; the measuring system may be significantly improved if fixing fibre splices are used. Further improvement can also be achieved by applying numeric data accumulation and averaging procedures. The full OTDR curves recorded with the 120 m long fibre as fabricated, sensitized by reagent R within the range (104-110) m, and exposed to 10000 ppm ammonia gas within the same range were dominated by two Fresnel reflections (Fig. 6a). The pulse dispersion along the fibre length is clearly demonstrated by the broadening of the distant (second) reflection. The reflection at x~0 comes from the front end of an internal fibre within the Photodyne unit; the second main maximum corresponds to the reflection from the free end of the tested fibre. The tiny side-maximum of the first reflection (at x~8 m in Fig. 6a) is caused by the splice connecting the tested fibre to the measuring unit. The fibre sensitization resulted (i) in a slight increase in the intensity back-scattered from the sensitized region (Fig. 6b), followed (ii) by a steeper signal decay from the more distant fibre part combined with a well-resolved reduction in the second Fresnel reflection intensity (Fig. 6, curve 2) compared to the unsensitized state (Fig. 6, curve 1). The first effect may likely be ascribed to the increase in polarizability within the sensitized range enhancing the Rayleigh term S R (x) and reducing NA(x) (cf Eq. (3, 4)). The local drop of the numeric aperture elicits leakage of some part of the guided modes into the cladding, where their scattering and absorption level is much higher than in the fibre core. The second effect results from the enhanced fibre attenuation within the sensitized region (cf Eq. 2, 3, 5).
The character of the OTDR signal variation observed after exposure to concentrated ammonia gas (Fig. 6, curve 3) was opposite to the reaction following the sensitization procedure. The second Fresnel reflection grew, the back-scattered signal coming from the sensitized/exposed region decreased, and the slope of the signal that originated just after the exposed region rose. The behaviour is again in accord with the basic OTDR model represented by Eq. 2-5 and with reaction mechanism Eq. 1. Decomposition of the reagent complexes and the creation of much smaller ions [(NH 3 ) 4 Cu] 2+ led to a local reduction of parameters a(x) and ¢ a (x), causing the observed signal changes. The two intersections of curves 2 and 3 (Fig. 6b) conform very well with the boundaries of the exposed region.
Conclusions
The results show the principal feasibility of fabricating an ammonia sensing fibre using the selected reagent and fibre sensitization procedure employed here. The sensing parameters obtained with a short fibre sample (r » 50 ppm, t 90 » 30 s) are comparable with the figures required for detecting an extensive ammonia gas leakage. We anticipate that comparable sensing parameters would also be achieved with long sensing fibres; the concentration resolution can likely be further improved by using a better optical coupling of the tested fibre to the measuring unit, and numeric signal accumulation and averaging procedures.
The OTDR measurements were performed with the aim to demonstrate the principal feasibility of distributed ammonia gas detection using the proposed fibre design. Two fea- Curve 1 -before sensitization; curve 2 -after sensitization with reagent R; curve 3 -after exposure to 10000 ppm ammonia gas in nitrogen.
tures of the observed OTDR signal are important for the subsequent design of the prototype: (1) Variation of the Fresnel reflection coming from the free fibre end can be instrumental in detecting any ammonia leak along the fibre length. (2) The absorbing reagent embedded in the fibre cladding not only enhances the local fibre attenuation (the main factor restricting the maximum length of absorption--based sensing fibres), but also contributes to growth of the local back-scattered light intensity, thus slightly improving the signal-to-noise ratio of the resulting signal.
Our forthcoming research will focus on tests of alternative reagents (such as L1-complex with cobalt) followed by the fabrication of an optimized distributed fibre optic sensor. The static and dynamic characteristics and the long-term stability in field conditions of the sensor will be analysed with reference to the influence of ambient temperature and variations in relative humidity. | v3-fos-license |
2020-04-28T13:02:06.234Z | 2020-04-26T00:00:00.000 | 216556904 | {
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} | pes2o/s2orc | miR‐145 inhibits the proliferation and migration of vascular smooth muscle cells by regulating autophagy
Abstract miR‐145, the most abundant miRNA in the vascular smooth muscle cells (VSMCs), regulates VSMC function in intimal hyperplasia. It has been reported that autophagy participates in the regulation of proliferation and migration of VSMCs. However, the effect of miR‐145 on autophagy and related mechanism in the proliferation and migration of VSMCs remains unclear. Therefore, we aimed to determine the effect of miR‐145 on autophagy and the mechanism in VSMCs. Cell autophagy was determined by transmission electron microscope, mRFP‐GFP‐LC3 assay and Western blotting. A recombinant lentivirus containing miR‐145 was used to construct VSMCs with miR‐145 overexpression. We found that miR‐145 expression was decreased, and autophagy was increased in the carotid arteries of C57BL/6J mice with intimal hyperplasia and TGF‐β1‐stimulated VSMCs. Furthermore, miR‐145 overexpression inhibited cell autophagy, whereas miR‐145 inhibition promoted autophagy in TGF‐β1‐stimulated VSMCs. Meanwhile, miR‐145 inhibited the proliferation and migration of VSMCs. More importantly, our study showed that autophagy inhibition augmented the inhibitory effect of miR‐145 on the proliferation and migration of VSMCs. In addition, we found that the sirtuins are not direct targets of miR‐145 in the proliferation and migration of VSMCs. These results suggest that miR‐145 inhibits the proliferation and migration of VSMCs by suppressing the activation of autophagy.
| INTRODUC TI ON
microRNA-145 (miR-145) is a 22-nt, highly conserved miRNA. It is generally accepted that miR-145 has a strong inhibitory effect on the proliferation and migration of cancer cells and is a tumour suppressor. 1, 2 Cheng et al found that miR-145 is the most abundant miRNA in vascular walls, and miR-145 is selectively expressed in the vascular smooth muscle cells (VSMCs) of vascular walls. 3 Subsequent studies demonstrated that miR-145 participates in the regulation of VSMC function including the proliferation and migration in intimal hyperplasia. 4,5 Autophagy is an important biological process and plays a crucial role in cellular homeostasis in cardiovascular diseases. 6 Autophagy is generally recognized as an important mediator of VSMC function. 7 Several studies have indicated that the activation of autophagy contributes to the proliferation and migration of VSMCs. Li et al showed that sonic hedgehog induced cell autophagy and resulted in an increase in VSMC proliferation, which plays a key role in the pathogenesis of neointima formation. 8 Another study showed that platelet-derived growth factor (PDGF) induced autophagy and that inhibition of autophagy by 3-methyladenine (3-MA) reduced PDGF-induced proliferation and migration of VSMCs. 9 However, the effect of miR-145 on autophagy and the underlying mechanism in the proliferation and migration of VSMCs remains unclear.
miR-145 exerts biological functions, including the modulation of VSMC proliferation and migration, via its multiple target genes. It has been reported that Krüppel-like factor 5 (KLF5), TGFβ receptor II (TGFBR2) and CD40 were the direct targets of miR-145 in the proliferation and phenotypic modulation of VSMCs. 3,10,11 Sirtuins are a family of evolutionally conserved class III histone deacetylases.
The mammalian sirtuin family includes seven members (SIRT1-7). 12 Emerging evidence indicates that sirtuins are also the targets of miR-NAs in cardiovascular diseases. A recent report showed that SIRT1 was the target of miR-34a in the differentiation of SMCs from pluripotent stem cells. 13 Zhu et al reported that miR-195 augmented palmitate-induced apoptosis of cardiomyocytes by targeting SIRT1. 14 In addition, miR-497 inhibited cardiac hypertrophy by targeting SIRT4. 15 Therefore, we speculate that miR-145 is likely able to regulate the proliferation and migration of VSMCs by targeting sirtuins.
In this study, we first determined the change of miR-145 and autophagy in mice with intimal hyperplasia and VSMCs stimulated with TGF-β1. Then, we investigated the effect of miR-145 on autophagy and the related mechanism in the proliferation and migration of VSMCs.
| Animals and treatment
Male C57BL/6J mice, weighing 22-24 g, were provided by the Laboratory Animal Center of Xi'an Jiaotong University. Mice were anesthetized, and the carotid arteries were dissected after a midline cervical incision. For the ligation model, the carotid arteries just proximal to the bifurcation were ligated with silk sutures. 16
| VSMC culture and characterization
Male Sprague Dawley rats ranging from 150 to 180 g were from Laboratory Animal Center of Xi'an Jiaotong University. The primary VSMCs were isolated from the thoracic aorta of rats by the tissue explant method. 17 Briefly, the thoracic aorta was removed and longitudinally opened, and it was then washed with cold phosphatebuffered saline (PBS) under sterile conditions. The adventitia and intima were separated from the media, and the isolated media was cut into pieces and placed in DMEM supplemented with 20% foetal calf serum. The cells were characterized by immunofluorescence staining with α-actin antibody. Cells at passages 3-9 were used for experiments.
| Cell proliferation assay
VSMC proliferation was evaluated by cell counting and the CCK-8 assays. Cell counting was performed by the trypan blue exclusion method. 18 For the CCK-8 assay, VSMCs was added 10 μL of CCK-8 to each well of 96-well plates for 2 h, and the absorbance at 450 nm was measured using a microplate reader (Thermo Fisher, Waltham, MA, USA). 19
| Cell migration assay
Cell migration was assessed by the wound-healing assay. 20 VSMCs were cultured in 6-well plates at 90% confluence. The monolayer was scratched with a pipette tip. Images at time zero (t = 0 h) were photographed by a microscope (Nikon, Tokyo, Japan) to record the initial area of the wounds. The cells were then cultured for 24 h, and the widths of the wounds were also captured (t = 24 h). The wound-healing width was determined by the IPP software (Media Cybernetics, Bethesda, MD, USA). Wound diameter was measured at two time points (0 h and 24 h) to assess % wound healing at 24 h using the formula: (Width at 0 h -Width at 24 h)/ (Width at 0 h) X 100%
| Western blotting
The total protein of VSMCs was extracted with RIPA lysis buffer containing protease inhibitor cocktail. Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% nonfat dry milk, the membranes were incubated with primary antibodies overnight. The primary antibodies used
| Quantitative real-time PCR
Total RNA was extracted from cells using the RNAiso Plus reagent.
cDNA was synthesized using the PrimeScript RT Master Mix. cDNA was amplified using the SYBR Premix Ex Taq II. GAPDH was used as an endogenous control. Data were normalized to the GAPDH mRNA level.
miR-145 expression was determined by the mir-X™ miRNA First-Strand Synthesis and SYBR qRT-PCR kits according to the manufacturer's instructions. U6 was used as an endogenous control, and relative gene expression was quantitatively analysed by the comparative Ct method (2 −△△CT ). Data were normalized to the U6 mRNA level.
Twelve hours after adenovirus infection, the cells were treated with
TGF-β1 for 24 h. The results were visualized using super-resolution confocal microscope (Leica, Mannheim, BW, Germany).
| Transmission electron microscope
The cells were collected by trypsinization and centrifugation and then were fixed with 2.5% glutaraldehyde and 1% osmium tetroxide followed by dehydration in an increasing series of ethanol. The samples were embedded in Durcopan ACM for 6 h, and ultrathin sections were cut using a Leica Ultramicrotome EM UC6, The sections were then stained with uranyl acetate and lead citrate, and examined with a Tecnai G 2 12 transmission electron microscope (FEI Company, Holland).
| miR-145 inhibition
The miR-145 inhibitor was obtained from GenePharma Co., Ltd. in a CO 2 incubator prior to experimental use.
| Ingenuity pathway analysis
Ingenuity pathway analysis was performed by Shanghai Cloud Scientific Technology Co., Ltd. (Shanghai, China).
| Luciferase reporter assay
A luciferase reporter assay was performed to confirm the target genes of miR-145. Briefly, HEK-293T cells were transfected at 60% confluency in 24-well plates with wild-type or mutant 3'-UTR vectors and miR-145 or miR-145 control vectors. A co-transfected Renilla luciferase reporter vector was used as an internal control for the normalization of luciferase activity in each sample. Cells were analysed at 48 h after transfection. Firefly and Renilla luciferase activities were quantified in lysates using the luciferase reporter assay kit. The firefly luciferase enzyme activity was normalized to the Renilla luciferase enzyme activity.
| Statistical analysis
Data are presented as the mean ± SEM from three independent experiments. Statistical significance was determined using one-way analysis of variance. P < 0.05 was considered statistically significant.
| The changes of miR-145 and autophagy in mice with intimal hyperplasia
The left carotid arteries were ligated, and the right carotid arteries were used as the control in C57BL/6J mice. To observe the intimal hyperplasia, the carotid arteries were stained with H&E at 28 days after ligation. Our results showed that there was a significant intimal thickening in the ligated carotid arteries ( Figure 1A,B). Moreover, the protein expression of PCNA in the ligated carotid arteries was increased compared with the control carotid arteries (P < 0.01) ( Figure 1C). More importantly, miR-145 expression was decreased in the ligated carotid arteries ( Figure 1D). Our results also showed that the conversion of LC3 I to LC3 II and Beclin1 protein expression were increased in the ligated carotid arteries compared with that of the control carotid arteries ( Figure 1E,F). In addition, we found that the p62 protein expression was decreased in the ligated carotid arteries ( Figure 1G). These results suggest that miR-145 expression is decreased and autophagy is increased in the carotid arteries of mice with intimal hyperplasia.
| The effect of TGF-β1 on autophagy in VSMCs
To investigate the effect of TGF-β1 on autophagy, VSMCs were treated with TGF-β1 (5 ng/mL) for 24 h, and the conversion of LC3 I to LC3 II and Beclin1 expression were determined by Western blotting. We found that TGF-β1 increased the conversion of LC3 I to LC3 II and Beclin1 protein expression in VSMCs (Figure 2A,B).
Meanwhile, TGF-β1 decreased p62 protein expression of VSMCs ( Figure 2C). Then, we performed the mRFP-GFP-LC3 assay to observe the autophagic flux in VSMCs. When autophagy was induced by TGF-β1, the red puncta were accumulated in VSMCs compared with the control group (P < 0.01) ( Figure 2D,E). The result showed that TGF-β1 activated the autophagic flux in VSMCs. To further confirm these results, the autophagosome formation was assessed by transmission electron microscopy. We found that TGF-β1 increased the autophagosome formation in VSMCs compared with the control group ( Figure 2F,G). These data demonstrate that TGF-β1 induces VSMC autophagy by increasing the autophagic flux and the conversion and expression of LC3 and Beclin1, and autophagosome formation.
Given our findings that TGF-β1 promoted cell autophagy and de-
| TGF-β1 promotes the proliferation and migration of VSMCs
To confirm the effect of TGF-β1 on VSMC proliferation, the VSMCs were treated with TGF-β1 (5 ng/mL) for 24 h. Cell proliferation was evaluated by cell counting and the CCK-8 assays. The expression of PCNA protein was determined by Western blotting. As shown in Figure 4A,B, TGF-β1 increased both the cell number and cell viability compared with the control group (P < 0.01). In addition, the expression of PCNA protein was also increased ( Figure 4C). These results collectively show that TGF-β1 promotes the proliferation of VSMCs.
The effect of TGF-β1 on the migration of VSMCs was determined by the wound-healing assay. As shown in Figure 4D,E, TGF-β1 decreased the width of the scratched wound and increased the wound-healing width, compared with the control group (P < 0.01).
The result show that TGF-β1 promotes the migration of VSMCs.
| miR-145 inhibits the proliferation and migration of VSMCs
The above results showed that TGF-β1 decreased miR-145 expression and promoted the proliferation and migration of VSMCs. Next, we investigated whether miR-145 could regulate VSMC proliferation and migration. VSMCs infected with LV-miR-145 or LV-NC were then treated with TGF-β1 (5 ng/mL) for 24 h. The result of CCK-8 assay showed that miR-145 overexpression decreased the cell viability of VSMCs stimulated with TGF-β1 ( Figure 5A). Meanwhile, miR-145 overexpression inhibited the migration of VSMCs ( Figure 5B,C).
To further verify the effect of miR-145 on VSMC proliferation and migration, the miR-145 inhibitor was transfected into VSMCs.
The result showed that the miR-145 inhibitor promoted TGF-β1induced proliferation of VSMCs ( Figure 5D). Furthermore, the result of the wound-healing assay also showed that the miR-145 inhibitor further increased the wound-healing width in VSMCs stimulated with TGF-β1 (P < 0.05) ( Figure 5E,F).
| miR-145 inhibits the proliferation and migration of VSMCs through autophagy
To evaluate whether miR-145 regulates the proliferation and migration of VSMCs through autophagy, the VSMCs were pretreated F I G U R E 4 TGF-β1 promotes the proliferation and migration of VSMCs. The VSMCs were treated with TGF-β1 (5 ng/mL) for 24 h. A, B, Cell proliferation was evaluated by cell counting and the CCK-8 assays. C, The expression of PCNA protein was determined by Western blotting. D, E, The migration of VSMCs was determined by the wound-healing assay. The results are expressed as the mean ± SEM, n = 3. Statistical significance was determined using ANOVA by Student's t test. **P < 0.01 vs. the control group with the autophagy inhibitor 3-MA (5 mM) for 1 h prior to TGF-β1.
Compared with the miR-145 overexpression group, 3-MA further decreased cell viability and the expression level of PCNA ( Figure 6A,B).
Moreover, 3-MA decreased the wound-healing width in VSMCs stimulated with TGF-β1 ( Figure 6C). These results suggest that autophagy inhibition augmented the inhibitory effects of miR-145 on the proliferation and migration of VSMCs. In addition, we also found that the promotive effects of miR-145 inhibitor on the proliferation and migration of VSMCs were significantly attenuated by 3-MA ( Figure 6D-F). These data demonstrate that miR-145 regulates the proliferation and migration of VSMCs through autophagy.
| Sirtuins are not the direct targets of miR-145
To investigate the role of sirtuins in neointimal hyperplasia and VSMC function, the expression levels of SIRT1, SIRT3, SIRT5 and SIRT6 in carotid arteries of C57BL/6J mice were determined at 28 days after ligation. The results showed that the protein and mRNA levels of SIRT1, SIRT3, SIRT5 and SIRT6 in the ligated carotid arteries were markedly decreased compared with that of the control arteries ( Figure 7A-C). Then, we found that treatment of VSMCs with different concentrations of TGF-β1 decreased the protein levels of SIRT1, SIRT3, SIRT5 and SIRT6 ( Figure 7D,E). These findings suggest that SIRT1, SIRT3, SIRT5 and SIRT6 may be involved in the proliferation and migration of VSMCs.
To clarify the regulatory effect of miR-145 on sirtuins, the VSMCs with miR-145 overexpression were stimulated with TGF-β1 (5 ng/ mL). The results showed that miR-145 overexpression increased the expression levels of SIRT1, SIRT3, SIRT5 and SIRT6 in VSMCs, compared with the TGF-β1 group ( Figure 7F and G). Given the above results, we then proposed the hypothesis that miR-145 may regulate the proliferation and migration of VSMCs through sirtuins. First, ingenuity pathway analysis was performed to analyse the relationships between microRNAs and sirtuins in cardiovascular diseases. The results showed that SIRT3 and SIRT5 may be the targets of miR-145 ( Figure 8A). Then, the luciferase reporter assay was performed to further validate whether miR-145 interacts with SIRT5 directly. We found that there was no significant difference in the relative luciferase activity between cells cotransfected with miR-145 and with the control oligonucleotide ( Figure 8B). These results show that sirtuins are not the direct targets of miR-145.
| D ISCUSS I ON
miR-145 is considered as a potential biomarker and prognostic marker for progressing stages of cardiovascular diseases. 21,22 miR-145 is the F I G U R E 5 miR-145 inhibits the proliferation and migration of VSMCs. The VSMCs infected with LV-miR-145 or LV-NC were treated with TGF-β1 (5 ng/mL) for 24 h. A, Cell proliferation was evaluated by the CCK-8 assay. B, C, Cell migration was determined by the woundhealing assay. **P < 0.01 vs. the LV-NC group. ## P < 0.01 vs. the LV-NC + TGF-β1 group. The miR-145 inhibitor or miR-NC were transfected into VSMCs for 48 h, prior to treatment with TGF-β1 for an additional 24 h. D, Cell viability was determined by the CCK-8 assay. E, F, Cell migration was determined by the wound-healing assay. *P < 0.05 and **P < 0.01 vs. the miR-NC group. # P < 0.05 vs. the miR-NC + TGF-β1 group. The results are expressed as the mean ± SEM, n = 3. Statistical significance was determined using ANOVA by Tukey's post hoc test most abundant miRNA in VSMCs, and it controls vascular neointimal lesion formation. [3][4][5] In the current study, we first observed that miR-145 expression was decreased in the ligated carotid arteries compared with the control carotid arteries of mice. A previous study showed that miR-145 expression was down-regulated in the balloon-injured rat carotid arteries. 4 In addition, miR-145 overexpression significantly reduced the neointimal thickness in a rabbit model of vein graft disease. 23 We observed that TGF-β1 decreased miR-145 expression and promoted the proliferation and migration of VSMCs. It has been reported that miR-145 expression was decreased in PDGF-induced VSMC proliferation, and miR-145 overexpression markedly inhibited VSMC proliferation. 3 In the present study, we also found that miR-145 overexpression inhibited the proliferation and migration of VSMCs, while the miR-145 inhibitor caused the opposite effects on the proliferation and migration of VSMCs. These results suggest that miR-145 regulates the proliferation and migration of VSMCs in intimal hyperplasia.
Autophagy is an evolutionarily conserved mechanism and linked to several cellular pathways, impacts the survival and function of VSMCs. 7 Several studies have showed that the activation of autophagy contributes to the proliferation and migration of VSMCs. 8,9 The conversion of LC3, Beclin1 and p62 expression have been widely used to indicate the changes of autophagy. 24 We first found that autophagy was activated by increasing the conversion of LC3 I to LC3 II and Beclin1 expression as well as decreasing p62 expression in the ligated carotid arteries of mice. Li et al also found that the conversion of LC3 I to LC3 II was significantly increased in neointimal lesions of mouse carotid arteries, 8 which was consistent with our results. Then, our study indicated that TGF-β1 promoted VSMC autophagy by increasing the autophagic flux and the conversion and expression of LC3, Beclin1 and p62, and autophagosome formation.
It has been reported that miR-145 regulated the autophagy of cardiomyocytes and then improved cardiac function and remodelling. 25 F I G U R E 6 miR-145 regulates the proliferation and migration of VSMCs through autophagy. A-C, The VSMCs with miR-145 overexpression were pretreated with the autophagy inhibitor 3-MA for 1 h before stimulating with TGF-β1 (5 ng/mL), and the cell viability, PCNA protein expression and cell migration were determined. **P < 0.01 vs. the LV-NC + TGF-β1 group. # P < 0.05 vs. the LV-miR-145 + TGF-β1 group. D-F, The VSMCs were transfected with the miR-145 inhibitor for 48 h, followed by application of 3-MA for 1 h before stimulating with TGF-β1 and the cell viability, PCNA protein expression and cell migration were determined. *P < 0.05 vs. the miR-NC + TGF-β1 group. # P < 0.05 and ## P < 0.01 vs. the miR-145 inhibitor + TGF-β1 group. The results are expressed as the mean ± SEM, n = 3. Statistical significance was determined using ANOVA by Tukey's post hoc test Therefore, we speculate that miR-145 may regulate the proliferation and migration of VSMCs via autophagy. Further study found that miR-145 overexpression inhibited cell autophagy, whereas miR-145 inhibition promoted autophagy in VSMCs stimulated with TGF-β1.
More importantly, autophagy inhibition augmented the inhibitory effects of miR-145 on the proliferation and migration of VSMCs. Wu et al found that overexpression of miR-145 significantly attenuated the proliferation and induced the autophagy and apoptosis of osteosarcoma cells. 26 Another study showed that curcumin sensitized prostate cancer cells to radiation partly via miR-143-mediated autophagy inhibition. 27 These studies provide further evidence that miR-145 inhibits the proliferation and migration of VSMCs through autophagy.
It is generally accepted that miR-145 exerts biological functions via its multiple target genes, including KLF5, TGFBR2 and CD40. 3,10,11 Emerging evidence indicates that sirtuins are also the targets of miRNAs in cardiovascular diseases. [13][14][15] In this study, we found that the expression of SIRT1, SIRT3, SIRT5 and SIRT6 was down-regulated in the ligated carotid arteries of mice and VSMCs stimulated with TGF-β1. Additionally, our study showed that miR-145 overexpression increased the expression of SIRT1, SIRT3, SIRT5 and SIRT6 in VSMCs. It has been reported F I G U R E 7 The expression of sirtuins in the carotid arteries and VSMCs. A-C, The expression levels of SIRT1, SIRT3, SIRT5 and SIRT6 in carotid arteries were determined by Western blotting and real-time PCR, respectively. D, E, The VSMCs were treated with TGF-β1 (1.25, 2.5, 5, 10 and 20 ng/mL) for 24 h, and the expression levels of SIRT1, SIRT3, SIRT5 and SIRT6 in VSMCs were determined by Western blotting. Statistical significance was determined using ANOVA by Student's t test. *P < 0.05 and **P < 0.01 vs. the control group. (F and G) The effect of miR-145 on the expression of SIRT1, SIRT3, SIRT5 and SIRT6 in VSMCs was determined by Western blotting. Statistical significance was determined using ANOVA by Tukey's post hoc test. **P < 0.01 vs. the LV-NC group. # P < 0.05 and ## P < 0.01 vs. the LV-NC + TGF-β1 group. The results are expressed as the mean ± SEM, n = 3 that SIRT1 plays a pivotal role in the regulation of cellular proliferation and invasion in atherosclerosis and angiogenesis. 28,29 The studies showed that SIRT1 was also the target of miR-34a and miR-138 in the proliferation, migration and differentiation of VSMCs. 13,30 Therefore, miR-145 may regulate autophagy through sirtuins in the proliferation and migration of VSMCs. However, further studies found that sirtuins are not the direct targets of miR-145. It has been reported that miR-216a controls autophagy of vascular endothelial cells, but the autophagy associated gene 5 (ATG5) is not a direct target of miR-216a. 31 Le et al found that p53 is not a direct target of miR-125b in mice; however, miR-125b can indirectly affect p53 expression by upstream regulators. 32 Therefore, we suppose that miR-145 regulates the proliferation and migration of VSMCs through sirtuins. However, sirtuins are not the direct targets of miR-145 in the proliferation and migration of VSMCs.
ACK N OWLED G M ENTS
This study was supported by the National Natural Science University for their help in the detection of super-resolution confocal microscope.
CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.
AUTH O R CO NTR I B UTI O N S
WRW, LFC and CXS performed the experiments and summarized the results. ZJ, FY and RW assisted in performing the experiments.
LB and SHZ assisted in interpreting the data. WRW wrote the manuscript. EQL provided the supervision and assisted in writing the manuscript. All authors read and approved the final manuscript.
DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.
F I G U R E 8
The results of the luciferase reporter assay. A, The relationships between microRNAs and sirtuins in cardiovascular diseases were examined by ingenuity pathway analysis. B, The luciferase reporter assay was performed to validate the direct targets of miR-145 | v3-fos-license |
2016-05-04T20:20:58.661Z | 2010-07-21T00:00:00.000 | 35935417 | {
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} | pes2o/s2orc | Methyl 1-{4-[(S)-2-(methoxycarbonyl)pyrrolidin-1-yl]-3,6-dioxocyclohexa-1,4-dien-1-yl}pyrrolidine-2-carboxylate
The complete molecule of the title diproline ester quinone, C18H22N2O6, is generated by a crystallographic twofold axis, which passes through the centre of the benzene ring. Both –CO2Me groups are orientated to the same side of the benzene ring, with the carbonyl groups pointing roughly towards each other. The conformation of the proline residue is an envelope. In the crystal, a three-dimensional network is sustained by C—H⋯O interactions involving both the quinone and carbonyl O atoms.
The complete molecule of the title diproline ester quinone, C 18 H 22 N 2 O 6 , is generated by a crystallographic twofold axis, which passes through the centre of the benzene ring. Both -CO 2 Me groups are orientated to the same side of the benzene ring, with the carbonyl groups pointing roughly towards each other. The conformation of the proline residue is an envelope. In the crystal, a three-dimensional network is sustained by C-HÁ Á ÁO interactions involving both the quinone and carbonyl O atoms.
Comment
Oxidative nucleophilic addition of amines to quinones results in the formation of aminoquinone products (Lyons & Thomson, 1953). As part of a study into concise methodology for the synthesis of heterocyclic systems, we envisaged that oxidative addition of α-amino acid derivatives to benzoquinone could yield a suitably functionalized precursor for cyclization to yield pyrroloindole quinones, a structural motif present in the mitomycin anticancer drugs (Tomasz, 1995). The title diproline ester quinone, (I), was synthesized in this context.
The molecule of (I), Fig. 1, exists about a crystallographic 2-fold axis of symmetry passing through the centre of the benzene ring. This has the result that the two -CO 2 Me groups are orientated to the same side of the benzene ring. The carbonyl groups are tucked in under the benzene ring. The conformation of the proline residue is an envelope with the C5 atom lying above the plane through the remaining atoms. The conformational descriptors (Cremer & Pople, 1975) The crystal packing features C-H···O contacts, Table 1. The quinone-O1 atom accepts two such interactions, one from a methylene-H and the other from a methyl-H, whereas the carbonyl-O2 accepts a quinone-H. The C-H···O interactions combine to give a 3-D network, Fig. 2.
The maximum and minimum residual electron density peaks of 0.64 and 0.63 e Å -3 , respectively, were located 1.59 Å and 0.85 Å from the H5b and C8 atoms, respectively. In the absence of significant anomalous scattering effects, 750 Friedel pairs were averaged in the final refinement. However, the absolute configuration was assigned on the basis of the chirality of the L-proline starting material. Fig. 1. The molecular structure of (I) showing displacement ellipsoids at the 50% probability level. Unlabelled atoms are generated by (-x, y, -z).
Special details
Geometry. All s.u.'s (except the s.u. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell s.u.'s are taken into account individually in the estimation of s.u.'s in distances, angles and torsion angles; correlations between s.u.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell s.u.'s is used for estimating s.u.'s involving l.s. planes.
Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > 2σ(F 2 ) is used only for calculating Rfactors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger. | v3-fos-license |
2018-04-03T06:01:59.405Z | 1999-04-30T00:00:00.000 | 45396657 | {
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} | pes2o/s2orc | Characterization of a Neutrophil Cell Surface Glycosaminoglycan That Mediates Binding of Platelet Factor 4*
Platelet factor 4 (PF-4) is a platelet-derived α-chemokine that binds to and activates human neutrophils to undergo specific functions like exocytosis or adhesion. PF-4 binding has been shown to be independent of interleukin-8 receptors and could be inhibited by soluble chondroitin sulfate type glycosaminoglycans or by pretreatment of cells with chondroitinase ABC. Here we present evidence that surface-expressed neutrophil glycosaminoglycans are of chondroitin sulfate type and that this species binds to the tetrameric form of PF-4. The glycosaminoglycans consist of a single type of chain with an average molecular mass of ∼23 kDa and are composed of ∼85–90% chondroitin 4-sulfate disaccharide units type CSA (→4GlcAβ1→3GalNAc(4-O-sulfate)β1→) and of ∼10–15% di-O-sulfated disaccharide units. A major part of these di-O-sulfated disaccharide units are CSE units (→4GlcAβ1→3GalNAc(4,6-O-sulfate)β1→). Binding studies revealed that the interaction of chondroitin sulfate with PF-4 required at least 20 monosaccharide units for significant binding. The di-O-sulfated disaccharide units in neutrophil glycosaminoglycans clearly promoted the affinity to PF-4, which showed a K d ∼ 0.8 μm, as the affinities of bovine cartilage chondroitin sulfate A, porcine skin dermatan sulfate, or bovine cartilage chondroitin sulfate C, all consisting exclusively of monosulfated disaccharide units, were found to be 3–5-fold lower. Taken together, our data indicate that chondroitin sulfate chains function as physiologically relevant binding sites for PF-4 on neutrophils and that the affinity of these chains for PF-4 is controlled by their degree of sulfation.
Several members of the ␣-chemokine family like interleukin-8 (IL-8), neutrophil-activating peptide 2, or melanoma growth stimulatory activity have been shown to act as potent activators of PMN by binding to common IL-8 receptors (1). Such binding elicits diverse biological responses such as chemotaxis, degranulation, or adhesion. PF-4, another member of the ␣-subgroup of the chemokine family, is released in high concentrations from activated platelets (2,3). The functional role of PF-4 is intriguing. Highly purified PF-4 lacks any apparent biological activity for PMN but will in the presence of tumor necrosis factor ␣ stimulate these cells to exocytose secondary granule markers or adhere tightly to different surfaces (4). These PF-4-induced functions are not elicited through binding to IL-8 receptors but by interaction with distinct binding sites different from all other chemokine receptors known so far (4,5). The action of PF-4 on PMN was shown to be sensitive to chondroitinase ABC treatment and could be inhibited by soluble chondroitin sulfate (CS), indicating that the potential receptor is of CS proteoglycan type (5).
CSs are galactosaminoglycans composed of alternating glucuronic acid and galactosamine units (34GlcA133GalNAc13) n that are O-sulfated on one or both units. 2 In contrast to the glucosaminoglycans heparin and heparan sulfate (HS), they do not contain N-sulfate groups or L-iduronic acid units (except for CSB), which have been particularly implicated in protein binding to HS chains (6). The expression of glycosaminoglycans (GAGs) on neutrophils has been described previously by several authors. Pioneering work by Olsson and co-workers showed that PMN predominantly express chondroitin 4-sulfate (CSA) (7,8), and Levitt et al. demonstrated a minor proportion of HS in these cells (9). However, as all of these analyses were done with total cell extracts, little is known about the composition and function of cell surface-expressed GAGs in PMN. Gardiner and colleagues showed that the majority of metabolically 35 S-labeled compounds occurs as proteoglycans in neutrophil granules where they may enable proper storage of granule contents or exert protective functions against cellular damage (10,11). Here, we provide evidence that surface exposed CS chains serve as physiologically relevant receptors for PF-4 on PMN, and propose that this function is critically dependent on the content of sulfate groups.
* This work was supported in part by Deutsche Forschungsgemeinschaft Sonderforschungbereich 367, Projekt C4; by Swedish Medical Research Council Grant 2309; and by Polysackaridforskning AB (Uppsala, Sweden). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
CSA from bovine nasal cartilage, dermatan sulfate (CSB) from porcine skin, and CSC from bovine nucleus pulposus cartilage were generous gifts from Dr. Anders Malmström (University of Lund, Lund, Sweden). CSD from shark cartilage and CSE from squid cartilage were obtained from Seikagaku (Tokyo, Japan), while bovine lung heparin was received from The Upjohn Co. Human aortic HS (15) was a gift from Dr. Emadoldin Feyzi (University of Uppsala, Uppsala, Sweden). Heparin oligosaccharides of defined length used as size-defined standards were prepared as described previously (16) Iodination of PF-4 and PF-4 Receptor Binding Assay-PF-4 was iodinated using the chloramine-T method as described for IL-8 (17). To control the integrity of the labeled chemokine, iodinated PF-4 was tested for its capacity to induce an exocytosis response in PMN (4), its ability to form tetramers (5), and its capacity to bind to different GAGs (filter binding assay, see below). No differences to unlabeled PF-4 were seen in these assays. Specific receptor binding of 1 M [ 125 I]PF-4 to PMN was determined as reported previously (5).
Preparation and Metabolic Labeling of Human Neutrophils-PMN cells were routinely isolated from citrated whole blood or fresh buffy coats of healthy single donors by gradient centrifugation on Ficoll-Hypaque to a purity consistently greater than 95% as described previously (21). Viability was examined by trypan blue exclusion and exceeded 98% in all experiments. Metabolic labeling was performed as described by Gardiner et al. (10). Briefly, PMN cells were washed twice with PBS and subsequently incubated at a concentration of 10 7 cells/ml in sulfate-free Dulbecco's modified Eagle's medium, supplemented with 5% fetal calf serum, 1% glutamine, and 100 Ci/ml Na 2 35 SO 4 or 10 Ci/ml [ 3 H]glucosamine for 5 h at 37°C in humidified air. Cells were washed three times with an excess of PBS, and cell clumps were removed by digestion of cells with 100 g/ml DNase I in 10 mM Tris/ HCl, 10 mM MnCl 2 , pH 7.5, for 45 min at 37°C under gentle agitation.
Preparation of Surface-expressed PMN-GAGs-Neutrophil surface-GAGs were proteolytically released from the cell surface by digestion with protease type XIV. Cells were suspended to 5 ϫ 10 7 cells in 1 ml of 0.1 M Tris/HCl, pH 8.0, 1 mM CaCl 2 and incubated with 500 g of enzyme at 37°C under agitation. After 18 h of incubation, the same amount of enzyme was added a second time and the incubation was continued for another 18 h. The suspension was clarified by centrifugation (16.000 ϫ g, for 20 min at 4°C), and proteolytic activity in the supernatant was heat-inactivated at 98°C for 20 min. GAGs were isolated from the supernatant by ion-exchange chromatography on a DEAE-Sephacel column (1 ϫ 10 cm), equilibrated with 0.2 M NH 4 HCO 3 . After removal of contaminants by washing with 0.3 M NaCl and re-equilibration of the column with 0.2 M NH 4 HCO 3 , GAGs were eluted with 2 M NH 4 HCO 3 and subsequently lyophilized. GAGs were quantified by colorimetric determination of hexuronic acid using the meta-hydroxydiphenyl method (22) with purified CSA as a standard. Depending on the respective donor, about 14 -16 g of GAGs/10 8 cells could be isolated with a specific 35 S-radioactivity of ϳ3 ϫ 10 4 dpm/g of GAG.
To examine a possible contamination of surface GAGs with granulederived material, the integrity of primary and secondary granules was monitored. Therefore, freshly isolated PMN as well as PMN incubated for 36 h in the presence or absence of protease type XIV were lysed with detergent and contents of the azurophilic granule marker elastase as well as the specific granule marker lactoferrin were determined as described elsewhere (17). In addition, these markers were assayed also in the supernatant of PMN after 36 h of incubation in the absence of protease.
Composition of GAG Preparations-Size determination of intact 35 Slabeled GAG chains was performed by analytical gel filtration on a Superose 6 column, equilibrated with 50 mM Tris/HCl, pH 7.4, 1 M NaCl, 0.1% Triton X-100, and calibrated with heparin fragments of defined size (4-mer ϳ 1.3 kDa, 10-mer ϳ 3.3 kDa, 20-mer ϳ 6.6 kDa, and 26-mer ϳ 8.6 kDa) as well as with hyaluronan-fragments (11.9, 18.9, 30, and 43 kDa). The nature of neutrophil GAGs was deduced from the susceptibility to deaminative cleavage and to digestion with chondroitinases as examined by gel chromatography on Superose 12. Deamination with nitrous acid was carried out at pH 1.5 according to the method of Shively and Conrad (23), which results in degradation of HS to oligosaccharides. Digestions with chondroitinase ABC or AC were performed with 0.1 unit/ml of the respective enzyme in 50 mM Tris/HCl, pH 8.0, 50 mM sodium acetate, 0.1% BSA for 16 h at 37°C. About 1-2 ϫ 10 4 dpm (ϳ0.5 g) 35 S-labeled PMN-GAGs were used in all incubations in a 200-l final volume.
Preparation and Analysis of CS Disaccharides- 35 S-Labeled neutrophil GAGs (2 ϫ 10 5 dpm; ϳ6.6 g), were digested with chondroitinase ABC (0.1 unit/ml) in 200 l of chondroitinase buffer for 16 h at 37°C, and the resulting disaccharides were purified on a Sephadex G-15 column (1 cm ϫ 170 cm) equilibrated with 0.2 M NH 4 HCO 3 . The samples were freeze-dried and dissolved in water before analysis. PMNderived GAGs were quantitatively converted into disaccharides, Ͻ5% of the degradation products being tetrasaccharides. Disaccharides were separated further with regard to charge by preparative high voltage electrophoresis on Whatman 3MM paper in 1.6 M formic acid (pH 1.7; 40 V/cm) for 80 min (24,25), and labeled fractions were recovered by elution with water. Monosulfated disaccharides (⌬Di-S) were subsequently analyzed by paper chromatography as described (25). For analysis of the disulfated disaccharides (⌬Di-diS), the glycosidic linkage between the unsaturated hexuronic acid residue and the galactosamine residue was cleaved by treatment with 10 mM mercuric acetate as described (26) and followed by separation of the products by high voltage electrophoresis. Papers from high voltage electrophoresis and chromatography were dried, cut into 1-cm segments, and extracted with water, and the radioactivity was determined by -scintillation counting.
Chondroitinase Protection Assay-35 S-Labeled neutrophil GAGs (ϳ 0.5 g) were preincubated with increasing concentrations of PF-4 for 20 min at room temperature in 200 l of chondroitinase buffer and were then digested with 0.1 unit/ml chondroitinase ABC for 16 h at 37°C as described above. Proteins and GAG chains were dissociated by heating samples at 96°C for 10 min in buffer containing 50 mM Tris/HCl, pH 7.4, 1 M NaCl, 0.1% Triton X-100, and the products were separated on a Superose 12 column.
Filter Binding Assay-Approximately 0.2 g of radiolabeled intact GAGs or size-defined fragments were incubated with PF-4 at the indicated concentrations in 200 l of PBS, 0.1% BSA for 2 h at 37°C. Unbound GAG was removed by filtration through a nitrocellulose filter, while protein-bound GAG was trapped on the filter surface (16). The protein-bound GAG was dissociated from the membrane-trapped proteins in 2 M NaCl and analyzed for radioactivity in a -scintillation counter. In some assays, binding affinities of PF-4 for different GAGs were assessed by transformation of the data according to Scatchard. As the molar amounts of PF-4 even at the lowest dosages used in the assay exceeded those of the GAGs by at least 5-fold, the amount of free PF-4 was set to total PF-4.
RESULTS
Characterization of Neutrophil Surface GAGs-In previous experiments we had shown that PMN proteoglycans responsible for PF-4 binding were rather resistant toward proteolytic digestion by trypsin or chymotrypsin (5). For this reason, a more unspecific protease (Streptomyces protease type XIV) was used for the digestion of neutrophils. In a first approach, experiments were designed to explore the time course for the elimination of PF-4 binding sites. A constant concentration of 500 g/ml protease was used and the residual capacity for binding of iodinated PF-4 was monitored at different timepoints as described (5). Binding of 1 M [ 125 I]PF-4 to PMN at 4°C decreased over time with 20% residual binding remaining after 10 h, and 5% residual binding after 18 h of incubation (data not shown). Therefore, the incubation time for proteolytic digestion of neutrophil PGs was extended to 36 h, when PF-4 binding was decreased to background levels. Under these conditions, about 40 -50% of the total metabolically 35 S-labeled material could be mobilized, while 50 -60% remained cell-associated. No difference in the enzymatic activity of the primary granule marker elastase or content of the secondary granule marker lactoferrin (both determined in detergent-treated lysates) was seen between non-treated and protease-treated cells after 36 h. Furthermore, no marker protein was found in the cell-free supernatant (data not shown), indicating that neither primary nor secondary granules had leaked. These results support the notion that the integrity of granules after the proteolytic treatment was conserved such that the remaining 35 Slabeled material presumably would be located in neutrophil granules (10,11).
The released 35 S-labeled surface GAGs were further purified by ion-exchange chromatography, after which the molecular size was determined by gel filtration on a Superose 6 column. PMN-GAGs eluted as a single broad peak representing molecules of a wide range of molecular sizes with an average of ϳ23 kDa (Fig. 1). This value corresponds to a GAG chain length of about 90 monosaccharide units (calculated with an average mass of 500 Da for a disaccharide unit), similar in size to a bovine nasal cartilage CSA preparation used as a standard. In order to characterize the GAG chains, the metabolically labeled PMN-GAGs were treated with either nitrous acid at pH 1.5 or with chondroitinase ABC and AC, respectively, followed by gel chromatography of the products. Nitrous acid treatment at pH 1.5 did not affect the elution behavior of the sample, indicating the absence of N-sulfated hexosamine residues as found in HS ( Fig. 2A). In contrast, digestion with either chondroitinase ABC (Fig. 2B) or chondroitinase AC (Fig. 2C) resulted in the quantitative conversion of the chains into small saccharides. From these data, we conclude that neutrophil surface GAGs consist predominantly of CS and lack detectable amounts of HS or CSB.
Composition of Neutrophil Surface CS-For compositional analysis, the surface PMN-GAGs were extensively digested with chondroitinase ABC and the resulting fragments separated by gel filtration on a Sephadex G-15 column. More than 90% of the labeled material eluted as disaccharides which were further separated by high voltage paper electrophoresis in order to characterize their sulfation degree. The migration profile of the disaccharides revealed two peaks (Fig. 3A). The major peak (P I) with approximately 74% of the total radioactivity comigrated with a monosulfated disaccharide (⌬Di-S) standard, while about 26% of the radioactivity appeared with the disulfated disaccharide (⌬Di-diS) fraction. As the radioactivity of this second peak (P II) represents two [ 35 S]sulfate groups per disaccharide molecule, the molar proportion of ⌬Di-diS corresponds to half of the amount of 35 units in neutrophil surface CS appeared to be a general phenomenon as the analysis of disaccharides from two other donors revealed similar disulfated disaccharide contents of 12 and 13% with minor variations between individuals (data not shown). Also, [ 3 H]glucosamine labeling was performed to as-sess the amount of non-sulfated disaccharide units in the whole population. No non-sulfated disaccharides could be detected by chondroitinase ABC digestion of these preparations followed by high voltage electrophoresis, indicating that the chains are composed essentially of sulfated disaccharide units (data not shown). 3 Paper chromatography of the monosulfated disaccharide fraction P I showed a single peak corresponding to the standard ⌬Di-4S disaccharide (Fig. 3B), whereas no material co-migrating with the ⌬Di-6S standard was seen. In order to assign the sulfation pattern of the disulfated disaccharide in fraction P II, the disaccharides were treated with mercuric acetate (26) and the resulting products were separated by high voltage electrophoresis. About 85% of the labeled material displayed an increased migration after Hg-acetate cleavage as compared with the untreated control (Fig. 3C). This peak corresponds to the disulfated monosaccharide GalNAc(4,6-OSO 3 ), originating from the disaccharide ⌬HexA133GalNAc(4,6-OSO 3 ) (⌬Di-diS E ). The remaining 15% of the generated monosaccharides migrated slower than the original disaccharide and correspond to a monosulfated monosaccharide, originating from either ⌬HexA(2-OSO 3 )133GalNAc(4-OSO 3 ) (⌬Di-diS B ) or ⌬HexA(2-OSO 3 )133GalNAc(6-OSO 3 ) (⌬Di-diS D ). In summary, the neutrophil surface GAGs contain predominantly (up to 90%) chondroitin-4-sulfate disaccharide sequences, while 12-15% of the chains consists of disulfated disaccharide units, mainly due to the presence of GalNAc-4,6-disulfate residues.
PF-4 Binding to Neutrophil Surface CS Chains-In order to characterize the putative PF-4 binding sites on the CS chains, enzyme protection assays were performed. Purified 35 S-labeled PMN-CS was preincubated with increasing concentrations of PF-4 and subsequently digested with chondroitinase ABC. At a concentration of 10 M PF-4, more than 95% of the PMN CS-chains were protected against digestion with the bacterial eliminase (Fig. 4). However, a stepwise decrease of the PF-4 concentration led to a corresponding decrease in the amount of protected polysaccharide: at 1 M PF-4 59% and at 0.2 M only 13% of the total radioactivity remained in the high molecular weight fraction. At 0.04 M PF-4, protection of the CS chains was completely abrogated and all of the labeled carbohydrates eluted in a second peak, representing the breakdown products. Therefore, at sufficiently high concentrations PF-4 can bind to and protect all PMN-derived CS. Corresponding control experiments performed with the bovine [ 3 H]CSA revealed a similar PF-4-mediated protection of these chains against digestion with chondroitinase ABC. However, compared with the PMN-GAGs, the concentration of PF-4 required for complete protection was significantly higher (20 M) indicating a lower affinity of the chemokine for the bovine CSA. Notably, decreasing the concentrations of PF-4 led to a reduction in the total amounts of protected CS chains but did not affect the size of these chains (Fig. 4). Thus, binding of PF-4 to the polysaccharide protected the entire chains from digestion but did not reveal any limited fragment that could be identified as a binding site for the chemokine.
Structural Requirements of CS-Binding to PF-4: The Importance of Chain-length and Disulfated Disaccharide Units-As PF-4 appeared to protect the entire neutrophil CS chain from lyase digestion, our next approach was to identify the minimal fragment size for PF-4 binding. Binding of size-defined, 3 Hlabeled CSA fragments to PF-4 was examined using a nitrocel- 3 Metabolic labeling of PMN-GAGs with [ 3 H]glucosamine is hampered by the short metabolical labeling period possible and by the reduced metabolic turnover of these terminally differentiated cells. A general use of this labeling procedure for analytical purposes is therefore practically unfeasible.
FIG. 3. Disaccharide composition of neutrophil-derived [ 35 S]CS.
A, disaccharides were obtained by complete digestion of PMN-GAGs with chondroitinase ABC, desalted by gel filtration, and analyzed by high voltage paper electrophoresis at pH 1.7 as described under "Experimental Procedures". Migration positions of 3 H-labeled standard disaccharides run in parallel lanes are indicated by arrowheads: ⌬Di-S, monosulfated disaccharide, ⌬ 4,5 HexA133GalNAc(OSO 3 ); mono-S, monosulfated monosaccharide GalNAc(OSO 3 ); ⌬Di-diS, disulfated disaccharide, ⌬ 4,5 HexA133GalNAc(4,6-di-OSO 3 ). Peaks I (P I) and II (P II) were eluted from the paper stripes as indicated. B, unsaturated, monosulfated disaccharides in peak I from panel A were subjected to paper chromatography. Arrowheads indicate the migration positions of 3 Hlabeled standards: ⌬Di-4S, ⌬ 4,5 HexA133GalNAc(4-OSO 3 ), ⌬Di-6S, ⌬ 4,5 HexA133GalNAc(6-OSO 3 ). C, mercuric acetate-treated (⅐q⅐) or non-treated (-E-) disaccharides from peak II in panel A were separated by high voltage electrophoresis at pH 1.7. Arrowheads indicate the migration positions of 3 H-labeled standards run in parallel lanes: mono-S, GalNAc(4-OSO 3 ); mono-diS, GalNAc(4,6-di-OSO 3 ); and ⌬Di-diS, ⌬ 4,5 HexA133GalNAc(4,6-di-OSO 3 ). lulose filter binding assay (16). PF-4 (5 M) was incubated with constant quantities of CSA fragments of various chain lengths, and protein-GAG complexes were retained by passing the solution through a nitrocellulose filter. As shown in Fig. 5A, interaction of PF-4 with CSA-fragments did not exceed background levels (ϳ 2.5% bound oligomer) until a fragment length of ϳ 8 monosaccharide units (12.8% bound oligomer). Binding further increased with increasing oligomer size. In an alternative approach to defining the minimal PF-4-binding region, the chemokine was mixed with [ 3 H]CSA that had been partially degraded by extended hydrazinolysis (27), and the mixture was subjected to digestion with chondroitinase ABC. Analysis of the undigested [ 3 H]CSA preparation by gel chromatography showed a broad peak from 10.5 to 18.5 ml (Fig. 5B), representing chain lengths varying between dimers and ϳ48-mers, with an average approximate size of a 20-mer (14.5 ml). The same material that had been treated with chondroitinase ABC in the presence of PF-4 emerged as two clearly separated peaks. The first peak, representing the protected fraction, started to elute in the same range as the untreated control, indicating full protection of the largest fragments. Fragments were protected down to ϳ22-26-mer size. However, fractions corresponding to smaller fragment size showed a sharp decrease in radioactivity, indicating that chains shorter than ϳ20 monosaccharide units (eluting after 14.5 ml) were not protected by PF-4. These smaller fragments were found to be completely degraded by chondroitinase and eluted in the second peak, representing the breakdown products. The same labeled GAG fragments in control digestions lacking PF-4 were completely degraded and eluted exclusively in the second peak (data not shown). Taken together, the results of the two experiments indicate that PF-4 binding to CSA requires a minimal saccharide sequence of ϳ20 monosaccharide units.
The affinity of CS to PF-4 is considered low as compared with that of heparin or HS (28). As the affinity of GAGs for chemokines appears related to the charge of the carbohydrate chain (29), we wondered whether the presence of disulfated disaccharide units in neutrophil-derived CS would influence the binding to PF-4. Filter binding assays thus were performed with la-beled PMN-CS or CSA at constant concentration (ϳ1 g/ml), mixed with purified PF-4 at increasing concentrations. The binding curves indicated that PMN-CS bound with higher affinity to PF-4 than did CSA (Fig. 6). Scatchard analysis of the data (Fig. 6, inset) revealed an unusual binding pattern composed of essentially two phases. Linear plots were obtained only above certain minimal concentrations of PF-4 (1.25 and 2.5 M, respectively, in interactions with PMN-CS and CSA). Based on these data, PF-4 exposed to both CS types a single class of binding sites with apparent K d values of ϳ0.8 M for PMN-CS and ϳ4.4 M for CSA, hence more than 5-fold different. However, at concentrations below 1 M PF-4, the affinity of the GAG chains for the chemokine ligand decreased dramatically. Almost identical non-linear binding patterns were described for the interaction of PF-4 with binding sites on intact neutrophils, suggesting similar mode of binding of PF-4 to isolated CS and to intact cells (5). The cellular receptors were shown to preferentially bind tetrameric PF-4, which was found to occur only at concentrations exceeding 50 nM.
As the major structural difference between PMN-CS and CSA was the presence of disulfated disaccharide units in the former species, the possible influence of these groups on the binding to PF-4 was considered. For this purpose, binding assays were performed with various 3 H-labeled CSs from different sources and affinity constants were determined as before. Furthermore, all CS preparations were analyzed for their content of disulfated disaccharide units. Bovine intestinal heparin as well as human aortic HS served as references. As shown in Table I, all of the GAGs tested bound to PF-4, but with significantly different affinities. CSA, CSB, and CSC, which did not contain any detectable disulfated disaccharide units, showed relatively low affinities for PF-4, K d values ranging from 2.9 to 4.4 M. However, CSD, which yielded about 8.7% ⌬Di-diS upon chondroitinase digestion, bound to PF-4 with an affinity comparable to that of PMN-CS (K d values of 0.6 and 0.8 M, respectively). Moreover, CSE, with the highest content of ⌬Di-diS (30.7%), scored the lowest K d : 0.3. Interestingly, binding of PF-4 to HS from aorta revealed an approximately 2.5-fold lower affinity (K d ϳ 2.3 M) as compared with that of PMN-CS. By contrast, heparin, the most negatively charged carbohydrate tested, bound PF-4 with appreciably higher affinity than any of the other GAGs tested (Table I). DISCUSSION The composition of neutrophil GAGs has been investigated by several groups over the last decades. However, the GAGs expressed at the PMN surface remain poorly defined with regard to structure as well as functional role(s). The present study was initiated by our observation that PF-4 binds to a CS proteoglycan on human PMN cells (5). We therefore aimed at characterizing surface exposed GAGs and their binding to PF-4. Proteolytic release of cell surface-associated GAGs under conditions that removed all binding sites for PF-4 but conserved the macroscopic appearance of the cells resulted in the isolation of a GAG pool that contained CS, essentially of the CSA type, but no HS (Fig. 2). These findings are in accordance with earlier findings that CSA constitutes the major part of neutrophil GAGs (7,8). Under the conditions of isolation, only about 40 -50% of the total metabolically 35 S-labeled material was released from the cells, whereas the rest remained cellassociated. Assessment of cellular integrity by means of marker proteins from either primary or secondary granules indicated that no leakage had occurred from these intracellular compartments. It therefore seems reasonable to assume that the remaining radioactivity would be localized intracellular, in agreement with earlier publications localizing the majority of intracellular GAGs to the granules (10,11). Up to 25% of GAGs from total PMN extracts was identified as HS, based on susceptibility to nitrous acid treatment (9). Although we cannot exclude the presence of HS in PMN, we conclude that HS is not expressed at the cell surface and therefore does not participate in the recognition of PF-4 by these cells. Such recognition is mediated by CS chains.
PF-4 binds to CS as well as to heparin-related GAGs (12,13,28,30,31), and is stored as a CS proteoglycan/PF-4 complex in ␣-granules of platelets (32,33). The affinity of PF-4 for different GAGs has been postulated to decrease in the order heparin Ͼ Ͼ HS Ͼ Ͼ DS Ͼ CSC Ͼ CSA (28). In previous work, PF-4 displayed the highest affinity for heparin of all the chemokines tested (13); a K d of 30 nM (12) is in fair agreement with the value (60 nM) determined in the present study. However, the postulated generalized order of affinities for GAGs needs to be modified to account for the effects of minor variations in the degree of sulfation. Although PMN-derived CS contained only ϳ13% di-O-sulfated disaccharides, it bound PF-4 with an affinity (K d ϳ 0.8 M) more than 5-fold higher than that of the strictly monosulfated CSA from nasal cartilage (K d ϳ 4.4 M), and more than 2-fold higher than that of HS from human aorta HS (K d ϳ 2.3 M). The positions of the additional sulfate residues appear to be less important for the increased affinity, as in CSD, with an affinity for PF-4 (K d ϳ 0.6 M) comparable to that of PMN-derived CS, the disulfated disaccharides have the GlcA(2-OSO 3 )133GalNAc(6-OSO 3 ) and not the GlcA133-GalNAc(4,6-OSO 3 ) structure as predominantly found in PMN-CS.
PF-4 is a member of the CXC chemokine family, with a three-dimensional structure very similar to that of other mem- a The content of disulfated disaccharides was determined by high voltage electrophoresis of labeled disaccharides prepared by complete digestion of GAGs with chondroitinase ABC as described in Fig. 3A.
b Affinity constants were determined from data of binding assays after transformation according to Scatchard. Assays were performed as described for PMN-CS and CSA in Fig. 6. The data represent mean Ϯ S.D. of three independent experiments, each performed in duplicate.
c The data represent mean Ϯ S.D. of results obtained from GAGs of three different healthy donors. d ND, not determined.
bers of this family. The monomeric unit, consisting of a Cterminal aliphatic ␣-helix lying on top of a three-stranded antiparallel -sheet (34), forms dimers and tetramers (35). Basic amino acid residues implicated with GAG binding are predominantly located in the ␣-helix, but also in loops of the -sheets (12,36), in such a way that a PF-4 tetramer will display a belt of positively charged residues around the entire molecule (37). This arrangement may help to explain some intriguing observations pertaining to the molecular dimensions of GAG-PF-4 interactions. Although a short heparin fragment of ϳ6/8-mer size is shown to bind PF-4 (16), maximal affinity is attained only for Ͼ20-mers (16,38). These extended sequences are believed to interact with both dimer subunits of a tetramer (38), as has been found for HS (14). The interaction between CS and PF-4 is too weak to show up at the 6/8-mer level, but is clearly evident for ϳ20-mer sequences (Fig. 5A), thus suggesting similar modes of binding for CS, HS and heparin. Moreover, even more extended, ϳ9-kDa (ϳ40 monosaccharide units), sequences of HS were protected against heparitinase digestion in the presence of PF-4 (14). These large fragments were shown to contain sulfated domains positioned at both ends, separated by a central, mainly N-acetylated region, and would be expected to wrap around the entire circumference of a PF-4 tetramer (14). CS fragments of similar initial size were also protected by PF-4 against digestion with chondroitinase ABC (Fig. 5B). Unexpectedly, however, more extended CS chains (average ϳ19 kDa) were either completely degraded by the enzyme or remained seemingly intact, depending on the relative proportions of CS and PF-4 (Fig. 4). A likely explanation to this finding is that the extended CS chain interacts with more than one PF-4 tetramer, in such a manner that essentially the entire length of the chain is engaged in protein binding. Provided that sufficient amounts of PF-4 are present to saturate all CS chains in the mixture, these will be completely protected against enzymatic cleavage; unbound CS chains will be completely degraded. A HS chain will behave differently, due to its less homogeneous structure, highly sulfated domains being interspersed by essentially unsulfated sequences (39). The latter structures will be less prone to protein binding, hence protection, and will therefore be preferentially cleaved during incubation with the appropriate endoglycosidase. Composite domain structure for HS fragments interacting with cytokines have been postulated not only for PF-4 (14), but also for interferon-␥ (40) and IL-8 (29).
The results of the present study are clearly relevant to the mode of action of PF-4 at the PMN cell surface. The K d for PF-4 interaction with isolated PMN-CS chains (ϳ0.8 M) was in a range similar to that determined for the binding to intact PMN (ϳ0.65 M). Moreover, the binding curves obtained with whole cells (5) and with isolated CS showed similar sigmoidal shapes and non-linear Scatchard plots, indicating a decrease in the affinity for PF-4 binding sites at low concentrations of the chemokine. This phenomenon is most likely caused by the selectivity of neutrophil GAGs for binding to the tetrameric form of the chemokine. As we have shown previously, tetramerization of PF-4 takes place only at concentrations exceeding 50 nM PF-4, and in the absence of PF-4 tetramers neither binding to cellular receptors, nor functional activation of PMN is detected (5). Finally, the effects of "oversulfation" of the PMN-CS on PF-4 binding should be considered. The increase in binding strength caused by the presence of disulfated disaccharide units would seem to be of key importance in the control of PF-4 binding and PF-4-mediated cellular activation. As about halfmaximal occupation of the implicated receptors on PMN is required for the induction of a measurable cellular response (4), whereas the serum concentrations of PF-4 do not exceed 1.0 -2.5 M (41), a receptor substituted simply with CSA (K d ϳ 4.4 M) would hardly recruit sufficient amounts of the chemokine to mediate a cellular response. Although it seems likely that the signaling component of the PF-4 receptor is a protein constituent, receptiveness is determined by the composition of the associated CS chains. We cannot exclude that a PMN-CS proteoglycan serves as a co-receptor that is coupled to a secondary receptor function. An important step toward the elucidation of the signaling mechanism will be the isolation and characterization of the putative receptor core protein. | v3-fos-license |
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} | pes2o/s2orc | Isolation of the Subunits of Transcarboxylase and Reconstitution of the Active Enzyme from the Subunits
Abstract The separation of the 12 SH central subunit, the 5 SE peripheral metallo subunit, and the 1.3 SE biotinyl carboxyl carrier protein which are formed on the dissociation of transcarboxylase has been accomplished by molecular sieving on Bio-Gel. The 12 SH and 5 SE subunits have been obtained in nearly homogeneous form as judged by the sedimentation velocity profiles and by acrylamide gel electrophoresis. The 1.3 SE carboxyl carrier protein has given less consistent results; sometimes a single band of molecular weight of approximately 12,000 is obtained on gel electrophoresis in sodium dodecyl sulfate but sometimes there is an additional band of lower molecular weight of approximately 11,000. This lower molecular weight component may result from limited proteolytic degradation in spite of efforts to prevent it. Two or more bands are obtained in the absence of dodecyl sulfate. This heterogeneity may result from aggregation. subunits. The most effective reconstitution is accomplished by a two-step process. First, the 1.3 SE carboxyl carrier protein and 5 SE metallo subunit are combined to form the 6 SE complex; then this product is combined with the 12 SH subunit to yield active enzyme. With a limiting amount of the 6 SE complex and an excess of the 12 SH subunit the resulting enzymatic activity is proportional to the concentration of 6 SE complex. Likewise, with an excess of the 6 SF, complex and the 12 SH subunit limiting, the enzymatic activity is proportional to the concentration of the 12 SH subunit. The maximum specific activities of the reconstituted 6 SE complex and the 12 SH subunit were approximately 60% and 50%, respectively of the value estimated for their specific activities in the native 18 S form of the enzyme with three peripheral 6 SE subunits. Because assay of the 6 SE complex is done using an excess of the 12 SH subunit, this may yield enzyme with a single peripheral subunit. The 6 SE subunit may be less effective in this form. In the case of the 12 SH subunit, the activity is most likely low because the reconstituted 6 SE complex does not contain the full complement of 1.3 SE biotinyl carboxyl carrier proteins. The carboxyl carrier protein provides the groups which link together the 5 SE peripheral subunits with the central subunit. Evidence is presented that the 1.3 SE biotinyl carboxyl carrier must first combine with the 5 SE subunit to assume a form which effectively provides the combining groups for association with the 12 SH subunit.
This heterogeneity may result from aggregation.
Active enzyme is readily reconstituted from the isolated subunits.
The most effective reconstitution is accomplished by a two-step process. First, the 1.3 SE carboxyl carrier protein and 5 SE metallo subunit are combined to form the 6 SE complex; then this product is combined with the 12 SH subunit to yield active enzyme. With a limiting amount of the 6 SE complex and an excess of the 12 SH subunit the resulting enzymatic activity is proportional to the concentration of 6 SE complex. Likewise, with an excess of the 6 SF, complex and the 12 SH subunit limiting, the enzymatic activity is proportional to the concentration of the 12 SH subunit.
The maximum specific activities of the reconstituted 6 SE complex and the 12 Sn subunit were approximately 60% and SO%, respectively of the value estimated for their specific activities in the native 18 Cleveland, Ohio 44166. assay of the 6 SE complex is done using an excess of the 12 SH subunit, this may yield enzyme with a single peripheral subunit. The 6 SE subunit may be less effective in this form.
In the case of the 12 SH subunit, the activity is most likely low because the reconstituted 6 SE complex does not contain the full complement of 1.3 SE biotinyl carboxyl carrier proteins.
The carboxyl carrier protein provides the groups which link together the 5 SE peripheral subunits with the central subunit.
Evidence is presented that the 1.3 SE biotinyl carboxyl carrier must first combine with the 5 SE subunit to assume a form which effectively provides the combining groups for association with the 12 SH subunit.
It catalyzes the following reaction: CH&H (COO-)COSCoA + CHICOCOO-F! CH&H&OSCoA + -OOCCH&OCOO-Transcarboxylase dissociates to subunits which can be reconstituted forming an active enzyme as diagrammed in Fig. 1. The accumulated evidence (l-8) including electron microscopy (1) shows that three peripheral subunits (designated 6 SE) are attached loosely to one face of a cylindrical shaped central subunit (designated 12 S,).
The enzyme has a molecular weight of 790,000 and ~~0,~ = 18 S and dissociates at pH 8 and low ionic strength with sequential loss of the peripheral 6 SE subunits (1,6,7). The isolated enzyme frequently contains a mixture of the 18 S form with three peripheral subunits together with a 16 S form of the enzyme with two peripheral subunits (1,6).
The 6 SE subunit of molecular weight 144,000 dissociates slowly at pH 8 and rapidly at pH 9 to a dimeric metallo subunit of molecular weight 120,000 (designated 5 SE) (1,6) and to two biotinyl subunits of molecular weight of approximately 12,000 (designated 1.3 SE) (2,3). In the presence of denaturing agents, the dimeric 5 SE subunit dissociates to its constituent peptides (designated 2.5 S,) (1). The 1. Subunits of transcarboxylase are formed at low ionic strength and alkaline pH and they reassociate at high ionic strength in phosphate buffer, pH 6.5, or in acetate buffer, pH 5. The sedimentation coefficients are given with subscripts H indicating the "head" or central subunit or E indicating the "ear" or peripheral subunit (1). SDS, sodium dodecyl sulfate. slowly at pH 8 and more rapidly at pH 9 to dimeric subunits of molecular weight 120,000 (designated 6 Sn). The 12 Sn subunit does not contain either metals (Co2+ or Zn*) or biotin (1,3,5).
In the presence of sodium dodecyl sulfate or urea, the dimeric 6 Sn subunits dissociate to the constituent peptides (designated 2.5 Sn) (1). The 18 S form of the enzyme thus is made up of 18 peptides of three different types; 6 contain the metals (cobalt and zinc) ,I 6 are the 1.3 SE biotinyl carboxyl carrier proteins, and 6 make up the central 12 Sn subunit.
Glycerol (207,) retards the dissociation of the 12 Sn subunit to 6 Sn dimers and of the 6 SE subunit to 5 SE and 1.3 SE subunits. Furthermore, glycerol appears to maintain the native configuration of the 12 Sn and 6 SE subunits because they readily reconstitute to the active enzyme under appropriate conditions. Subunits formed in the absence of glycerol do not reconstitute as readily and give rise to an active enzyme2 with a sedimentation coefficient of approximately 24 S with the peripheral subunits attached to both faces of the central subunit (1,6).
The 12 Sn subunit has been isolated by glycerol gradient centrifugation and recombined with the 6 SE biotinyl metallo subunit in 0.25 M acetate at pH 5.0 to 5.2 or in 0.75 M phosphate at pH 6.5 to 6.8 to form active enzyme (1,3 All of the Bio-Gel columns were maintained in 0.02% sodium azide to inhibit bacterial growth. The other methods and reagents were as described previously (1,5,6,12). It then had a specific activity of 1.5 and Fig. 2B shows that it consisted of some ~16 S material and -12 S material but the major peak was -6 S material. This dissociated enzyme was placed on a Bio-Gel A-1.5m column (4.5 x 172 cm) which had been equilibrated with 0.05 M phosphate buffer (pH 7.0) and was eluted with the same buffer over a period of about 40 hours at 4'. Three protein peaks which were not completely resolved were obtained and were separately Because the 2.5 Sn and 2.5 SE peptides are separated by this technique (7), this material contained little or no 6 Sn subunit.
Isolation of Subunits
It was, however, a mixture of 6 SE and 5 SE subunits; the latter being formed by partial dissociation of the 6 SE subunit.
That this had occurred was evident because there was a small peak of radioactivity in the eluate from the Rio-Gel column where the 1.3 SE subunit occurs. It is evident that there was considerable reconstitution to active enzyme during the chromatography on the Rio-Gel column because the dissociated enzyme (480 mg) had a specific enzymatic activity of 1.5, equivalent to 816 units whereas there were -4320 units in the 140 mg of protein in the pool from the first peak and it did not include all of the active enzyme eluted from the column.
Isolation of 12 SH Central Subunit and 5 SE Metallo Subunit from "Dead" Transcarboxylase-Our first successful large scale isolation of both the 12 Sn and 5 SE subunits was made possible by a fortuitous result during the preparation of a large batch of transcarboxylase.
On this occasion, all of the enzymatic activity was lost during the purification of transcarboxylase on a cellulose phosphate column.
The protein peak of the eluate with 0.3 M phosphate buffer (pH 6.8) which usually contains the transcarboxylase had a sedimentation coefficient of approximately 16 S, but there was no radioactivity associated with the protein, which indicated that the tritiated biotin had somehow been removed from the enzyme. This inactive protein, designated dead transcarboxylase, was stored at -20" for 2 years, until we learned that mild treatment with trypsin rapidly removes biotinyl peptides from normal transcarboxylase leaving the remaining portion of the molecule intact but inactive (8). The dead transcarboxylase was then examined and found to have a distinct advantage because dissociated dead transcarboxylase did not reassociate during Rio-Gel chromatography.
The dissociation was done as described above for the native enzyme and 420 mg of the dissociated dead transcarboxylase was fractionated on the Rio-Gel A-1.5m column.
Two protein peaks which were not completely resolved were obtained.
The peak fractions were carboxylase, the dissociated protein, and fractions after chromatography of the dissociated protein on a Bio-Gel A-1.5m column.
A, the original dead transcarboxylase, 15.7 S; B, the dissociated dead transcarboxylase, 13.0 S, and 6.9 S; C (top), first nrotein neak eluted from the Bio-Gel column. 11.8 S; C (bottom). protein irom fractions between the two peaks, 11.8 S and 5.5 St and D, protein from the second protein peak after dialysis at pH 9 and chromatography to remove any residual portion of the 1.3 SE subunit which might remain attached to the 5 Sx subunit, 5.8 S. Sedimentation was at 52,000 rpm at 4" for 56 min from right to left in a 30-mm double sector cell in the experiments of A, B, and D and a double sector wedge cell for C. The milligrams per ml of protein were: A, 2.5 mg per ml; B, 2.1 mg per ml; C (lop), 2 mg per ml ; C (bottom) 2 mg per ml ; and D, 2.2 mg per ml.
pooled separately and also the valley fractions between the two peaks. Sedimentation velocity profiles of the original dead transcarboxylase, of the dissociated dead transcarboxylase, and of the protein fractions from the Bio-Gel A-1.5m column are shown in Fig. 3. The material from the first peak ( Fig. 3C, lop) had an s20,W = 11.8 S. That from the second peak was used for isolation of 5 SE subunit as described below.
The protein from the valley fractions had values of 11.8 S and 5.5 S. It is apparent from the comparison of Fig. 2, C and D with Fig. 3C that unlike the subunits from normal transcarboxylase, those from dead transcarboxylasa did not reconstitute during the chromatography on Bio-Gel A-1.5m. The protein from the second peak was combined with another similar preparation from dead transcarboxylase for isolation of the 5 SE subunit.
The material (171 mg of protein in 10 ml) was dialyzed at room temperature for 23 hours with three changes of 1 liter each at 12, 17, and 23 hours against 0.05 M Tris-HCl (pH 9.0) containing 10m4 M phenylmethylsulfonylfluoride. This treatment dissociates from the 5 SE subunit any residual portion of 1.3 SE subunit that might remain attached to the 5 SE subunit. The material was then chromatographed on a Bio-Gel A-1.5m column (4.7 X 171 cm) using 0.05 M phosphate buffer (pH 7.0) containing low4 M phenylmethylsulfonylfluoride for the elution. A single symmetrical protein peak was obtained from the column. The pooled protein fractions were precipitated by 80% saturation with ammonium sulfate and then subjected to ultracentrifugation. The protein had an ~~0,~ = 5.8 S (Fig. 3D).5 Polyacrylamide gel electrophoresis of this protein and that from the first peak (Fig. 3C, top) in gels containing 8 M urea and sodium dodecyl sulfate (7) gave single bands showing that there was little cross contamination of the 5 SE subunit by the 12 Sn or 6 Sn subunits or of the 12 Sn subunit by the 5 SE subunit.
These two preparations of subunits in combination with the normal 1.3 SE subunit were found to be effective in reconstituting active transcarboxylase (see "Reconstitution of Transcarboxylase from Subunits").
Isolation of ld SH and 5 SE S&units by Other Methods-Mild treatment of transcarboxylase wit.h trypsin removes biotinyl peptides leaving the remaining portion of the enzyme intact but 6 The sedimentation coefficient of the 5 SE subunit which arises from the 6 SE subunit had not been determined previously because it had not been isolated.
It has been designated 5 SE for convenience in differentiating it from other subunits of similar sedimentation coefficients.
However, the yields have not been as good as with dead transcarboxylase because there is some recombination of the dissociated subunits even though a portion of the 1.3 SE subunit is removed by the trypsin treatment.
These results are described in detail by Ahmad et al. (8).
A method for isolation of 5 SE and 12 Sn subunits from transcarboxylase by complexing native enzyme with avidin-Sepharose has been developed by Berger and Wood (17). The complex is dissociated at pH 8 to liberate the 12 Sn subunit and then at pH 9 to obtain the 5 SE subunit. The 1.3 SE subunit remains in a complex with the avidin, thus the problem of reconstitution is eliminated and the eluated subunits may be collected under conditions which prevent their dissociation.
This procedure is useful in obtaining the 5 SE and 12 Sn subunit but is of no use in obtaining the 6 SE or 1.3 SE subunits.
Isolation of 1.3 SE Biotinyl Carboxyl Carrier Protein-We have not had consistent results in isolating a homogeneous form of this subunit.
Isolation of the 1.3 S, subunit as described previously (2) involved dissociation of transcarboxylase at 4" in 0.05 M Tris-HCl (pH 8.8) for 72 hours followed by a stepwise elution with an increasing concentration of KC1 from a DEAE-Sephadex A-50 column equilibrated with 0.05 M Tris-HCl (pH 8.8). The 1.3 SE subunit was obtained in a small breakthrough peak as well as in the 0.1 M KC1 eluate and gave a single band on polyacrylamide gel electrophoresis at pH 9 and had a molecular weight of approximately 12,000. Subsequently, this and other procedures have not given consistent results because more than one band is often observed on polyacrylamide gel electrophoresis. One method which we have adopted involves denaturation of transcarboxylase at 100" in 6 M urea plus 10ea M dithiothreitol and then chromatography on I&Gel A-1.5m in 6 M urea plus low4 M dithiothreitol.
As shown in Fig. 4, the resulting 2.5 S, and 2.5 SE peptides in Fractions 23 to 28 are easily observed by ultraviolet absorbtion at 280 nm and are well separated from the radioactive 1.3 SE subunit in Fractions 47 to 57. The 1.3 SE subunit, by virtue of its low content of aromatic amino acids, has a low ultraviolet absorbtion and is observed only by the presence of the aH label. The results of analytical polyacrylamide gel electrophoresis of the 2.5 Sn and 2.5 SE preparation in urea and of the 1.3 SE preparation in dodecyl sulfate are shown in the insets of Fig. 4. Two major bands were observed in the fractions containing the 2.5 SE and 2.5 Sn peptides and these had no radioactivity.
Fractions 47 to 56 containing the 1.3 SE subunit gave one major band which was highly radioactive and contained about 85y0 of the total recovered radioactivity.
There was a nearby smaller band with about 15y0 of the radioactivity. Some preparations obtained by this method give two bands in these positions almost equally stained and radioactive.
Both types of preparations are effective for the reconstitution of active enzyme in combination with 12 Sn and 5 SE subunits.
A second method for isolation of the 1.3 SF: subunit involves dissociation of transcarboxylase to the 6 Sn, 5 SE, and 1.3 SE subunits in 0.1 M Tris-HCl, pH 9, plus 10% glycerol. This mixture is then chromatographed on Bio-Gel A-1.5m (Fig. 5). The 6 Sn and 5 SE subunits are eluted in Fractions 60 to 76 which is consistent with their molecular weights (approximately 120,000) but the 1. The fractions were monitored for 280-nm absorbance and for radioactivity. Fractions 23 through 28 and 47 through 56 were pooled separately and each was dialyzed against 2000 ml of 0.02 M sodium phosphate buffer (pH 7) with three changes of buffer. Pool 23 to 28 was concentrated by rotary evaporation to 7.2 ml (~55 mg per ml) and Pool 47 to 56 tb 2.2 ml (some turbiditv develoned in this nool). The 1.3 SF nrenaration'was subjected to gel eiectrophoresis in dodecyl suliiie 6) and the 2.5 SE and 2.5 SH preparation to gel electrophoresis in urea (17). The results are shown in the insets. and/or (c) dissociation and reassociation of the 5 SE and 1.3 S3 subunit during the chromatography.
The protein in Fractions 60 to 76 was precipitated with ammonium sulfate and the protein in Fractions 80 to 102 was concentrated by lyophilization. Gel electrophoresis in the absence of denaturing agents of the preparation containing 6 Sn and 5 SH subunits gave two major bands and gel electrophoresis in sodium dodecyl sulfate of the 1.3 SE preparation gave a single band aside from a small band near the origin which may be an aggregate.
Some preparations of 1.3 SE have given two radioactive bands by this procedure.
A similar procedure has been used to obtain the 1.3 SE and 5 Sn subunits from the 6 SE subunit shown in Fig. 20, bottom.
Gel electrophoresis of preparations of the 1.3 SE subunit in the absence of dodecyl sulfate often yields multiple bands. This observation is considered under "Discussion." The concentration of the 1.3 SE subunit has been calculated from its biotin content on the basis that it contains 1 mol of biotin per mol of molecular weight of approximately 12,000 (2). For this purpose, the specific radioactivity (counts per min per nmol of biotin) of a given batch of transcarboxylase is determined from its total radioactivity and biotin content. Then knowing the total radioactivity of the 1.3 SE preparation, its biotin content is calculated from the specific radioactivity of the biotin. Reconstitution of Transcarboxylase from PurifLed 12 SH, 5 SE, and 1 .S XB Subunits-The reconstitution of transcarboxylase has been accomplished previously with the unresolved subunits of dissociated transcarboxylase either by adjustment to pH 5 with acetate buffer or by addition of a high concentration of phosphate buffer, pH 6.5 to 6.8 (3, 7) A similar experiment in which the 12 Sn subunit was limiting isolated 1.3 SE, 5 SE, and 12 Sn subunits does combine to form the and the reconstituted "6 SE" subunit was in excess is shown in active enzyme, but better yields are achieved when the conversion Fig. 7. Four concentrations of 12 Sn subunit were used with an is done by a two-step process. First, the 5 SE and 1.3 S, subunits excess of the reconstituted "6 SE" complex which was prepared in are combined to form the "6 SE" subunit,4 then the resulting acetate buffer at pH 5. The reconstitution was done in 0.75 M "6 SE" subunit is combined with the 12 Sn subunit to yield active phosphate buffer (pH 6.8) and the enzymatic activity was transcarboxylase.
The extent of the conversion is determined by assayed in O.Ol-ml portions. The results of Fig. 7 show that at measurement of the enzymatic activity of the reconstituted 27 hours, the enzymatic activity increased linearly with the enzyme by the usual spectrophotometric assay which measures concentration of the 12 Sn subunit up to 3.2 pg. the oxalacetate formed from pyruvate and methylmalonyl-CoA Determination of Specijic Activity of Subunits-It is evident using malate dehydrogenase.
from the experiments of Figs. 6 and 7 that the specific activity of Linearity of Enzymatic Activity with "6 SE" Subunit Limiting-a given subunit in forming active transcarboxylase could be The results of two experiments are shown in Fig. 6 in which the determined by making the one subunit limiting and the other 5 SE subunit was combined with the 1 3 SE subunit using phos-subunit in excess, much as is usually done in assaying an enzyme. phate buffer at pH 6.5 in one case and acetate buffer at pH 5 A comparison between the activities of the buffer (pH 6.5) which were held at 0" and assayed over a period of reconstituted subunit with that of the native enzyme, therefore, several days. The mixtures contained 0.205 nmol of the 12 Sn requires an estimation of the number of 1.3 S, subunits bound to subunit and 0.029, 0.058, or 0.116 nmol (3.5, 7, and 14 pg) of each 5 SE subunit.
For this purpose, the [3H]biotinyl 1.3 SE the 5 SE subunit, which had been converted in part to the "6 SE" subunit and 5 SE subunit were reconstituted and then the unsubunits.
Thus, the 12 Sn subunit was in large excess. bound 1.3 SE subunit was separated by glycerol gradient cen- Fig. 6 shows that the enzymatic activity (AA per min per 0.01 trifugation from the 5 SE subunits containing bound 1.3 SE ml) was linear at 96 hours with 3.5 and 7 pg of 5 SE subunit when subunits.
The results of such an experiment are shown in Fig. 8. either phosphate at pH 6.5 or acetate at pH 5 was used in the Two peaks of radioactivity were obtained; the first coinciding reconstitution.
The Pool 21 to 28 was used without further purification for the reconstitutions of the experiment of Fig. 9.
923
Pools 21 to 28 from the glycerol gradient were used directly for reconstitution with the 12 Sn subunit in 0.75 M phosphate buffer (pH 6.5). The results are shown in Fig. 9. The proportion of 12 Sn subunits to "6 SE" subunits was varied so as to obtain ratios of sites which varied from 17 to 0.13 assuming a 12 Sn subunit has six sites for binding a 1.3 SE subunit and that one 1.3 SE subunit bound to one 5 SE subunit yields one "6 Se" site. The specific activity of the "6 SE" subunit was calculated from the observed enzymatic activity of the reconstituted mixture and it was assumed that 1 nmol of 1.3 SE subunit bound to a 5 SE subunit is equivalent to 0.072 mg of "6 SE" protein (this follows because 1 nmol of 6 SE subunit containing 2 nmol of 1.3 SE subunits is equal to 0.144 mg). If the enzymatic activity is the same for the 1.3 SE subunit, whether one or two are bound to the 5 SE subunit, then this calculation is correct. 924 activity of 27 for the 18 S form of the enzyme with a full complement of 3 complete 6 SE subunits (50 X 3 X 1.44 X 105/7.90 x lo5 where 1.44 x lo5 is the molecular weight of the 6 SE subunit and 7.90 x lo5 of the 18 S form of the enzyme). The observed specific activity for the 18 S form of the enzyme is approximately 45, thus, the "6 SE" subunit had about 60 y0 of the activity in the reconstituted enzyme as the same amount of this subunit has in the native enzyme. Fig. 9B gives the specific activities based on the content of the 12 Sn subunit.
A maximum specific activity of approximately 40 was reached when the ratios of the "6 SE" subunit sites to 12 Sn subunit sites were 7.6 and 3.0. A specific activity of 40 for the 12 Sn subunit is equivalent to an activity of 18.2 for the 18 S form of transcarboxylase (40 x 3.6 x 105/7.9 X 10" where 3.6 x lo5 is the molecular weight of the 12 Sn subunit and 7.9 x lo5 of the 18 S transcarboxylase).
Thus, the 12 Sn subunit had about 40% of the activity in the reconstituted enzyme as the same amount of this subunit has in the native enzyme.
In other experiments this value has been as high as 50%.
Competition of Subunits with "6 SB" Subunit for Binding with 12 SH Subunit-The 1.3 SE subunit is required to form a complex between the 5 SE and 12 Sn subunits in transcarboxylase (8) and apparently provides the groups that promote this linkage. We have noted that more complete reconstitution with the 12 Sn subunit is obtained if the reconstitution is done in two steps in which the 5 SE plus 1.3 SE subunits are first combined to "6 SE" subunits which are then combined with the 12 Sn subunit. These results suggested that the 1.3 SE subunit, when combined with the 5 SE subunit, might undergo a conformational change which would facilitate combmation with the 12 Sn subunit. If this were so, the free 1.3 SE subunit might not compete effectively with the "6 SE" subunit in forming a complex with the 12 Sn subunit during reconstitution.
To test this hypothesis, the ability of the 1.3 SE subunit to compete with the "6 SE" subunit for 12 Sn sites was investigated in experiments in which the amount of "6 SE" subunit and 12 Sn subunit were maintained about equal on the basis of "sites" and a large excess (approximately go-fold) of 1.3 SE subunit was added during the reconstitution in 0.75 M phosphate buffer.
In addition, the effect of the non-biotinyl peptide was tested. This is the portion of the 1.3 SE subunit which remains combined with transcarboxylase when the biotinyl peptides are cleaved from it by trypsin. This peptide has been isolated and contains the portion of the 1.3 SE subunit which provides the combining groups for linkage of the 5 SE and 12 Sn subunits (8). The effect of an excess of 5 SE subunits was also tested.
The results of these experiments are presented in Table I, which are expressed as enzymatic activity of the reconstituted mixture on the basis of the specific activity of the 12 Sn subunit. It is seen that neither the 1.3 SE subunit nor the nonbiotinyl peptide had a significant influence on the amount of enzyme reconstituted. This lack of inhibition by a large excess of the 1.3 SE subunit compared to the "6 SE" and 12 Sn subunits makes it very likely that the 1.3 SE subunit has a much stronger affinity for the 12 Sn subunit when if forms a complex with the 5 SE subunit than does the free 1.3 SE subunit or the nonbiotinyl pcptide.
Most likely, the 1.3 SE subunit undergoes a conformational change during the formation of the complex which increases its capacity to combine with the 12 Sn subunit.
The 5 SE subunit, as expected, had no influence on the reconstitution of active enzyme.
DISCUSSION
Transcarboxylase is a monofunctional enzyme catalyzing only a single metabolic reaction but in many respects it resembles the multi-enzyme complexes with components capable of catalyzing two or more metabolic reactions.
Ciassical examples of the multiple enzyme complexes are the cr-keto acid dehydrogenases (18) and the fatty acid synthetases (19-21) in which the lipoyl moiety serves as an arm to link the various enzymes of the a-keto acid dehydrogenases and the 4'-phosphopantetheine to link the enzymes of the fatty acid synthetascs.
Transcarboxylase is similar because it involves two partial reactions (shown below) which in combination make up the over-all reaction. 12 complex in which the lipoyl or the phosphopantetheine group serves as the link. There are numerous biotinyl enzymes in which the biotinyl group serves as a carboxyl carrier (see review by Moss and Lane (23)) but transcarboxylase is the only one which has been isolated as an intact complex, dissociated to its subunits, the subunits isolated, the partial reactions studied with the subunits, and the active enzyme complex reassembled from the isolated subunits.
925
Similar studies have been done with acetyl-CoA carboxylase of E. coli (24) but in this case the complex has not been isolated nor have the constituent subunits been reassembled into a complex. In this regard, acetyl-CoA carboxylase of E. coli resembles the fatty acid synthetase from E. coli, which has not as yet been obtained as a multi-enzyme complex (24, 25). Thus far, we have devoted most of our efforts to isolating the 12 Sn, 6 SE, 5 SE, and 1.3 SE subunits by chromatography on Bio-Gel; separation of the 6 Sn, 6 SE, and 5 SE subunits by this technique is not feasible because of their nearly identical size. Although studies with the 6 Sn subunit have been very limited, it seems likely that the 6 Sn subunit does reassociate to form the 12 Sn subunit.
For example, a major portion of the dissociated enzyme of the experiment of Fig. 2 had a sedimentation coefficient of approximately 6 S (Fig. 2B). When this material was passed through a 13io-Gel column much of it was recovered in the form of the reconstituted -18 S enzyme and the 12 Sn subunit (Fig. 2C). Clearly, the 6 Sn subunits were reconverted to the 12 Sn subunit during the chromatography and subsequently combined with 6 SE subunits to form the -18 8 enzyme.
The 12 Sn subunit has been isolated from dead, normal (17), and trypsinizcd transcarboxylasc (8). Each preparation has been found to be active with 6 SE subunits in yielding reconstitutcd active enzyme.
The 12 Sn subunit from trypsinizcd transcarboxylase has had a somewhat lower specific enzymatic activity on reeonstitution than the other 12 Sn subunits. The 5 SE subunits from all three sources have been found to be active but the activities of the different preparations have not been compared extensively.
Both the 12 Sn and 5 SE subunits have consistently been obtained in nearly homogeneous form as judged by sedimentation velocity and by gel electrophoresis.
The same has not been true of the 1.3 SE subunit.
Gel electrophoresis in sodium dodecyl sulfate has yielded a single band with a number of preparations but some preparations have given two major bands. Gel electronhoresis in the absence of dodecvl sulfate but in the presence of mercaptoethanol frequently yields multiple bands which may result from aggregation.
Likewise, there is evidence of a self-associating system during sedimentation equilibrium studies of the 1.3 SE subunit6 A further difficulty with the 1.3 SE subunit is that with storage it frequently loses its capacity to promote formation of active enzyme in combination with the 5 SE and 12 Sn subunits.
Most of the studies reported here were done with preparations that had been stored frozen no more than 2 or 3 weeks (usually less).
Vagelos and co-workers in a series of publications (26-30) have described multiple forms of the biotinyl carboxyl carrier protein from E. coli. Molecular weights of 9,065, 10,267, -22,500, and -45,000 have been observed and prolonged dialysis of the homogeneous protein resulted in polydisperse mixtures with molecular weights ranging from 20,000 to greater than 200,000 (29). They propose that the protein of molecular weight 45,000 is a dimer and is the native form of the carboxyl carrier protein and that the forms smaller than 22,500 arise by proteolysis which occurs during the isolation of the protein (30).
We have attempted to prevent proteolysis by use of benzamidine-HCl as employed by Fujikawa et al. (31) and also of phenylmethylsulfonyl fluoride (32). The cells were broken in the presence of these inhibit.ors and they also were included at each stage of purification of the enzyme. The dissociation was done by addition of the enzyme to boiling 6 M urea plus lop3 M 6 F. Ahmad, unpublished observations. dithiothreitol. Gel electrophoresis in the presence of dodecyl sulfate gave a major single band of the 1.3 SE subunit.
Our preparations have molecular weights of 11,000 to 13,000.
The present study is the first that the authors are aware of in which it has been possible to determine the activity of a subunit in forming the active enzyme by making the given subunit limiting and adding the other subunits to it in cxccss. The comparison of the activity of the given subunit with its activity in the intact enzyme is made somewhat uncertain, however, because various preparations of transcarboxylase, albeit pure as judged by their sedimentation behavior, possess varying specific activities.
There are two factors which may influence the specific activity.
The first factor is the number of peripheral biotinyl subunits that are attached to the central subunit.
The 18 S form of the enzyme has three peripheral subunits and has a specific activity of approximately 45 but values in the 50s have been observed occasionally.
The 16 S form has only two peripheral subunits and has a correspondingly lower activity. In addition, there is a -24 S form of the enzyme2 (1, 6) which may have six peripheral subunits attached to the central 12 Sn subunit. This form has not been isolated and its specific activity is not known but may be greater than that of the 18 S form. The second factor influencing specific activity is manifest by a loss of enzymatic activity (sometimes quite rapidly) which is not accompanied by a change in the sedimentation coefficient or the dissociation to subunits (6,12). Sometimes this loss in activity can be restored by incubation in 1.5 M (NH&S04 at 25" (6). The above factors make it difficult to evaluate the efficiency of reconstitution of active enzyme from the isolated subunits. We have chosen as our standard of comparison a specific activity of approximately 45 for the 18 S form of the enzyme and have compared the observed specific activity of the subunits with this value. Thus, the theoretical maximum specific activity for the 12 Sn subunit is 98.8 (45 X 7.9 x 105/3.6 X 105) and for the 6 SE subunit is 82.3 (45 X 7.9 x 105/3 X 1.44 X 105) where 7.9 x lo5 is the molecular weight of the 18 S form of the enzyme, 3.6 x 10" of the 12 Sn subunit and 1.44 X lo5 of the 6 SE subunit (1). On this basis, when the 12 Sn subunit was made limiting and the reconstituted "6 SE" subunit4 was added in excess, the specific activity of the 12 Sn subunit had about 507, of its potential activity.
There are several factors which may cause the specific activity to be lower than that calculated from the standard.
One is the uncertainty of the value to be used for the standard as explained above. A more important factor is the fact that the "6 SE" subunit formed by reconstitution from the 5 SE and 1.3 SE subunits only carried about 50 y0 (Fig. 8) of the full complement of 1.3 SE subunits which is two per 5 SE subunit. Thus, when the 12 Sn subunit is made limiting, three peripheral "6 SE" subunits which are deficient in the 1.3 SE subunits may form a complex with the 12 Sn subunit.
In this case, some of the 12 Sn sites would be ineffective in the transcarboxylation reaction because they would lack the biotinyl carboxyl carrier protein which is essential for the activity of that site to become evident. It is not clear why we have been unsuccessful in obtaining a complete conversion of the 5 SE subunit to a 6 SE subunit with its full complement of 1.3 SE subunits.
Perhaps a larger excess of the 1.3 SE subunit is required than was used in the reconstitution of the "6 S n" subunit.
The situation with regard to the specific activity of the "6 SE" subunit is somewhat different.
Here, the calculation of its specific activity has been done on the basis of each nanomole of complexed 1.3 SE subunit being equivalent to 0.072 mg of "6 SE" subunit (0.144 mg = 1 nmol of 6 SE with 2 nmol of 1.3 SE).
926
Thus, if there is only one 1.3 SE subunit in combination with a 5 SE subunit, only one-half of the weight of 6 SE is considered in calculating the specific activity and as noted above, a theoretical maximum specific activity of 82 would be anticipated for the 6 SE subunit.
The observed value was about 50 (Fig. 9) when the 12 SH subunit was in large excess. Under these conditions, the reconstituted enzyme may consist of forms with only one peripheral "6 SE" subunit combined with a 12 SH subunit.
Possibly there is cooperativity and a single "6 SE" subunit bound to a 12 SH subunit is less effective than when two or more are bound on the same 12 SH subunit.
A more detailed study is required to obtain information about this possibility. | v3-fos-license |
2017-10-18T17:06:26.992Z | 2017-10-18T00:00:00.000 | 28911164 | {
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} | pes2o/s2orc | Uncovering Active Constituents Responsible for Different Activities of Raw and Steamed Panax notoginseng Roots
Although Panax notoginseng (PN) roots in raw and steamed forms were historically supposed to be different in the efficacies, the raw materials and steamed ones were often undifferentiated in the use and market circulation, which might bring unstable curative effects or even adverse reactions. To uncover chemical constituents responsible to different activities of raw and steamed PN, chemometrics analyses including partial least squares regression (PLSR) and multi-linear regression analysis (MLRA) were used to establish the relationships between the chromatographic fingerprints and activities of PN samples. Chemical fingerprints of PN were determined by HPLC. Anticoagulant and antioxidant activities were evaluated by the thromboplastin inhibiting test and hydroxyl radical scavenging assay, respectively. Results showed that there was a significant difference in the chemical composition between raw and steamed PN, which could be discriminated by principle component analysis according to different steaming temperatures. Compared with the steamed PN, raw PN exhibited stronger anticoagulation and weaker antioxidation. By chemometrics analyses, notoginsenoside R1, ginsenosides Rg1, Re, Rb1, and Rd were found to be the major active constituents of raw PN, whereas ginsenosides Rh1, Rk3, Rh4, and 20(R)-Rg3 had the key role in the activities of steamed PN, which could be used as new markers for the quality control (QC) of steamed PN.
INTRODUCTION
The processing of herbal medicines, including special crafts of steaming, baking, cooking, and other methods with liquid or solid supplementary materials, plays an important role in the application of traditional Chinese medicine (TCM). The purposes of processing include transforming the properties of medicines, strengthening the curative efficacy, generating new effects and reducing the toxicity or side-effects Li et al., 2013). In recent decades, it has been found that the main mechanism underlying the property changes of herbs is mainly related to the alteration in the chemical composition and/or bioactivity of constituents in herbs (Cao et al., 2011;Li S. L. et al., 2012). For example, diester diterpene alkaloids responsible for the toxicity of Aconitum Radix could be decomposed into less or non-toxic derivatives by boiling the raw materials at 100 • C for 8 h before drying it (Sun et al., 2012). Thus, the uses of raw and processed medicines cannot be mixed up for different curative or toxic effects.
Panax notoginseng (PN) Burk., a plant in genus Panax (Araliaceae), is a well-known medicinal herb used to treat blood disorders for more than 400 years (Ge et al., 2015;Li et al., 2016). Based on the US Dietary Supplement Health and Education Act of 1944, PN and the relative products were also classified as dietary supplements (103rd Congress, 1994). Traditionally, there are two forms of PN, namely raw PN and steamed PN, of which the former one is used as a hemostatic, traumatic, and cardiovascular medicine removing blood stasis, while the steamed one is used as a tonic for nourishing the body and improving the health (Ge et al., 2015;Gu et al., 2015). Pharmacologic studies have also shown that the effects of PN changed when steamed. Lau et al. (2009) reported that the treatment of raw PN extract resulted in shorter bleeding time compared with rats treated with steamed PN, which is consistent with the traditional use of raw PN as a hemostatic. While for steamed PN, it could significantly increase the levels of hemoglobin and white blood cells, as well as the organ index of mice with blood deficiency caused by cyclophosphamide, which were inapparent when treated with raw PN (Zhou et al., 2014). Triterpenoid saponins including ginsenosides and notoginsenosides are considered to be the major bioactive constituents of PN, among which ginsenosides Rg 1 , Rb 1 , Re, and notoginsenoside R 1 show higher levels than others in raw PN (Kim, 2012).
Despite the difference in traditional uses and pharmacological effects, trade in raw and steamed PN has still been complicated by the mixed use. Until now, only raw PN material or powder is recorded in the pharmacopeias of different countries or regions, in which the standard of processed PN is not included yet (British Pharmacopoeia Commission, 2014;Chinese Pharmacopoeia Commission, 2015; European Pharmacopoeia 8.0 1 ; U.S.A Herbal Medicines Compendium 1.0 2 ), indicating that the difference between the raw and steamed PN has not been authorized by codex standards. Even for some institution standards involving steamed PN, the marker constituents for its quality control (QC) are consistent with those of raw PN, despite the significant change in the chemical composition of PN during the steaming process reported in several studies Ge et al., 2015). For example, in the China Food and Drug Administration (2012), the marker constituents for the QC of steamed PN powder are ginsenoside Rb 1 , ginsenoside Rg 1 , and notoginsenoside R 1 , which are same as the markers of raw PN involved in Chinese Pharmacopoeia of 2015 edition. Due to the lack of respective quality standards to differentiate raw and steamed PN, there would be risks of unstable curative effects or even adverse reactions for customers. Therefore, besides the investigation on changes in the chemical composition and pharmacological effects of PN during the steaming process, constituents responsible to different activities should also be uncovered for the individualized QC of raw and steamed PN, which have not been reported yet.
Since the traditional efficacy of removing blood stasis and promoting blood circulation of raw PN could be related to the anticoagulant effect , and the body tonifying function of herbal medicines is partly attributed to their antioxidant and immunomodulatory effects by modern pharmacological researches (Yim and Ko, 2002), the anticoagulation (obtained by thromboplastin inhibiting test) and antioxidation (obtained by hydroxyl radical scavenging assay) activities of PN during the steaming process were studied in this research. Meanwhile, we developed the HPLC chromatographic fingerprints of PN under different steaming conditions, and investigated the correlation between the activities and fingerprints of PN samples by using multivariate regression techniques including principal component analysis (PCA), partial least squares regression (PLSR), and multi-linear regression analysis (MLRA). Constituents/peaks predicted to be responsible for different activities of PN were then identified, of which the activities were finally verified by pharmacologic tests.
Sample Preparation
Samples were obtained from a single batch of PN root in Yunnan, China. Steamed PN samples were prepared by steaming the crushed raw PN in an autoclave (Shanghai, China) for 2, 4, 6, 8, and 10 h at 105, 110, and 120 • C, respectively. The steamed powder was then dried in a heating-air drying oven at about 45 • C to constant weight, then powdered and sieved through a 40-mesh sieve.
Animals
Kunming mice, male and female, weighing 18-22 g, were purchased from Tianqin Biotechnology Co. Ltd., Changsha, Hunan (SCXK (Xiang) 2014-0011). Before the experiments, the mice were given 1-week acclimation period in a laboratory at room temperature (20-25 • C) and constant humidity (40-70%), and fed with standard rodent chow and tap water freely. Animal experimental procedures in the study were strictly conformed to the Guide for the Care and Use of Laboratory Animals and related ethics regulations of Kunming University of Science and Technology. The protocol was approved by the Experimental Animal Welfare and Ethics Committee, Kunming University of Science and Technology.
HPLC Analyses
The sample solutions were prepared according to the method described in Chinese Pharmacopoeia Commission (2015). HPLC analyses were done on an Agilent 1260 series system (Agilent Technologies, Santa Clara, CA, USA) consisting of a G1311B pump, a G4212B DAD detector and a G1329B autosampler. A Vision HT C 18 column (250 × 4.6 mm, 5 µm) was adopted for the analyses. The mobile phase consisted of A (ultra pure water) and B (acetonitrile). The gradient mode was as follows: 0-20 min, 80% A; 20-45 min, 54% A; 45-55 min, 45% A; 55-60 min, 45% A; 60-65 min, 100% B; 65-70 min, 80% A; 70-90 min, 80% A. The flow rate was set at 1.0 mL/min. The detection wavelength was set at 203 nm. The column temperature was set at 30 • C and sample volume was set at 10 µL.
Anticoagulation Test in Vitro
Blood was collected from healthy mice and directly transferred into citrated tubes (0.109 mol citrate, 9:1). The supernatant platelet-poor plasma (PPP) was obtained by centrifuging the blood samples above at 3,000 rpm for 10-15 min. The mixture of PPP and thrombokinase of various concentrations at the proportion of 2:1 (v/v) of total 50 µL was added into the test cup and incubated for 3 min at 37 • C in a Blood Coagulation Instrument (XN06 series, Diagnostic Technology Ltd of Wuhan Jingchuan, China). 10 U/mL thrombokinase of 100 µL dissolved in 0.1 mol/L Tris-HCl buffer solution (pH 7.4) was subsequently added and incubated at the same condition. The prothrombin time (PT) was determined in accordance with the manufacturer's recommended protocols. The prolongation rate of PT was calculated according to the following equation: where PT 0 was the PT of control (blank, the normal saline replaced of thrombokinase), PT was the PT in the presence of thrombokinase.
The standard curve was drawn with the concentration of thrombokinase (U i ) as the X axis and the lg [prolongation rate of PT (%)] as the Y axis. PN samples of 5 g, in the powdered form, were extracted with pure water (50.0 mL) by refluxing twice for 2 h at 80 • C. The combined solution was filtered and concentrated under reduced pressure to the extract containing 0.1 g/mL of PN. The extract was then diluted with the normal saline to different concentrations. The prothrombin time of the mixed plasma sample containing PPP and PN extract (PT') of different concentrations was determined. The prolongation rate of PT' was calculated according to the following equation: Prolongation rate of PT'(%) = (PT' − PT 0 ')/PT 0 '×100% (2) where PT 0 ' was the prothrombin time of control (blank, the normal saline replaced of extracts), PT' was the prothrombin time in the presence of extracts.
The corresponding concentration of thrombokinase (U i ) was determined according to the standard curve. And the thromboplastin inhibition rate (%) was calculated according to the following equation: Thromboplastin inhibition rate (%) = (U i −10)/10×100% (3) where U i was the concentration of thromboplastin determined by the standard curve.
Antioxidation Test in Vitro
The extracts prepared in "Anticoagulation Test in vitro" were diluted with normal saline to 0.5, 1, 1.5, 2, 2.5, 3, and 3.5 mg/mL, respectively. The scavenging activity for hydroxyl radicals was measured according to the procedure described by Zhao et al. (2006). Reaction mixture contained 60 µL of 1.0 mmol FeCl 2 , 90 µL of 1 mmol 1,10-phenanthroline, 2.4 mL of 0.2 mol phosphate buffer (pH7.8), 150 µL of 0.17 mol H 2 O 2 , and 1.5 mL of extracts prepared. The reaction was started by adding H 2 O 2 . After incubation at room temperature for 5 min, the absorbance of the mixture at 560 nm was measured with a spectrophotometer. The scavenging activity for hydroxyl radicals was calculated according to the following equations: Where A 0 was the absorbance of the control, A 1 was the absorbance in the presence of the extract and A 2 was the absorbance without 1,10-phenanthroline.
Pharmacologic Verification
Constituents/peaks predicted to be responsible for different activities of PN were then identified by reference standards, of which the activities were finally verified by pharmacologic evaluation using the methods described in section "Anticoagulation Test in vitro" and "Antioxidation Test in vitro."
Statistical Analyses
All data were expressed as means ± SD. SPSS 21.0 software (Statistical Program for Social Sciences, SPSS inc, Chicago) was applied to carry out the two-tailed unpaired t-test and PCA. DPS 9.50 software (Data Processing System, China) was used for MLRA. Umetrics SIMCA-P 11.5 software (Sartorius Stedim Biotech, Sweden) was applied for PLS analysis. A value of P < 0.05 was considered to be significant difference. A value of P < 0.01 was considered to be highly significant difference. EC 50 value was fitted by Probit regression with Origin 7.5 for windows (OriginLab Corporation, USA) software.
THEORY PCA
PCA is applied for data compression and visualization. PCA produces so-called latent variables, here called principal components (PCs), which are linear combinations of the original manifest variables. The orthogonal PCs are constructed in such way that they maximize the description of the data variance in the n × p data matrix X. The projections of the objects (PN samples) on PC i are the scores on PC i , and the projections of the variables (HPLC fingerprint signals here) on PC i are the loadings on PC i . Thus, score plots give information related to the (dis)similarity of the objects, while on loading plots information about the contribution of the original variables to a given PC i can be found (Nguyen Hoai et al., 2009).
MLRA MLRA attempts to model the relationship between two or more variables and a response by fitting a linear equation to observed data (Noori et al., 2010;Placca et al., 2010). The general purpose of MLRA is to learn about the relationship between several independent variables and a dependent variable. MLRA can be generally represented in the following form: where Y is the estimated value and represents the dependent variable. X 1 , X 2 , X 3 ,..., X n are measures of not correlated variables that may help in estimating Y. For example, X 1 is the known score of the first independent variable, X 2 is the known score of the second independent variable, etc. The coefficient b 0 is the estimated constant, and b 1 , b 2 , b 3 ..., b n are called the regression coefficients (Hair et al., 1999).
PLSR
PLSR is used to find the inner relationship between independent variables (X) and dependent variables (Y), which are simultaneously modeled by taking into account not only X variance, but the covariance between X and Y (Martens and Naes, 1989). In our study, the X matrix is composed of the enhanced fingerprints and the Y vector is constructed with the reference values of anticoagulation and antioxidation activities (EC 50 ) obtained by the thromboplastin inhibition rate and hydroxyl radicals assay, respectively. Then, X and Y are decomposed in a product of another two matrices of scores and loadings; as described by the following equations: where TP T approximates to the chromatographic data and UQ T to the true Y values; notice that the relationship between T and U scores is a summary of the relationship between X and Y. The terms E and F from the equations are error matrices. Hence, the PLS algorithm attempts to find factors (called Latent Variables) that maximize the amount of variation explained in X that is relevant for predicting Y; i.e., capture variance and achieve correlation (Brereton, 2007).
HPLC Fingerprints
The results of methodology validation showed that the relative standard deviation values for precision, reproducibility and storage stability were less than 3.0, 4.0, and 3.0%, respectively. All the results indicated that the method of HPLC for the fingerprint analyses was valid and satisfactory. The optimized conditions for the 90-min HPLC fingerprints were described in sections "HPLC Analyses." The chromatograms were generated for all batches of PN samples (Figure 1A), and for a typical raw PN sample and a steamed PN sample ( Figure 1B). Peaks with good segregation, which also occupied large areas from consecutive peaks, were determined as the common peaks of PN samples. Therefore, fifteen peaks were selected by comparing their ultraviolet spectra and HPLC retention time. The method used to identify common peaks refers to reports in similar researches (Zheng et al., 2014;Shi et al., 2016). Along with the duration of steaming time and rise of temperature, the area and height of major peaks (peaks 1-3, and 6-8) in the raw PN were decreased gradually, while other peaks (peaks 5, 9-15) were increased or formed ( Figure 1B). The areas of 15 peaks in 18 batches of PN samples were listed in Table 1. The peak area was defined as 0 for peaks lacked in chromatograms. The coefficients of variance for almost all common peaks were higher than 46.6%. This is due to the diversity in the levels of constituents contained in samples under different process conditions. The areas of 15 common peaks were used for the following analysis.
Clustering Results by PCA
PCA is a classical technique to reduce the dimensionality of the data set by transforming to a new set of variables, named PCs to summarize the features of the data. Since PCs are uncorrelated and ordered, the first few PCs, which contain most of the variations in the data, are usually used in cluster analysis (Yeung and Ruzzo, 2001). As shown in Figure 2, the fingerprints of PN samples were separated into four clusters according to the peak area. Samples 1-3 in cluster one were raw PN. Samples 4-8 in cluster two were PN steamed at 105 • C. Samples 9-13 in cluster three were PN steamed at 110 • C. Samples 14-18 in cluster four were PN steamed at 120 • C. It indicated that PN samples steamed at the same temperature had similar chemical fingerprints. And PCA could initially separated raw and steamed PN samples at different temperatures from the chemical level.
The results also suggested that compared with the steaming time, the steaming temperature had a more important role in the change of chemical composition of PN. Based on the results, a combination of PCA and HPLC methods could, at least roughly, discriminate raw and PN samples under different process conditions.
Anticoagulation Test
PT is used to evaluate the overall efficiency of extrinsic clotting pathway. A prolonged PT indicates a deficiency in coagulation factors V, VII, and X (Chan et al., 2007). In the study, the EC 50 determined by the logarithm to base 10 of PT prolongation rate was applied to evaluate the anticoagulant effect of PN. The standard curve between the concentration of thrombokinase and logarithm of PT prolongation rate showed a good linearity (R = −0.9991). As shown in Figure 3A, raw PN samples (S1-S3) exhibited lower EC 50 values of anticoagulation, suggesting that the anticoagulant effect of raw PN was stronger compared with steamed PN samples. The EC 50 values of PN steamed at the same temperature were generally increased along with the increase of steaming time. For PN steamed for the same time, the higher the steaming temperature, the higher was the EC 50 values of samples. Among these samples, S18 steamed for the longest time of 10 h at the highest temperature of 120 • C showed the highest EC 50 value, suggesting that its anticoagulant activity was the weakest compared with other ones.
Antioxidation Test
PN roots are mostly consumed as popular food tonic in the soup form by people in the southern region of China. Various studies have suggested that the tonifying functions of Chinese herbal medicines could be due to, at least partially, the protective effects against oxidation (Yim and Ko, 2002). Hydroxyl radical is very reactive which can be generated in biological cells through the Fenton reaction. Meanwhile, hydroxyl radical scavenging assay is commonly used for the determination of antioxidant activities of plant extracts. And PN showed higher sensitivity of scavenging hydroxyl radicals than other ones like 1,1-diphenyl-2-picrylhydrazyl free radicals in our previous work ( Figure S1). Therefore, the method was applied to investigate the antioxidant effect of raw and steamed PN roots, with results shown in Figure 3B, where EC 50 was the concentration of PN scavenging 50% hydroxyl radicals. According to the results, raw PN samples (S1-S3) exhibited much higher EC 50 values of antioxidation, suggesting that the antioxidant effect of raw PN was much weaker compared with steamed PN samples. PN steamed at 120 • C showed general lower EC 50 values than samples steamed at lower temperatures, suggesting that higher steaming temperature was related to stronger antioxidation activity of PN.
MLRA
The relationship between the fifteen independent variables X 1 , X 2 , X 3 ,..., X 15 (the values of normalized peak areas) and the dependent variable Y of each activity was established by fitting a linear equation to observed data with multiple linear regression model. The regression equations of anticoagulation and antioxidation were shown as follows:$$ where Y anticoagulation was the EC 50 of lg [prolongation rate of PT (%)], Y antioxidation was the EC 50 of hydroxyl radicals scavenging activity, X 1 -X 15 were the normalized peak areas of peaks 1-15 (Figure 1), respectively. The F-values for the two equations were 8.82 and 13.16, respectively. And the corresponding Pvalues were <0.01 (R 2 = 0.9996), and <0.05 (R 2 = 0.9999), respectively, showing that the established MLRA models were satisfied. According to the equations, EC 50 values of a new PN sample could be obtained by inputting the corresponding peak areas to preliminarily evaluate the anticoagulant and antioxidant activities of this sample. For the anticoagulant activity, values of Pr > |T| of X 2 , X 3 , X 5 , X 6 , X 7 , X 10 , and X 11 were all <0.05, suggesting that constituents corresponding to peaks 2, 3, 5, 6, 7, 10, and 11 had more important role in the anticoagulation of PN. From Table 1 and Figure 1B, peaks 2, 3, 5, 6, and 7 were observed in the chromatographic fingerprints of raw PN. Among them, only peak 5 showed an increase trend along with the increase of steaming temperature and time. For steamed PN, peaks 10 and 11 were exclusively existed in the fingerprints. Thus, constituents corresponding to peaks 2, 3, 6, and 7 might play the major role in the anticoagulation of raw PN, whereas constituents corresponding to peaks 5, 10, and 11 could be the major active ones for the anticoagulation of steamed PN.
For the antioxidant activity, values of Pr > |T| of X 2 , X 3 , X 5 , X 7 , X 10 , and X 13 were all <0.05, indicating that constituents corresponding to peaks 2, 3, 5, 7, 10, and 13 had more significant influence on the scavenging activity of hydroxyl radicals. From Table 1 and Figure 1B, peaks 2, 3, 5, and 7 were observed in the chromatographic fingerprints of raw PN. And peaks 10 and 13 are exclusively existed in the fingerprints of steamed PN. The area of peak 5 was increased along with the increase of steaming temperature and time. Therefore, constituents corresponding to peaks 2, 3, and 7 had the major role in the antioxidation of raw PN, whereas constituents corresponding to peaks 5, 10, and 13 were the major active ones for the antioxidation of steamed PN. The variations in the contents and contribution degrees of above constituents to the activities of PN may lead to the difference in the anticoagulant and antioxidant effects of raw and steamed PN samples.
PLSR
The PLSR models to correlate chromatographic data and the activities of PN were constructed with the 18 batches of PN samples. Since the total number of samples (18) was small and since the prediction for new samples was not our first concern, no division was made into a calibration set to build a PLSR model and a test set to validate the predictive properties. Our main concern was to focus on the indication of anticoagulant and antioxidant peaks from the modeling results. PLSR models were built from the normalized data matrix X containing the 18 PN fingerprints and the response matrix Y, i.e., either the EC 50 of lg [prolongation rate of PT (%)] or the EC 50 of hydroxyl radicals scavenging activity. For the anticoagulation model, two principle components were achieved, accounting for an explained variance of 89.9% for X variable, 84.3% for Y variable, and a predictive ability (Q 2 ) of 85.3% (Table S1), indicating the obtained model was excellent. As shown in the regression coefficients plot (Figure 4A), peaks 4, 5, and 9-15 were positively correlated with the EC 50 of lg [prolongation rate of PT (%)], whereas peaks 1-3 and 6-8 were negatively correlated with the EC 50 value. It should be noted that the predicted EC 50 values could not be defined if these variables increased or decreased, because a negative coefficient did not necessarily mean that the relevant variable has the opposite effect on the anticoagulant activity. Besides, the importance of the X-variables for the model could be summarized by variable importance for the projection (VIP) values (usually with a threshold >1.0). Thus, constituents corresponding to peaks 1, 2, 5, 10, and 11, of which the VIP values were >1.0 (Table S2) with high absolute values of coefficients were considered to be highly related to the anticoagulant activity of PN samples. For the antioxidant model, two principle components were achieved, accounting for an explained variance of 89.9% for X variable, 83.1% for Y variable, and a predictive ability (Q 2 ) of 65.8% (Table S1), indicating the obtained model was excellent. As shown in the regression coefficients plot (Figure 4B), peaks 1, 2, 4, 6, 8, and 11 were positively correlated with the EC 50 of lg [prolongation rate of PT (%)], whereas peaks 3, 5, 7, 9, 10, and 12-15 were negatively correlated with the EC 50 value. Besides, the VIP value of each peak was shown in Table S2. Therefore, constituents corresponding to peaks 3, 5, 10, and 13, of which the VIP values were >1.0 with high absolute values of coefficients were considered to be highly related to the antioxidant activity of PN samples.
Next, the contents of those constituents in raw and steamed PN were determined, as shown in Figure 6. The contents of all the constituents were significantly different between the raw PN and PN samples steamed at 120 • C, and could be markers for the QC of raw and steamed PN.
Pharmacological Verification
In order to verify the predicted active constituents and determine their contributions to each activity, the anticoagulation and antioxidation of the nine identified constituents were tested. As shown in Figure 7A, the sequence of the anticoagulant activity of the constituents was ginsenoside Rd > ginsenoside Rg 1 > ginsenoside Re > ginsenoside Rb 1 > ginsenoside Rh 1 > ginsenoside Rh 4 > notoginsenoside R 1 > ginsenoside Rk 3 > 20 (R)-Rg 3 . Meanwhile, Figure 7B showed that the antioxidant activity in descending order was ginsenoside Rg 1 > ginsenoside Rd > ginsenoside Rk 3 > ginsenoside Rh 1 > ginsenoside Re > ginsenoside 20 (R)-Rg 3 > notoginsenoside R 1 > ginsenoside Rh 4 > ginsenoside Rb 1 . According to the results, ginsenosides Rd, Rg 1 , Re , and Rb 1 , and notoginsenoside R 1 , with stronger anticoagulant activities than other constituents, and higher levels in raw PN than steamed ones, were the major active constituents for the anticoagulation of raw PN, which was consistent with the predicted result of chemometrics analyses. Among the five constituents, Rg 1 , Rd, and Re also showed certain degrees of antioxidant activity, which should be the major antioxidant constituents of raw PN. Conversely, the levels of the three constituents were decreased in PN along with the increase of steaming temperature and duration of time. And other constituents of ginsenosides Rk 3 , Rh 1 , 20 (R)-Rg 3 , and Rh 4 with higher levels or exclusively existed in steamed PN should have more important role in the activities of steamed PN.
DISCUSSION
PN is widely used as a herbal medicine or food tonic in the global market. In order to control the quality of PN, several constituents including notoginsenoside R 1 , and some or all of ginsenosides Rg 1 , Re, Rb 1 , and Rd are determined as markers in the quality standards of different countries or regions (British Pharmacopoeia Commission, 2014;Chinese Pharmacopoeia Commission, 2015; European Pharmacopoeia 8.0; U.S.A Herbal Medicines Compendium 1.0). PN in raw and steamed forms are considered to be different in the medicinal qualities by practitioners of Oriental medicine: the raw materials eliminate and the steamed ones tonify. The so-called "eliminate" means raw PN can move stagnant blood, promote blood circulation, stopping bleeding, and resolving swelling. And the "tonify" means steamed PN can tonify the blood, enhance the immunity and anti-aging (Ge et al., 2015;Gu et al., 2015). Besides the differentiated use of raw and steamed PN by traditional medicine practitioners, the differences in the chemical composition and pharmacologic effects between raw PN and steamed PN have also been verified by modern researches (Lau et al., 2009;Wang et al., 2012). In our research, along with the duration of steaming, some peaks in the chromatograms of raw PN were decreased, whereas other ones were increased or formed. That transformation might be due to the hydrolyzation or dehydration of constituents induced by high temperature. Besides, raw PN was found to be much better in the anticoagulation than the antioxidation compared with the steamed PN, suggesting that raw PN was more suitable to treat coagulation disorders. With the increase of steaming time and temperature, the anticoagulant activity of PN weakened and the antioxidant effect strengthened, which was consistent with the traditional description of medicinal properties of raw and steamed PN. The difference in the pharmacologic effects between raw and steamed PN could be attributed to the change in the chemical composition of PN during the steaming process. However, such differences have still not been acknowledged by national statutes, in terms of the fact that QC markers for raw and steamed PN were undifferentiated at present. This may be due to that the relationships between efficacies and specific constituents of raw and steamed PN remain ambiguous.
The typical approach to select bioactive chemical markers in herbal medicines usually involves two sequential procedures: phytochemical isolation and purification to obtain pure compounds, in vitro and/or in vivo bioactivity evaluation of the individual isolates. There are some shortfalls of this reductionist methodology, such as ignorance of the possible synergism of multiple constituents, missing of the minor components, and too much time-and labor-consumption (Jiang et al., 2010). To overcome these shortcomings, the analysis of fingerprinteffect relationship has been applied to screen characteristic constituents with bioactivity related to the efficacy of a herbal FIGURE 6 | The contents of notoginsenoside R 1 , ginsenosides Rg 1 , Re, Rh 1 , Rb 1 , Rd, Rk 3 , Rh 4 , and 20 (R)-Rg 3 in raw and steamed Panax notoginseng (PN) roots at different steaming temperatures. The points on the scatter plots represent the quantities of the nine constituents in 18 batches of PN samples. Error bars represent mean ± s.e.m. From a two-tailed unpaired Student's t-test, *P < 0.05, **P < 0.01. Frontiers in Pharmacology | www.frontiersin.org medicine. Multivariate regression techniques such as PLSR, PCR, and MLRA are often used to correlate the bioactivity of a herbal medicine and its fingerprint. Regression is a generic term for all methods intended to adjust a model to the observed data, with the purpose of quantifying the relationship between two groups of variables. The adjusted model can then be used either to describe the relationship between the two variables or to predict new variables. Among these techniques, MLRA and PLSR are frequently used to specify a linear relationship between a set of dependent variables from a large set of independent variables, especially when the sample size is small relative to the dimension of these variables (Garza-Juárez et al., 2011;Wu et al., 2015). In this study, the two chemometrics modeling methods, MLRA and PLSR, were preliminarily applied to predict bioactive constituents of raw and steamed PN. Combined with the verification of pharmacologic tests, constituents responsible to different bioeffects of PN were rapidly uncovered. According to the results, nine constituents differently distributed in raw and steamed PN were predicted to be active ones related to different activities of raw and steamed PN. Notoginsenoside R 1 , ginsenosides Rg 1 , Re, Rb 1 , and Rd were predicted by MLRA and PLSR to be the major constituents related to the anticoagulation of raw PN, which was consistent with the measured EC 50 values of anticoagulation. Among them, ginsenosides Rg 1 , Re, and Rd also showed a certain degree of antioxidation, giving evidence for determining these constituents as QC markers of raw PN. Whereas, for steamed PN samples, we found that ginsenosides Rh 1 , Rk 3 , 20 (R)-Rg 3 , and Rh 4 with higher levels or exclusively existed in them could be the major constituents contributing to the activities of steamed PN. Conversely, notoginsenoside R 1 , ginsenosides Rg 1 , Re, Rb 1 , and Rd, as QC markers of raw PN, showed little or no contents in PN steamed at higher temperatures (110 and 120 • C) or for a longer time, which were samples with strong antioxidation (Table 1, Figure 3). Therefore, active constituents as markers for the QC of steamed PN should be different from raw PN, because QC markers of a herbal medicine should be correlated with its safety and efficacy (Capasso et al., 2000).
Ensuring the safety and efficacy of drugs involves multiple considerations and the quality of the drug must be fundamentally guaranteed. An important part of drug QC is to ensure consistent medical and biological effects are delivered by the same drug dosage (Busse, 2000). A differentiated QC standard of steamed PN from raw PN is necessary to ensure the accuracy and safety in clinic use. Meanwhile, the anticoagulation and antioxidation effects are just parts of the major medicinal properties of PN. For better uncovering bioactive constituents of raw and steamed PN, the relationships between chemical information and other efficacies such as anti-inflammation, hemostasis, blood-tonifying, and immunoregulation, need to be further studied.
CONCLUSIONS
In the research, there were divergences in the chemical composition and bioactivities between raw and steamed PN based on the fingerprints and pharmacologic results. Notoginsenoside R 1 , ginsenosides Rg 1 , Re, Rb 1 , and Rd with higher levels in raw PN were verified to be its active constituents, whereas ginsenosides Rh 1 , Rk 3 , 20 (R)-Rg 3 , and Rh 4 with higher levels or exclusively existed in steamed PN were found to be its active constituents. Ginsenosides Rh 1 , Rk 3 , 20 (R)-Rg 3 , and Rh 4 could be used in the future as new markers for the QC of steamed PN. Future research is needed to uncover bioactive constituents related to other efficacies of raw and steamed PN.
AUTHOR CONTRIBUTIONS
YX did the writing of paper and a part of statistical analysis; LC did the anticoagulation test and pharmacological verification as well as the statistical analysis; YH did the HPLC experiments and antioxidation test; YX and XC supervised the project. All the authors read and approve the final manuscript. | v3-fos-license |
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} | pes2o/s2orc | Safety Evaluation of Sous Vide-Processed Products with Respect to Nonproteolytic Clostridium botulinum by Use of Challenge Studies and Predictive Microbiological Models
ABSTRACT Sixteen different types of sous vide-processed products were evaluated for safety with respect to nonproteolytic group IIClostridium botulinum by using challenge tests with low (2.0-log-CFU/kg) and high (5.3-log-CFU/kg) inocula and two currently available predictive microbiological models, Food MicroModel (FMM) and Pathogen Modeling Program (PMP). After thermal processing, the products were stored at 4 and 8°C and examined for the presence of botulinal spores and neurotoxin on the sell-by date and 7 days after the sell-by date. Most of the thermal processes were found to be inadequate for eliminating spores, even in low-inoculum samples. Only 2 of the 16 products were found to be negative for botulinal spores and neurotoxin at both sampling times. Two products at the high inoculum level showed toxigenesis during storage at 8°C, one of them at the sell-by date. The predictions generated by both the FMM thermal death model and the FMM and PMP growth models were found to be inconsistent with the observed results in a majority of the challenges. The inaccurate predictions were caused by the limited number and range of the controlling factors in the models. Based on this study, it was concluded that the safety of sous vide products needs to be carefully evaluated product by product. Time-temperature combinations used in thermal treatments should be reevaluated to increase the efficiency of processing, and the use of additional antibotulinal hurdles, such as biopreservatives, should be assessed.
The term "sous vide" means "under vacuum" and describes a processing technique whereby freshly prepared foods are vacuum sealed in individual packages and then pasteurized at time-temperature combinations sufficient to destroy vegetative pathogens but mild enough to maximize the sensory characteristics of the product (39,40). After cooking, the products are chilled, stored refrigerated, and reheated before consumption. Sous vide foods are mainly used in mass catering and restaurants (30). Compared with traditional cooking methods, sous vide has many advantages (40,42). Economic benefits include better use of labor and equipment through centralized production and extended shelf life due to vacuum packaging, which by excluding oxygen inhibits oxidative processes and growth of spoilage organisms. The shelf life of a sous vide product can be as long as 42 days (42). In addition, the reduced need for preservatives and flavor enhancers, better preservation of vitamins, and retention of most of the original food juices all contribute to higher quality of sous vide foods over conventional meals.
Concerns associated with sous vide processing involve the microbiological safety of the products (40). The psychrotrophic food-borne pathogens and particularly nonproteolytic group II Clostridium botulinum bacteria are of concern due to the methods of preparing, distributing, and storing these products. Mild heat treatments in combination with vacuum packaging may actually select for C. botulinum and increase the potential for botulism. Sous vide products are generally formulated with little or no preservatives and frequently do not possess any intrinsic inhibitory barriers (pH, a w , or NaCl) that either alone or in combination would inhibit growth. Therefore, strict adherence to refrigerated storage below 3.3°C must be maintained to ensure the safety of sous vide products with respect to nonproteolytic C. botulinum (1). However, the temperature control in chill chains is often inadequate, and temperature abuse is common throughout distribution and retail markets and by consumers (8,16,27).
Recent research has identified combinations of mild heat treatment and subsequent refrigerated storage that, when combined with a specified shelf life, provide a defined safety margin with respect to nonproteolytic C. botulinum (10,15,46). Based on these research results, the Advisory Committee on the Microbiological Safety of Food (1) recommended certain procedures to ensure the safety of refrigerated processed foods of extended durability. According to these recommendations, heat treatments or combination processes should reduce the number of nonproteolytic C. botulinum bacteria by a factor of 10 6 (a 6-decimal [6-D] process). However, the capability of a combination process to consistently prevent growth and toxin production by C. botulinum in a particular product must be reliably demonstrated.
There are two main approaches that can be used to evaluate the stability and the safety of a product with respect to foodborne pathogens. Traditionally, the effect of thermal processing on pathogenic microorganisms, as well as the risk of their growth and possible toxin production in foods, has been determined through the use of inoculated pack studies. Now, however, there are too many products, alternate ingredients, and process variations to conduct a complete laboratory evaluation of each possible contingency and potential food-borne pathogen for each product. Therefore, predictive food microbiology, the modeling of microbial populations, particularly those of food-borne pathogens, has become an active field of research. Predictive models are equations which can use the information from a large database to predict inactivation or growth of microorganisms under defined conditions. However, current models cannot be used with confidence until their validation in various foods is tested by comparing the predictions to data obtained from inoculated pack studies (47).
The present study was performed to evaluate the safety of 16 different types of sous vide-processed products with respect to nonproteolytic C. botulinum. The efficiency of thermal processes to inactivate botulinal spores and the subsequent effect of mildly abusive storage temperatures on C. botulinum outgrowth and toxigenesis were studied by using inoculated pack studies and two currently available predictive microbiological programs.
MATERIALS AND METHODS
Products. Sixteen sous vide-processed products of various types were evaluated for safety with respect to nonproteolytic C. botulinum. The details of the products are described in Table 1. The ingredients of each product were obtained from local processors and were transported to the laboratory in refrigerated vacuum packages at 2°C.
Product inoculation and vacuum packaging. A mixture of five nonproteolytic C. botulinum strains was used in the inoculum: three type E strains (31-2570 E, 4062 E, and C-60 E), one type B strain (706 B), and one type F strain (FT 10 F). The strains were of North American and European origin and were isolated from seafood and meat products between the 1960s and the 1980s. The spore suspensions of individual strains were prepared according to the method recommended by the Food and Agricultural Organization (12), and the concentration of each spore suspension was determined as described by Doyle (9). The spore mixture used for inoculation contained an equivalent number of non-heat-shocked spores of each strain diluted in sterile distilled water; 5 ml of dilution contained the required spore load for one sample. Low (2.0-log-CFU/kg) and high (5.3-log-CFU/kg) levels of inoculum were used. The inoculation was performed in vacuum pouches by spraying the spore mixture evenly on the surfaces of solid products or by thoroughly mixing the spore mixture into liquid products. The vacuum pouches (250 by 500 mm) were prepared from nylon-polyethene multiple-layer laminate films (Wipak Oy, Nastola, Finland) with an oxygen permeability of 17 cm 3 /m 2 /24 h/atm (23°C, 50 to 70% rH) and a water vapor permeability of 1.3 g/m 2 /24 h (23°C). Samples were vacuum packaged (Multivac A 300/16 1986; Multivac Verpackungsmaschinen, Wolfertschwenden, Germany) immediately after inoculation.
Thermal processing. The samples were cooked in a process autoclave (Stock Pilot Rotor 900 G; Hermann Stock Maschinenfabrik GmbH, Neumünster, Germany) by using either full water immersion or water spray circulation cooking, depending on the product. Uninoculated samples were used to probe (Ellab; Ellab A/S, Roedovre, Denmark) measure the core temperature during the process. The temperatures measured at the coolest part of the product as a function of processing time were used to calculate the inactivation ratio (P/t) for each processing time by using the formula P/t ϭ 10 (T Ϫ Tref)/z (5), where P is the pasteurization value (minutes) and t is the processing time (minutes) at the actual temperature, T (degrees Celsius). T ref is the reference temperature related to pasteurization process. z value is the temperature change (degrees Celsius) necessary to change the decimal reduction value (D) by a factor of 10. Of the nonproteolytic serotypes, type B has the most heat-resistant spores. Accordingly, the T ref and z values used in the present study, 82.2 and 16.5°C (D ϭ 32.3 min), respectively, were those for type B (4). P values for each product (Table 1) were calculated by integrating the inactivation ratio curve over processing time. After the thermal process, the samples were cooled to 13 to 40°C in the autoclave with cold water either by spraying or by full immersion depending on the method used in the heat process. The final cooling to 2°C was accomplished in cold storage.
Storage conditions and sampling procedures. After processing, samples of each product were stored at 4 and 8°C. The samples were analyzed for the presence of C. botulinum type B, E, and F cells and botulinum neurotoxin after the shelf life typically recommended for a corresponding commercially available product and 7 days after that. The analyses were performed with three parallel samples for each storage temperature and inoculum level. pH was analyzed for four parallel inoculated samples immediately after processing and at both sampling times after storage.
Microbiological quality (aerobic plate count [APC], number of sulfite-reducing clostridia, lactic acid bacteria, and yeasts and molds) of selected products (no. 1, 3, 4, 5, 6, 7, 8, 10, and 11) was determined by using uninoculated samples in parallel with sensory evaluation. All analyses were performed on single samples in duplicate. The samples stored at 8°C were examined immediately after processing, in the middle of the recommended shelf life, after the recommended shelf life, and after the safe-storage time predicted by the Pathogen Modeling Program (PMP).
Detection of C. botulinum. Twenty grams of each sample was examined for the presence of C. botulinum type B, E, and F cells by PCR analysis as described by Hielm et al. (21), with some modifications. The quantification was based on a 1-dilution-level most-probable-number (MPN) series (11). Briefly, 20 tubes containing 10 ml of tryptone-peptone-glucose-yeast extract (Difco, Detroit, Mich.) broth with 625 IU of lysozyme (Sigma Chemical Co., St. Louis, Mo.) per ml were each inoculated with 1 g of thoroughly homogenized sample. Enrichment cultures were incubated at 26°C in an anaerobic cabinet with an internal atmosphere of 85% N 2 -10% CO 2 -5% H 2 (MK III; Don Whitley Scientific Ltd., Shipley, United Kingdom) for 3 days. Washed and boiled cells from overnight (18-h) cultures were used as a template for PCR. DynaZyme DNA polymerase (Finnzymes, Espoo, Finland) and a 96-well PTC-100 thermal cycler (MJ Research, Watertown, Mass.) were used. The sizes of the amplified PCR products were determined by agarose gel electrophoresis with comparison to standard DNA fragments (DNA molecular weight marker VI; Boehringer Mannheim GmbH, Mannheim, Germany).
Toxin analysis. The procedure for the assay of botulinum toxin followed the Nordic Committee on Food Analysis protocol (35), with modifications described by Hyytiä et al. (24). pH analysis. pHs of homogenates of minced sample and distilled water in a ratio of 1:1 (wt/vol) were determined with a digital Microprocessor pH 537 A trained laboratory panel of 10 judges evaluated the appearance and the aroma of the uninoculated samples heated to a service temperature typical of the product by using a five-point structured category scale (32). Each evaluation contained a marked reference sample that was obtained from a fresh production batch. In addition to a marked reference sample, in each session a fresh reference sample was hidden among the stored samples. A score of 2 on the category scale indicated that there was a "just-detectable" deterioration in sensory quality compared to that of the marked reference, a score of 3 indicated a "clearly detectable but not unacceptable" deterioration, and a score of 5 indicated that the judge had considered the sample unacceptable for human consumption. All samples were evaluated twice, and means of scores were calculated over replicates for each sample.
Predictive microbiological models. Food MicroModel (FMM), version 2.5 (Leatherhead Food Research Association, Leatherhead, Surrey, United Kingdom), was used to generate predictions for the lethal effect of each thermal process on nonproteolytic C. botulinum spores. FMM and PMP, version 5.0 (U. S. Department of Agriculture Eastern Regional Research Center, Wyndmoore, Pa.), were used to generate predictions for the safe-storage times after processing with the assumption that all or one part of the spores survived the thermal process.
The thermal death model for nonproteolytic C. botulinum type B by the FMM has temperature (80 to 95°C), water-phase NaCl level (0 to 5%), and pH (4.0 to 7.4) as controlling factors. The model predicts the estimated minimum decrease in the number of C. botulinum spores. The growth model for nonproteolytic C. botulinum types B, E, and F by the FMM has temperature (4 to 30°C), pH (5.1 to 7.5), and water-phase NaCl concentration (0 to 4.5%) as controlling factors. The minimum value for the initial number of organisms is 10 CFU/g. The estimate of the safe-storage time was based on the calculated lag time for growth. The time-to-turbidity predictive model for nonproteolytic C. botulinum type B by the PMP uses temperature (5 to 28°C), pH (5.0 to 7.0), water-phase NaCl level (0 to 4.0%), and initial number of organisms in the food (1 to 10 5 CFU/product unit) as controlling factors. The safe-storage time was reported as the lower 95% confidence limit of the tau, which is the time when the probability of growth reached half of the maximum probability of growth over the entire storage period.
The highest pH level measured from the samples of each product during the study and the water-phase NaCl level analyzed by the raw-material suppliers ( Table 1) were used as input values in all predictions. The thermal death predictions were based on the lowest temperature recorded at the center of the product, and heating and cooling times were taken into account. Since initial number of organisms is not a controlling factor in the FMM growth model, 5.3 log CFU/kg was used as an input value in growth predictions. PMP time-toturbidity predictions were generated by using the FMM thermal death model predictions as to the level of surviving spores in each product as an input value for initial number of organisms. In both growth models, 5°C was used as the lower storage temperature to enable the comparison of models.
RESULTS
During storage at 4 and 8°C, a distinct difference between low-and high-inoculum samples was observed, in that substantially fewer samples with a low inoculum were positive for C. botulinum in PCR analysis and no toxigenesis was observed (Tables 2 and 3). Storage temperature did not appear to have a considerable effect on the number of PCR-positive samples or on the number of C. botulinum bacteria in positive samples at either inoculum level. However, toxigenesis was detected only in samples stored at 8°C (products 1 and 8). Eighty-seven percent of the PCR-positive samples contained serotype E. The prevalence of serotypes B and F in positive samples was 15 and 9%, respectively.
The microbiological quality of the products studied remained unchanged during the storage period at 8°C with APCs being mainly Ͻ1.0 log CFU/g. The highest counts (3.0 log CFU/g) were detected in products 5 and 11. The numbers of sulfite-reducing clostridia, lactic acid bacteria, and yeasts and molds were below detectable levels (1.0 log CFU/g) in all products studied. The sensory quality remained good with 4 of the 126 evaluated samples having a mean score of 2 or below, indicating that difference from the fresh reference sample was just detectable in most cases, and all scores being below 3, showing no unacceptable deterioration in examined samples.
Two distinct groups could be discerned among the 16 products subjected to challenge testing. A high-risk group consisted of four products (no. 1, 7, 8, and 13) that had high numbers of C. botulinum bacteria in PCR analysis with one or both inoculum levels at both sampling times and/or that showed toxigenesis (Tables 2 and 3). Two products (no. 14 and 16) were designated safe since they were negative for C. botulinum cells and botulinum neurotoxin at both sampling times in all different treatment groups. Differences in P value, NaCl concentration, and pH were observed between high-risk and safe products ( Table 4). The remaining 10 products presented increased botulism risk by being positive in PCR analysis on one or several occasions.
According to the thermal death predictions by the FMM, 5 of the 16 products studied (no. 3, 9, 12, 14, and 15) had a thermal process that appeared to meet the Advisory Committee on the Microbiological Safety of Food requirement of a 6.0-log-unit reduction and would be adequate to eliminate the spores in the high-inoculum samples (Table 5). In seven products (no. 3, 9, 11, 12, 14, 15, and 16), the predicted reduction in spore numbers appeared to be adequate to achieve the 2.0-to 2.3-log-unit reduction required to eliminate the spores in lowinoculum samples. The thermal death predictions agreed well with the measured P values but not with the observed PCR results. The safe-storage times predicted by the FMM were in general substantially shorter than those predicted by PMP (Table 5). According to the FMM prediction, only three products (no. 13, 14, and 16) appeared to be safe within the limits of their recommended shelf life regardless of the storage temperature. According to the PMP, all products were safe at 5°C with the low inoculum level, but only 6 (no. 3, 9, 12, 14, 15, and 16) of the 16 products were safe within the limits of their recommended shelf life if storage temperature and initial number of surviving spores were increased.
DISCUSSION
Due to the ubiquitous spread of C. botulinum in nature (17,19,20), contamination of raw ingredients of food products by botulinal spores is possible and even probable. However, the number of spores in different foods has been generally reported to be low (17,18,23,26). The challenge tests of this study were designed to simulate as closely as possible the natural contamination level in foods (low-level inoculum) and to present a worst-case scenario to obtain an adequate margin of safety (high-level inoculum). The results gained from the inoculation studies question the current recommendations for safe processing set out by the Advisory Committee on the Microbiological Safety of Food. Based on the calculated P values, the thermal processes of five products (no. 3, 9, 12, 14, and 15) appeared to be adequate to achieve the 6-D reduction in botulinal spores (criterion: the ratio of P value to processing time required for 6-D reduction being Ն1) which is recommended to ensure the safety of refrigerated processed foods of extended durability with respect to nonproteolytic C. botulinum (1, 3). However, only one of these products (no. 14) was determined to be safe in challenge tests. The results of the inoculated pack studies revealed that the majority of thermal processes were inadequate to eliminate the spores even with the low inoculum level. The presence of botulinal spores in nonsterile low-acid vacuum-packaged foods must be considered a serious risk due to the high probability of temperature abuse and mishandling of these types of products (8,16,27). The botulism risk of sous vide products is additionally increased by the absence of spoilage flora and by the long sensory shelf life which allows toxigenesis before sensory spoilage occurs. However, to our knowledge, sous vide products have not been implicated as a cause of a botulism outbreak so far.
The comparison of high-risk products with safe products pointed out the factors contributing to the increased botulism risk. A low P value seemed to be the most significant single factor increasing the botulism risk in the products. A considerable overlap was observed in NaCl content and pH values between different risk groups, though high-risk products tended to have slightly lower NaCl concentrations and higher pHs than safe products. The growth and inactivation of microorganisms can be substantially affected by food type (42). The main ingredient of the four high-risk products was meat, while both of the two safe products contained a large amount of vegetables. It has been observed that the high fat content in meat-based foods can increase the heat resistance of microorganisms (2,22). Moreover, meat contains high amounts of osmoprotectants (e.g., proline, betaine, and carnitine), essential amino acids, and lytic enzymes (lysozyme) which either increase the heat resistance of botulinal spores or facilitate the germination and growth of the heat-injured survivors (42). On the other hand, certain vegetables have been reported to suppress the growth of nonproteolytic C. botulinum due to their inherent antibotulinal characteristics such as low pH, inadequate nutrient contents, or antimicrobial compounds (6,28). Overall, the results of the PCR analyses indicated that very little growth occurred during the storage. Toxin production by nonproteolytic C. botulinum has been shown to occasionally occur with very weak growth or no detectable growth (6,14,24,25). It is also possible that all true C. botulinum-positive samples were not detected due to the large size of the samples and the low inoculum levels. Despite thorough homogenization at the time of inoculation and sampling, the spores were probably unevenly distributed. Only a single 20-g aliquot from each 1,000-to 1,500-g sample was examined for the presence of botulinal spores, with the detection limit of the PCR method used being 0.4 log CFU/kg. Some products, on the other hand, were positive for botulinal spores at the first sampling but negative in the second. This correlates with the observation that the number of microorganisms is known to decline over time when placed in an adverse environment (31). None of the intrinsic factors (NaCl and pH) of the products alone was inhibitory to the growth of C. botulinum. However, when sublethal levels of NaCl and pH were combined with low storage temperature, the conditions may not have allowed for the survival of heat-injured spores (29).
To our knowledge, the predictive models used in the present study have not been previously validated in sous vide products with respect to nonproteolytic C. botulinum. The predictions by the FMM thermal death model were found to be unreliable in the 16 sous vide products studied. The model appeared to give high values for the logarithmic reduction of spores, since spores were observed even in those products with a low inoculum level which were predicted to have 6-D reduction in spore numbers. The FMM growth model predictions cannot be directly compared with the data obtained from the present challenge tests, since the model predicts lag time for growth and the observed results do not give evidence as to when growth began. The PMP time-to-turbidity model appeared to generate long safe-storage times for low-inoculum samples, since spores and/or slight growth was detected in most products. However, with the high inoculum level the model predicted considerably shorter safe-storage times for most products, including those that were considered to exhibit high risk. The failure of both growth models to predict safe-storage times for different types of vacuum-packaged fishery products with respect to C. botulinum type E and Listeria monocytogenes has been recently reported (7,25). The poor agreement of predicted and observed results in the above-mentioned studies and in this study was partly due to the limited number of controlling factors in the models. For example, the level and nature of the natural bacterial flora in the products and the product formulation have an effect on growth by food-borne pathogens. The models have been developed in broths under constant conditions and do not account for different changing variables in food products and characteristics of different bacterial strains that affect microbial behavior (41,47). Additionally, in many cases the levels of the controlling factors in the products studied were simply out of range or operated near the outer limits of those set by the models, which contributed to inaccurate predictions. With these types of products, models should not be used. Instead, safety evaluation should be done by inoculation studies. The results of the present study indicate that the safety of sous vide products with respect to nonproteolytic C. botulinum has to be carefully evaluated product by product. An increase in processing time and temperature would seem a logical solution in view of the difference in the P values observed between high-risk and safe products. However, the degree of benefit gained from increased thermal processing is obviously greatly dependent on the type of product. Additionally, adverse effects on sensory and nutritional qualities by increased thermal treatment are the opposite of the original idea of sous vide processing. Another alternative to improve safety would be to add additional hurdles to products. Biopreservatives, such as nisin and organic acids, are known to have an antibotulinal effect (33,43,45). However, even a slight change in formulation or processing conditions warrants a safety evaluation by challenge tests since the predictive models available to date appear to frequently provide misleading predictions. Furthermore, use of time-temperature indicators in individual product packages would record the storage history of a product (40,44) and might lead to enhanced temperature control in chill chains. Additionally, for evaluators to be able to make confident risk assessments and to avoid being unnecessarily overcautious, additional data on the prevalence and numbers of spores of psychrotrophic C. botulinum in different categories of sous vide-processed foods is needed (13). | v3-fos-license |
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} | pes2o/s2orc | Lactic Acid Bacteria Contribution to Wine Quality and Safety
Wine production is a complex biochemical process that brings into play different microorganisms. Among these, lactic acid bacteria (LAB) play a central role in the quality of the final wine. LAB are not only responsible for the malolactic fermentation that usually occurs after the alcoholic fermentation but also contribute for other important biochemical reactions such as esterase and glycosidase activities and citric acid and methionine metabolism. Nonetheless, LAB may also contribute negatively to wine quality by contributing to the production of volatile phenols, biogenic amines, and ethyl carbamate. This chapter aims to integrate the current knowledge about the role of LAB in wine flavor and quality.
Introduction
This review focuses on the current knowledge about the impact of lactic acid bacteria (LAB) in wine composition and flavor. In wine, LAB perform a second fermentation consisting of decarboxylating L-malic acid to L-lactic acid, designated by malolactic fermentation (MLF). This fermentation follows the alcoholic fermentation conducted by yeast (Saccharomyces spp.). MLF reduces wine acidity and provides microbiological stabilization by lowering nutrient content of wine.
Under favorable conditions, MLF occurs spontaneously after alcoholic fermentation by the growth of indigenous LAB population in wine. However, selected strains of LAB can be inoculated into wine to induce MLF. According to the types of wines produced, this biological deacidification may be considered beneficial or detrimental to wine quality. However, it containing hematin or related compounds, some strains may produce catalase or even cytochromes [3]. Though aerotolerant, they are a group of bacteria typical of non-aerobic habitats, very demanding from a nutritional point of view and tolerate very low pH values, with acidity tolerance being a variable trait among strains. LAB are present in very diverse environments (e.g., fermented foods and beverages, plants, fruits, soil, wastewater) and are also part of the microflora of the respiratory, intestinal, and genital tracts of man and animals [4,5].
Lactic acid bacteria (LAB) are naturally part of the microbiota of grapes, musts, and wines. In musts and wines, the LAB species that may be present and isolated are (i) heterofermentative cocci belonging to Leuconostoc and Oenococcus genera and homofermentative cocci belonging to Pediococcus of Streptococcaceae family and (ii) homofermentative, facultative, and strict heterofermentative bacilli belonging to Lactobacillus genus of Lactobacillaceae family [6][7][8]. In wine grapes, from several Australian vineyards, Bae et al. [9] were not able to isolate Oenoccoccus strains, but they detected strains of Enterococcus, Lactococcus, and Weissella; LAB more frequently associated to other food matrices. Although Oenococcus oeni is the predominant species in the final stage of wine production, it has rarely been isolated from grapes in the vineyard [10]. Recently, in a large survey of LAB isolation in grapes and wines from a Spanish region (Priorat Catalonia), Franqués et al. [11] were able to isolate 53 strains of Oenococcus oeni in a total of 254 LAB isolates from grapes. In Table 1, a list of LAB species isolated from grapes, musts and wines that undergone spontaneous MLF or from wines with alterations of different regions of the world is shown.
LAB metabolism in wine
Either complexity or multiplicity of LAB metabolic activities in wine demonstrates that MLF is more than a simple decarboxylation of L-malic acid into L-lactic acid, and thus this very special and important fermentation may affect positively and/or negatively the quality of wine [17].
Besides the immediate effect of decrease in acidity by the transformation of a dicarboxylic acid (L-malic acid) into a monocarboxylic acid (L-lactic acid), MLF also improves sensorial characteristics and increases wine microbiological stability [18,19]. Modifications in wine aroma induced by LAB are due to L-lactic acid, less aggressive to palate, and a huge number of other compounds such as diacetyl, acetoin, 2,3-butanediol, ethyl lactate and diethyl succinate esters, and some higher alcohols and aromatic aglycones that become free by the action of LAB β-glucosidases [20-23]. Although produced in lower concentrations, sulfur compounds, particularly 3-methylsulfanyl-propionic acid with chocolate and toasted odors, may contribute to aromatic complexity of wines [24]. Also the activity of taninoacil hydrolase enzyme, commonly termed tannase, reducing wine astringency and turbidity may increase the quality and result in a better and pleasant sensorial perception for consumers [25].
Although not well understood at that time, the knowledge of the negative role of LAB on wine quality comes from the first studies of Pasteur at the beginning of the twentieth century. Some wine defects due to microorganism development were accurately described and LAB were shown to be responsible for wine "diseases" such as "tourne," the degradation of tartaric acid; "bitterness," the degradation of glycerol; and "ropiness," the unacceptable increase in wine viscosity [26]. Although less frequent nowadays, due to better hygienic conditions in wineries and knowledge of microorganisms, these wine "diseases" together with others such as butter aroma due to excessive production of diacetyl, flocculent growth, mannitol taint, and the geranium odor, presented in Table 2, still may occur. Also, the formation of volatile phenols (4-ethylguaiacol and 4-ethylphenol) and mousy off-odor by acetamide production of tetrahydropyridines can be produced by some strains of LAB species responsible for malolactic fermentation (MLF). Other compounds, such as ethyl carbamate formed by the degradation of arginine and biogenic amines (histamine, tyramine, and putrescine) from the degradation of amino acids, contribute negatively to wine quality and may affect the consumer's health [18,29,30].
Production of volatile compounds by LAB
The main effect of malolactic fermentation is the decarboxylation of L-malic acid into L-lactic acid, catalyzed by the malolactic enzyme. However, lactic acid bacteria produce several volatile compounds that can significantly influence wine aroma. Acetic acid and acetoinic compounds (C4 compounds) are the major products of citric acid metabolism by LAB. Acetoinic compounds comprise diacetyl, acetoin, and 2,3-butanediol. The biosynthesis of these compounds depends on citric acid metabolism (Figure 1). Diacetyl (2,3-butanedione), one of the most important flavor compounds produced by LAB, imparts a distinct buttery or butterscotch aroma to wine. Diacetyl is formed as an intermediate metabolite of the reductive decarboxylation of pyruvate to 2,3-butanediol, associated with citrate metabolism by LAB. The precursor of diacetyl in this pathway is α-acetolactate, which is also an intermediate in the biosynthesis of the amino acids valine and leucine in prototrophic LAB. Pyruvic acid results from the metabolism of sugars and citric acid. To be capable to utilize citrate, LAB must possess the genes encoding permeases for citrate transport and citrate lyase for citrate metabolism [31].
Yeasts are also able to synthesize diacetyl in the course of alcoholic fermentation. However, most of this diacetyl is reduced by yeasts to acetoin and 2,3-butanediol, and only low concentrations of diacetyl remain at the completion of fermentation. Diacetyl reduction is further encouraged by the presence of yeasts or LAB after the conclusion of malolactic fermentation [32].
Salo [33] determined a sensory odor threshold level of 0.0025 mg/L for diacetyl in 9.4% (w/w) ethanolic solution. Yet, Guth [34] calculated 0.1 mg/L for diacetyl odor threshold in water/ ethanol (90 + 10, w/w). Moreover, Martineau et al. [35] showed that the diacetyl flavor threshold depends on the wine type. They found that the flavor detection threshold was 0.2 mg/L in a lightly aromatic Chardonnay wine, 0.9 mg/L in a low tannic aromatic Pinot noir wine, and 2.7 mg/L in a full-flavored, full-bodied Cabernet Sauvignon wine. These wines were made without oak contact.
Reports of diacetyl concentration in wine vary from 0.2 to 4.1 mg/L [36]. The final concentration of diacetyl in wine depends on the concentration of sulfur dioxide. Sulfur dioxide combines reversibly with diacetyl in wine, suppressing the buttery note of wine flavor [37].
LAB esterase activity
Wine esters are important contributors to wine aroma. They comprise ethyl esters of organic acids (e.g., ethyl lactate), fatty acids (e.g., ethyl hexanoate, ethyl octanoate, ethyl decanoate), and acetates of higher alcohols (e.g., ethyl acetate, isoamyl acetate). These compounds are not only produced by yeasts during alcoholic fermentation and LAB during MLF but can also be formed by slow chemical esterification between alcohol and acids during wine aging [38]. [12,27,28]).
Generation of Aromas and Flavours
of hydrolyzing ester substrates, with O. oeni showing the highest activity. But responses to pH, temperature, and ethanol concentration were strain-dependent [39]. LAB showed greater esterase activity toward short-chained esters (C2-C8) than long-chained esters (C10-C18). They present the highest esterase activity at a pH close to 6.0, though Oenococcus oeni retained appreciable activity even down to a pH of 3.0 and showed an increase in activity up to an
LAB glycosidase activity
Most volatile compounds that make the varietal aroma of wines are present in grapes in the form of glycoconjugated nonvolatile odorless molecules. These glycosides are β-D-glucose and diglycoside conjugates, with the latter consisting of glucose and a second sugar unit of α-L-arabinofuranose, α-L-rhamnopyranose, β-D-xylopyranose, or β-D-apiofuranose [42]. The aglycon moiety of these compounds belongs to different classes of volatiles, including monoterpenes and C 13 -norisoprenoids. The glycoconjugates can be slowly transformed into free volatile aroma compounds through acidic hydrolysis during wine aging. Yet, a faster enzymatic hydrolysis of these glycosides by wine microorganisms can also occur. The hydrolysis of the disaccharide glucosides requires the action of two enzymes in sequence: first the disaccharide (1 → 6) linkage is cleaved by the appropriate exo-glycosidase releasing the outermost sugar molecule and the corresponding β-D-glucoside; subsequently, liberation of the odorous aglycon takes place after action of β-D-glucosidase. Yet, the hydrolysis of monoglucosides only requires the action of a β-D-glucosidase.
Yeasts, mainly non-Saccharomyces species found on grapes, possess glycosidase enzymes capable of liberating aroma compounds, particularly volatile terpenes, from their glycosilated precursors. However, in a study by Rosi et al. [43] only one of 153 strains of Saccharomyces cerevisiae showed β-glucosidase activity.
O. oeni has the ability to hydrolyze grape-glycoconjugated aroma precursors, but large differences in the extent and specificity of this hydrolysis activity were observed [44].
The β-glucosidase activity of different strains of O. oeni was affected by pH, sugar, and ethanol content in variable degree [45]. β-glucosidase activity was optimal at a pH of 5.5 and decreased as pH was reduced: within a pH range of 3.5-4.0, O. oeni showed just 12-43% of the maximum activity. The β-glucosidase activity of some strains was strongly inhibited by even a low sugar content (10 g/L), while others were not affected by higher sugar contents (30 g/L). Ethanol concentration up to 10% v/v led to an increased O. oeni β-glucosidase activity, and for most strains higher concentrations (up to 14% v/v) did not affect or only slightly decreased this activity [45].
Lactobacillus plantarum isolated from Italian wines showed β-glucosidase activity and the ability to release odorant aglycones from odorless glycosidic aroma precursors [47]. Lactobacillus spp. and Pediococcus spp. possess varying degrees of β-D-glucopyranosidase and α-Dglucopyranosidase activities, influenced differently by ethanol and/or sugar concentration, temperature, and pH. But these activities are approximately one order of magnitude less than those seen for O. oeni [48]. propionic acid.
Production of off-flavors by LAB
When sorbic acid ((E,E)-2,4-hexadienoic acid) as potassium sorbate is added as an yeast inhibitor to wines containing residual sugar, LAB can degrade this compound in 2-ethoxyhexa-3,5-diene (2-ethoxy-3,5-hexadiene). 2-Ethoxyhexa-3,5-diene has an offensive crushed geranium leaves odor with a detection threshold of less than 1 ng/L [49,50]. Sorbic acid inhibits yeast growth, but it does not inhibit LAB growth at the levels allowed in wines for this compound, demonstrating the need for maintaining adequate levels of sulfur dioxide (an effective inhibitor of LAB) in such wines. When used together, sorbate and sulfur dioxide can prevent secondary fermentations and control the growth of LAB in sweet table wines, with a pH of 3.3-3.9, at levels as low as 80 mg/L sorbate and 30 mg/L sulfur dioxide [51].
Various LAB isolated from wine showed the ability to synthesize 4-vinylphenol, by decarbox-
Production of ethyl carbamate and biogenic amines by LAB
LAB use amino acids both as a strategy of survival particularly in nutrient limiting media and evidently in response to acid stress and as a source of energy. However, this may have implications for the quality and food safety of fermented products [53,54].
The metabolism of amino acids such as arginine and histidine does not affect taste but creates a problem at consumer's health level by increasing the concentrations of biogenic amine and ethyl carbamate precursors in the wine, which are toxic compounds, thus contributing negatively to wine safety [55].
Degradation of arginine and formation of ethyl carbamate
Arginine is one of the amino acids present in higher concentrations in grape musts and wines. LAB may use this amino acid by arginine deaminase pathway. This pathway involves three enzymes: arginine deiminase (ADI, EC 3.5.3.6), ornithine transcarbamylase (OTC, EC 2.1.3.3), and carbamate kinase (CK; EC 2.7.2.2) [56]. The presence of the three enzymes of the ADI pathway appears to occur in most heterofermentative lactobacilli, leuconostocs, and oenococci, although they have already been detected in homofermentative species of LAB isolated from wine. However, the arginine pathway presence in all species seems to be a straindependent phenotype [57]. By this pathway, 1 mole of L-arginine is converted into 1 mole of ornithine and 1 mole of carbon dioxide and 2 moles of NH 3. The intermediate products of this pathway, citrulline and carbamoyl phosphate, are precursors of the ethyl carbamate, a potentially carcinogenic compound. This compound is formed from a spontaneous chemical reaction involving ethanol and precursors including urea, citrulline, carbamoyl phosphate, N-carbamyl, α-and β-amino acids, and allantoin [58]. According to Ough et al. and Kodama et al. [59,60], the ethanolysis reaction of citrulline and urea for ethyl carbamate formation may occur at normal or elevated storage temperatures. Even though ethyl carbamate is produced in small quantities, its concentration in wine is subjected to international regulation and therefore must be carefully controlled. Maximum level in the European Union and Canada for table wines is 30 μg/L (100 μg/L for fortified wines in Canada), while in the USA the values are more restrictive, being 15 μg/L for table wines and 60 μg/L for dessert wines [55,60,61].
Some controversial information about the contribution of LAB for ethyl carbamate production is found in the scientific literature [8]. Tegmo-Larsson et al. [62] reported that malolactic fermentation did not affect the concentrations of ethyl carbamate in wine. However, more recent information suggests that some lactic acid bacteria, specifically O. oeni and L. hilgardii, can contribute to ethyl carbamate formation [61]. It must also be emphasized that in wine, prolonged contact of viable and viable but not cultivable LAB strains with residual lees from yeast should be considered as a significant risk factor for the increased formation of citrulline and therefore ethyl carbamate [63][64][65]. Therefore, it is not prudent to use Oenococcus oeni strains that excrete citrulline as starter cultures. Some of these authors further suggest that strains that possess only the first pathway enzyme (ADI +, OTC-) or strains that have ADI but low OTC activity should also be excluded in a starter selection process for MLF.
The formation of biogenic amines
Biogenic amines are low molecular weight organic bases, which can be formed and degraded during the normal metabolic activity of animals, plants, and microorganisms [29]. In the human body, these substances may play an important metabolic role, related to growth (polyamines) or to functions of the nervous and circulatory systems (histamine and tyramine). But when ingested in excess, they may be the cause of hypotension, hypertension, heart palpitations (vasoactive Generation of Aromas and Flavours amines), headaches (psychoactive amines), and various allergic reactions [30,66]. Biogenic amines are fundamentally formed from the decarboxylation of the precursor amino acids by the action of substrate specific enzymes [6,67,68]. Thus, the amines histamine, tyramine, tryptamine, serotonin, 2-phenylethylamine, agmatine, and cadaverine are formed from the amino acids histidine, tyrosine, tryptophan, hydroxytryptophan, phenylalanine, arginine, and lysine, respectively [69][70][71]. Putrescine can be formed from ornithine or agmatine, and spermidine and spermine are formed from putrescine by the binding of aminopropyl groups catalyzed by spermidine synthase and spermine synthase [72]. During the fermentative processes of many raw materials (milk, meat, vegetables, barley, and grapes) to obtain food and beverages, such as cheese, sausages, fermented vegetables, beer, and wine, the formation of biogenic amines by LAB may occur. Many bacteria present decarboxylase activities, which favor their growth and survival in acidic environments, by the increase of pH, as previously mentioned. In wine, several amino acids can be decarboxylated, and consequently, biogenic amines can be found, predominating histamine, tyramine, putrescine, isopentylamine, cadaverine, and α-phenylethylamine [29,30,[73][74][75][76][77][78][79][80][81]. However, their content in wine is much lower than that found in other foods [82], although ethanol may potentiate the toxic effect of histamine by inhibiting amino oxidases. Like ethyl carbamate, there are recommendations for the maximum histamine levels allowed in wine. EU countries and Canada recommend histamine levels not exceeding 10 mg/L, except Germany where the limit is 2 mg/L. Some biogenic amines, for example, putrescine and cadaverine, when in high concentrations, besides their toxicity, can confer sensory detectable unpleasant alterations, such as a fruit and rotten flesh odor, respectively. In wine, although biogenic amines may have other sources such as grapes, the metabolic activity of Saccharomyces and non-Saccharomyces yeasts and of acetic acid bacteria, they usually increase after MLF [30,76,79,[83][84][85][86][87]. Among LAB, the decarboxylase activity is strain-specific and is randomly distributed within the different species of Lactobacillus, Pediococcus, Leuconostoc, and Oenococcus.
So, the existing content of biogenic amines in wine will depend on the presence of precursor amino acids, LAB strains with decarboxylase activity, and environmental factors that affect the growth of these strains as well as some oenological practices [30,88,89]. In general, low pH and high concentrations of SO 2 and ethanol limit the growth of these strains and consequently the production of biogenic amines. On the other hand, factors favoring microbial growth such as high temperatures, availability of nutrients in must and wine (sugars, amino acids, organic acids), and inappropriate hygienic practices increase the probability of high amine concentrations [29]. As referred for the formation of ethyl carbamate, wines stored in prolonged contact with lees show higher levels of biogenic amines, attributed to viable but non cultivable LAB cells [30]. Generally, higher biogenic amine contents are found in red wines comparing to rosé, white, and fortified wines [86,90,91].
Conclusions
The contribution of LAB to wine flavor and composition has been described in this review. The difficulties in controlling and anticipating the effects of malolactic fermentation (MLF) on wine quality, given ample species and strain-dependent behavior, remark the importance of strain selection to explore the genomic diversity of LAB.
Selection of starter cultures for MLF should target good adaptation to the harsh wine conditions and potential for the production of flavor compounds, emphasizing in particular glycosidase and esterase activities. Also, the absence of arginine deaminase pathway and amino acid decarboxylases and ability to detoxify mycotoxins such as ochratoxin [92] and biogenic amine degradation [93,94] should be considered as criteria for LAB strain selection for using as starter cultures. | v3-fos-license |
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} | pes2o/s2orc | Soluble Dipeptidyl Peptidase-4 Induces Fibroblast Activation Through Proteinase-Activated Receptor-2
Fibroblasts are the chief secretory cells of the extracellular matrix (ECM) responsible for basal deposition and degradation of the ECM under normal conditions. During stress, fibroblasts undergo continuous activation, which is defined as the differentiation of fibroblasts into myofibroblasts, a cell type with an elevated capacity for secreting ECM proteins. Dipeptidyl peptidase-4 (DPP4) is a ubiquitously expressed transmembrane glycoprotein and exerts effects that are both dependent and independent of its enzymatic activity. DPP4 has been demonstrated to define fibroblast populations in human skin biopsies of systemic sclerosis. Shedding of DPP4 from different tissues into the circulation appears to be involved in the pathogenesis of the diseases. The mechanism underlying soluble DPP4–induced dermal fibrosis has not been clearly determined. The effects of DPP4 on murine 3T3 fibroblasts and human dermal fibroblasts were evaluated by measuring the expression of fibrotic proteins, such as α-SMA and collagen. Soluble DPP4 stimulated the activation of fibroblasts in a dose-dependent manner by activating nuclear factor-kappa B (NF-κB) and suppressor of mothers against decapentaplegic (SMAD) signaling. Blocking proteinase-activated receptor-2 (PAR2) abrogated the DPP4-induced activation of NF-κB and SMAD and expression of fibrosis-associated proteins in fibroblasts. Linagliptin, a clinically available DPP4 inhibitor, was observed to abrogate the soluble DPP4–induced expression of fibrotic proteins. This study demonstrated the mechanism underlying soluble DPP4, which activated NF-κB and SMAD signaling through PAR2, leading to fibroblast activation. Our data extend the current view of soluble DPP4. Elevated levels of circulating soluble DPP4 may contribute to one of the mediators that induce dermal fibrosis in patients.
Fibroblasts are the chief secretory cells of the extracellular matrix (ECM) responsible for basal deposition and degradation of the ECM under normal conditions. During stress, fibroblasts undergo continuous activation, which is defined as the differentiation of fibroblasts into myofibroblasts, a cell type with an elevated capacity for secreting ECM proteins. Dipeptidyl peptidase-4 (DPP4) is a ubiquitously expressed transmembrane glycoprotein and exerts effects that are both dependent and independent of its enzymatic activity. DPP4 has been demonstrated to define fibroblast populations in human skin biopsies of systemic sclerosis. Shedding of DPP4 from different tissues into the circulation appears to be involved in the pathogenesis of the diseases. The mechanism underlying soluble DPP4-induced dermal fibrosis has not been clearly determined. The effects of DPP4 on murine 3T3 fibroblasts and human dermal fibroblasts were evaluated by measuring the expression of fibrotic proteins, such as a-SMA and collagen. Soluble DPP4 stimulated the activation of fibroblasts in a dose-dependent manner by activating nuclear factor-kappa B (NF-kB) and suppressor of mothers against decapentaplegic (SMAD) signaling. Blocking proteinase-activated receptor-2 (PAR2) abrogated the DPP4induced activation of NF-kB and SMAD and expression of fibrosis-associated proteins in fibroblasts. Linagliptin, a clinically available DPP4 inhibitor, was observed to abrogate the soluble DPP4-induced expression of fibrotic proteins. This study demonstrated the mechanism underlying soluble DPP4, which activated NF-kB and SMAD signaling INTRODUCTION Fibrotic disorders encompass a wide spectrum of clinical entities such as systemic sclerosis, a systemic fibrotic disease that induces fibrosis of the skin and internal organs (Rosenbloom et al., 2017). Fibrotic disorders involve a complex and multistage process of tissue injury and inflammation (Lee and Kalluri, 2010). This process is constituted by extracellular matrix (ECM) expansion that is orchestrated by a network of cytokines, chemokines, growth factors, adhesion molecules, and signaling transduction processes (Lee and Kalluri, 2010). Fibroblasts are the chief secretory cells of the ECM (Biernacka and Frangogiannis, 2011). They remain quiescent under normal conditions and are responsible for the basal deposition and degradation of the ECM as well as the maintenance of the matrix network (Krenning et al., 2010). Under stress, stimulated by mediators released from injured and inflammatory tissue, fibroblasts undergo continuous activation, which is defined as the differentiation of fibroblasts into myofibroblasts (Kendall and Feghali-Bostwick, 2014), a cell type with an elevated capacity for secreting ECM proteins. TGF-b is one of the crucial mediators (Walton et al., 2017), which activates suppressor of mothers against decapentaplegic (SMAD), mitogen-activated protein kinase (MAPK), and nuclear factor-kappa B phosphorylated (NF-kB) signaling to induce the pathogenesis of fibrosis (He and Dai, 2015). Myofibroblasts are ultimately responsible for the replacement of healthy tissues with nonfunctional fibrotic tissues (McAnulty, 2007), which leads to increased tissue stiffness and ultimately organ failure (Tomasek et al., 2002). Because of the lack of effective therapeutic agents and insufficient knowledge regarding their pathogenesis in fibrotic diseases, identification of the mediator that regulates fibroblast activation and differentiation of fibroblasts into myofibroblasts is urgently required.
Dipeptidyl peptidase-4 (DPP4), also known as CD26 (Morimoto and Schlossman, 1998), is a type II transmembrane glycoprotein expressed in various cell types that has multifunctional properties (Rohrborn et al., 2015). DPP4 inhibitors, also commonly called gliptin, are being developed as a class of drugs for treating diabetes (Neumiller et al., 2010). In addition to its enzymatic activity, DPP4 itself participates in other cellular functions. Change in DPP4 expression is associated with disease progression (Trzaskalski et al., 2020). Increased DPP4 expression and activity have demonstrated an association with inflammation observed in obesity and metabolic disorders (Trzaskalski et al., 2020). DPP4 is highly expressed in bronchial epithelial cells of patients with asthma, and it increased cell proliferation in airway constitutive cells (Shiobara et al., 2016). Increased DPP4 levels were observed in fibroblasts isolated from individuals with systemic sclerosis relative to fibroblasts isolated from healthy individuals (Soare et al., 2020). Furthermore, the DPP4-positive fibroblast populations in the skin are highly proliferative and expand upon tissue injury to promote wound healing (Rinkevich et al., 2015). The action of DPP4 is complicated.
DPP4 may be released through a nonclassical secretory mechanism from the membrane that involves proteolytic cleavage near the flexible region for generating the soluble form (Rohrborn et al., 2014;Nargis and Chakrabarti, 2018). Soluble DPP4 has also been suggested to be a novel regulator, and elevated levels are indicative of several disorders in addition to diabetes, such as obesity, cardiovascular disease, and nonalcoholic fatty liver disease (dos Santos et al., 2013;Baumeier et al., 2017;Nargis and Chakrabarti, 2018). Soluble DPP4 is an adipokine (Lamers et al., 2011) and is positively associated with hemoglobin A1c levels and the insulin resistance index in type 2 diabetes (Sell et al., 2013;Nargis and Chakrabarti, 2018). It directly impairs insulin signaling in adipocytes, smooth muscle cells, and hepatocytes (Wronkowitz et al., 2014;Baumeier et al., 2017), whereas insulin-stimulated Akt phosphorylation was observed to be reduced when soluble DPP4 was administered (Baumeier et al., 2017). Soluble DPP4 also activates the MAPK and NF-kB signaling cascade involving proteinase-activated receptor-2 (PAR2), resulting in the induction of inflammation and proliferation of human vascular smooth muscle cells (Wronkowitz et al., 2014). Reports have suggested that soluble DPP4 not only possesses catalytic functions but also activates some receptors and signal pathways. Numerous reports have been published on membrane-bound DPP4; however, little information is available on soluble DPP4. The signaling pathway underlying DPP4 in the pathogenesis of fibrosis is still unclear, and revealing this mechanism could potentially lead to a greater understanding of the pathophysiology and treatments of fibrosis diseases. We hypothesized that soluble DPP4 plays a role in dermal fibrosis. Using cultured fibroblasts, we examined the mechanism of how soluble DPP4 enhances fibroblast activation.
Detection of Cell Viability
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay was used as the assessment of cell viability. MTT was then added in cell culture in a final concentration of 0.5 mg/ml. After discarding the supernatant, the purple formazan crystals were dissolved in DMSO. Solutions were then loaded in a 96-well plate, and determined on an automated microplate spectrophotometer at 570 nm.
Protein Extraction From Cell Culture
The proteins from cell culture were extracted using RIPA buffer (Thermo Fisher Scientific Inc., IL, USA) containing protease and phosphatase inhibitors (Sigma, St. Louis, MO, USA). A BCA protein assay kit (Thermo Fisher Scientific Inc.) was used to determine the protein concentration.
RNA Extraction and Reverse Transcription Quantitative Polymerase Chain Reaction
Total RNA was isolated from cells using TRIzol (Thermo Fisher Scientific, MA, USA). Total RNA was reverse transcribed with Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA) and SYBR Green was used for performing quantitative real time PCR. The following are the sequences of the primers used for amplification. PAR2: CGCACTGTAAAGCAGATGCAA and AATTCCCATCTGAGGACCTGG; ACTA2: GCCTGAGGG AAGGTCCTAA and GGAGCTGCTTCACAGGATTC; COL1A1: CACAGAGGTTTCAGTGGTTTGG and AGTAGCACC ATCATTTCCACGA; HPRT: CGTCTTGCTCGAGATGTGATG and GCACACAGAGGGCTACAATGTG.
DNA was amplified for 40 cycles of denaturation for 5 s at 95°C and annealing for 30 s at 60°C, using the TaKaRa Thermal Cycler Dice (TP900). The qPCR assays were performed and analyzed using the Thermal Cycler Dice Real Time System version 4.2 (TaKaRa). The expression level of each individual transcript was normalized to HPRT gene and expressed relative to the mean expression values of control samples.
Immunofluorescent Staining
The expressions of a-SMA, p-p65, and p-SMAD in cells were analyzed by immunofluorescence staining. Cells were cultured on glass coverslips at the density of 3,000 cells/cm 2 and treated with DPP4 with or without GB83 or linagliptin for 48 h. Following treatment for 48 h, cells in the basement layer were washed with PBS and fixed in 4% paraformaldehyde. Following three times washes with PBS for 5 min each, the cells were treated with 0.5% Triton X-100 for 10 min and blocked with bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature, and subsequently incubated with a-SMA antibodies (1:100 dilution; Abcam, USA) at 4°C overnight. Cells were washed three times for 5 min each with PBS and then incubated with secondary antibodies (1:200 dilution; Abcam, USA) for another 1 h. Following washing three times for 5 min each with PBS, cells were stained with DAPI (Beyotime Institute of Biotechnology) at room temperature to visualize the nuclei. Following washing with PBS, the slide was mounted with anti-fluorescence quenching agent (Abcam, USA) and coverslipped; digital images were captured using an inverted fluorescent microscope (magnification, ×400). Digital images were captured four fields per sample with the same exposure time. Three independent experiments were performed.
Lentiviral Transduction for Gene Knockdown
Lentiviral particles containing shRNAs pLKO.1 (#TRCN0000006770) was used to knockdown PAR2 in human dermal fibroblasts. Lentivirus containing scrambled shRNA, pLKO-shScr (#TRCN00001) was used as non-targeting control and served as wild-type. Human adult fibroblasts were transduced with lentiviral vectors with MOI of 3, along with 5 ug/ml polybrene (Sigma Aldrich, MO, USA. Transduced human dermal fibroblasts were treated with puromycin (2 ug/ml), for selection of transduced cells.
Statistical Analyses
All values are represented as mean ± standard error. The results were analyzed using one-way ANOVA, followed by Bonferroni post hoc tests. We considered that p < 0.05 to be significant.
Soluble DPP4 Enhanced Fibroblast Activation
Fibroblasts are key effector cells in tissue fibrosis (Krenning et al., 2010). TGF-b has been well recognized as a fibrotic cytokine that activates fibroblasts and drives their differentiation into myofibroblasts, leading to organ fibrosis. To determine the effect of soluble DPP4 on the pathogenesis of fibrosis, murine 3T3 fibroblasts were treated with recombinant DPP4 (80 and 200 ng/ml) or TGF-b (10 ng/ml) for 48 h and examined for the expression of fibrotic markers. Administration of TGF-b served as a positive control. Compared with control groups, DPP4 increased the expression of fibrotic markers ( Figure 1A), such as collagen I ( Figure 1B), elastin ( Figure 1C), and a-SMA ( Figure 1D), in a concentration-dependent manner, indicating that soluble DPP4 promoted the activation and transformation of fibroblasts into differentiated myofibroblasts.
Skin biopsies from patients with systemic sclerosis were highlighted by a prominent increase in the expression of ECM transcripts (Chadli et al., 2019). Activated dermal fibroblasts are considered to play a major role in the development of skin fibrosis in systemic sclerosis (Chadli et al., 2019). To confirm the results in human dermal fibrotic disease, primary human dermal fibroblasts were treated with DPP4 (200 ng/ml) or TGF-b (10 ng/ ml) for 48 h. Both DPP4 and TGF-b increased the expression of fibrotic gene expression, including ACTA2 ( Figure 1E) and COL1A1 ( Figure 1F), confirming the role of soluble DPP4 in human fibrotic disease.
Soluble DPP4 Activated the Transcription Factor Signaling Pathway in Fibroblasts
To determine the signaling pathway involved in soluble DPP4induced fibrosis and to compare it with TGF-b-induced signaling, we investigated the effect of soluble DPP4 and TGF-b on protein kinase and transcription factor in murine 3T3 fibroblasts. In our study, after 48 h of DPP4 or TGF-b exposure, only TGF-b but not DPP4 induced ERK phosphorylation (Figures 2A, B). By contrast, DPP4 enhanced the expression of NF-kB p-p65 (Figures 2A, C). In addition, both soluble DPP4 and TGF-b increased the expression of p-SMAD2/3 (Figures 2A, D). Soluble DPP4 induced a complex signaling pathway in the induction of the fibrotic signaling pathway.
NF-kB Stimulated by Soluble DPP4 Led to Fibroblast Activation
To confirm the role of NF-kB in soluble DPP4 signaling, an NF-kB inhibitor, Bay11-7082, was used in murine 3T3 fibroblasts. Bay11-7082 at a concentration of 1 mM had no effect on the cell viability of fibroblasts ( Figure 3A); however, it inhibited the DPP4-induced NF-kB p-p65 expression ( Figures 3B, C). Furthermore, NF-kB inhibition could completely abolish the soluble DPP4-induced expression of fibrosis-associated proteins ( Figure 3B), such as collagen I ( Figure 3D), elastin ( Figure 3E), and a-SMA ( Figure 3F).
The PAR2 Antagonist GB83 Abrogated the Soluble DPP4-Induced Response in Fibroblasts
PAR2 is a seven-transmembrane domain G protein-coupled receptor that is widely expressed in cells and regulates a variety of physiological and pathophysiological processes including fibrosis (Soh et al., 2010). To determine whether the DPP4induced fibroblast activation is associated with PAR2, murine 3T3 fibroblasts were treated with recombinant DPP4 in the presence or absence of GB83, a PAR2 antagonist. GB83 at a concentration of 10 mM did not affect the cell viability of fibroblasts and was used in the subsequent experiment ( Figure 4A). GB83 inhibited the soluble DPP4-induced expression of fibrotic proteins ( Figure 4B), such as collagen I ( Figure 4C), elastin ( Figure 4D), and a-SMA ( Figure 4E). Furthermore, the soluble DPP4-induced activation of transcription factor signaling pathways, such as NF-kB p-p65 ( Figure 4F) and p-SMAD2/3 pathways ( Figure 4G), could be prevented by GB83.
Linagliptin Prevented the Effect of Soluble DPP4 on Fibroblasts
We also investigated whether linagliptin, a clinically available enzyme inhibitor of DPP4, prevents DPP4-induced fibroblast activation. Murine 3T3 fibroblasts were treated with recombinant DPP4 in the presence or absence of linagliptin (30 nM). The concentration of linagliptin used in this study was observed to exert a protective effect that was achieved in the plasma of patients with type 2 diabetes treated with this drug (Heise et al., 2009). We observed that linagliptin abrogated the soluble DPP4-induced expression of fibrotic protein, such as elastin ( Figure 4D) and a-SMA ( Figures 4E, H). DPP4-induced activation of transcription factor signaling pathways, including NF-kB p-p65 ( Figure 4F) and p-SMAD2/3 ( Figure 4G) pathways, was also prevented by linagliptin.
Soluble DPP4-Induced p-p65 and p-SMAD in HDF
To determine the signaling underlying soluble DPP4 in HDF, p-p65 ( Figure 5) and p-SMAD ( Figure 6) were determined through immunofluorescence at different time points. Both DPP4 and TGF-b induced the activation of p-p65 and p-SMAD in 6 h and had a plateau effect in 24 h. TGF-b induced a more rapid activation than DPP4 did.
Knocking Down PAR2 Abrogated the Soluble DPP4-Induced Response in HDF
To determine whether PAR2 plays a role in human dermal fibrotic disease, PAR2 was knocked down in HDF. To confirm PAR2 expression in knocked-down HDF, the mRNA expression of PAR2 was measured using RT-qPCR ( Figure 7A). PAR2 mRNA expression was lower in knocked-down HDF than that in wildtype HDF. To investigate whether DPP4-induced fibrotic gene expression in HDF is mediated by PAR2, ACTA2, and COL1A1 mRNA expression in HDF was measured after DPP4 exposure ( Figures 7B, C). DPP4 induced ACTA2 and COL1A1 mRNA expression in HDF, whereas gene knocked-down PAR2 abrogated DPP4-induced fibrotic gene expression ( Figures 7B, C).
To confirm whether NF-kB and SMAD cause downstream signaling of PAR2, the expression of NF-kB p-p65 (Figures 7D, E) and p-SMAD ( Figures 7D, F) was investigated after DPP4 exposure in HDF. DPP4 induced NF-kB p-p65 and p-SMAD expression in HDF, whereas gene knocked-down PAR2 abrogated DPP4-induced signaling pathways.
DISCUSSION
Several growth factors and cytokine signaling molecules have been reported to be critical to the activation of cellular mechanisms in fibrotic diseases and the regulation of ECM protein production (Wynn, 2008;Rockey et al., 2015). Soluble DPP4 has physiological and pathological relevance beyond glycemic control. The concentrations of recombinant DPP4 protein used in this study were in the pathological range (Lee et al., 2013). This study observed that soluble DPP4 induced the expression of fibrosis-associated protein in fibroblasts, especially in primary human dermal fibroblasts, suggesting that soluble DPP4 induces the activation of dermal fibroblasts. This result coincides with that of a previous study, which demonstrated that DPP4-positive human dermal fibroblasts express higher levels of myofibroblast markers and collagen in systemic sclerosis (Soare et al., 2020). DPP4 is a functional requirement for fibroblast activation and tissue fibrosis and may serve as an activation marker (Soare et al., 2020).
Studies on the key signaling pathways that regulate fibrosis diseases have reported the following noteworthy findings. TGF-b was observed to be a profibrotic factor and acts as a crucial mediator in fibrogenesis (Walton et al., 2017). The SMAD2/3 intracellular pathway was heavily implicated in TGF-b-induced fibrosis and is known as the canonical pathway (He and Dai, 2015). Targeting the SMAD signaling pathway is a novel therapeutic approach to treating tissue fibrosis (Wojcik et al., 2013;He and Dai, 2015;Liu et al., 2016;Tee et al., 2018;Zhang et al., 2018). In addition to activating the SMAD-dependent pathway, TGF-b can signal in a noncanonical manner, as exemplified in MAPK and NF-kB signaling, which together induce a complete TGF-b response (Wu et al., 2019). The inhibition of a complete TGF-b response may exert beneficial effects that prevent myofibroblast formation and synthesis of ECM components (Luedde and Schwabe, 2011;Madala et al., 2012). NF-kB is the signaling molecule other than SMAD downstream of soluble DPP4. NF-kB acts as a double-edged sword, and the pronounced inhibition may negatively affect cell viability (Gieling et al., 2010). The concentration of the NF-kB inhibitor used in this study did not affect cell viability but abrogated the soluble DPP4-induced fibrotic response. The activation of SMAD and NF-kB signaling may contribute to the fibrotic response of soluble DPP4. Soluble DPP4 and TGF-b exerted different patterns in the activation of the signaling pathway in fibroblasts. In an experimental setting of NIH/3T3 fibroblasts, soluble DPP4 stimulated SMAD and NF-kB signaling, whereas TGF-b stimulated SMAD and ERK signaling. In HDF, both TGF-b and soluble DPP4 induced the activation of p-NF-kB and p-SMAD; in addition, TGF-b induced a more rapid signaling than DPP4 did. The key implication of our findings is that TGF-b possesses stronger and higher potency than soluble DPP4 does on inducing SMAD phosphorylation. To activate SMAD signaling in NIH/3T3 fibroblasts, the requisite concentration of soluble DPP4 is approximately 80-200 ng/ml, whereas the requisite concentration of TGF-b is less than 10 ng/ml. PAR2 plays crucial roles in tissue hemostasis, thrombosis, wound healing, inflammation-associated disorders, fibrosis, and (Ungefroren et al., 2018). PAR2 activation involves receptor cleavage by different serine proteases and exposure to an N-terminal tethered ligand (TL) that binds to and activates the cleaved receptor (Hollenberg et al., 1997). The activating sequence of PAR2 could be found in the cystein-rich region of DPP4 responsible for binding (Wronkowitz et al., 2014). Therefore, soluble DPP4 can activate PAR2 with its TL sequence and act as an agonist of PAR2 (Wronkowitz et al., 2014). PAR2 has been reported to be involved in soluble DPP4induced inflammation and dysfunction (Wronkowitz et al., 2014). The blockade of PAR2 prevented soluble DPP4-induced proliferation and inflammation of vascular smooth muscle cells (Wronkowitz et al., 2014) as well as the dysfunction of endothelial cells (Romacho et al., 2016). In addition, PAR2 is expressed on the surface of fibroblasts and has been suggested to play a role in tissue repair processes (Grandaliano et al., 2003;Wygrecka et al., 2011). PAR2 activation induces collagen synthesis and a-SMA expression (Asokananthan et al., 2015). Our results demonstrated that the PAR2 antagonist or knocking down PAR2 can prevent soluble DPP4-induced fibrotic marker expression in fibroblasts, indicating that PAR2 is a receptor of soluble DPP4 and participates in the stimulation of fibroblasts.
The signaling molecules downstream of PAR2 are complex. After being stimulated by soluble DPP4, PAR2 activated the ERK and NF-kB signaling cascades, consequently increasing the secretion of proinflammatory cytokines and stimulating the proliferation of vascular smooth muscle cells (Ervinna et al., 2013;Wronkowitz et al., 2014). Our results indicated that after PAR2 activation, soluble DPP4 stimulated SMAD and the NF-kB pathway to induce a complete response in the activation of fibroblasts because blocking PAR2 using pharmacological inhibitors or genetic knockdown of abolished DPP4 induced SMAD and NF-kB signaling as well as the expression of fibrosis-associated proteins. Membrane-bound DPP4 is essential for TGF-b-induced receptor heterodimerization and subsequent intracellular signal transduction (Shi et al., 2016). The stimulation of cultured dermal fibroblasts with TGF-b induced the upregulation of membrane-bound DPP4 expression (Soare et al., 2020), and the inactivation of DPP4 blocked the TGF-b-induced differentiation of fibroblasts into myofibroblasts and reduced the release of collagen in vitro (Soare et al., 2020). Further investigation is required for understanding whether soluble DPP4/PAR2/TGF-b induces a co-activation axis. Studies have reported that inhibition of DPP4 by pharmacological inhibitors alleviated fibrotic responses, such as in bleomycin-induced dermal and pulmonary fibrosis (Soare et al., 2020), CCl 4induced liver fibrosis (Kaji et al., 2014;Wang et al., 2017), and a high-salt-diet-induced cardiac failure and fibrosis (Esposito et al., 2017). In our studies, we observed that linagliptin prevented the effect of soluble DPP4 in fibroblasts. Linagliptin can block the interactions between DPP4 and ECM components, receptors, or plasma membrane components (Hasan and Hocher, 2017), thus ameliorating ECM and intracellular signal transduction. The effects of soluble DPP4 on fibroblast activation may be independent of its enzymatic activity.
CONCLUSION
We characterized the mechanisms underlying soluble DPP4, which activated NF-kB and SMAD signaling through PAR2, leading to the activation of dermal fibroblasts (Figure 8). Our data extended the current view of the effect of soluble DPP4 on dermal fibrosis. Elevated levels of circulating soluble DPP4 may contribute to one of the mediators that induce dermal fibrosis in patients.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. | v3-fos-license |
2018-06-07T13:35:13.390Z | 2018-05-28T00:00:00.000 | 44090842 | {
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} | pes2o/s2orc | Angucycline Glycosides from Mangrove-Derived Streptomyces diastaticus subsp. SCSIO GJ056
Nine new angucycline glycosides designated urdamycins N1–N9 (1–9), together with two known congener urdamycins A (10) and B (11), were obtained from a mangrove-derived Streptomyces diastaticus subsp. SCSIO GJ056. The structures of new compounds were elucidated on the basis of extensive spectroscopic data analysis. The absolute configurations of 6–9 were assigned by electronic circular dichroism calculation method. Urdamycins N7 (7) and N8 (8) represent the first naturally occurring (5R, 6R)-angucycline glycosides, which are diastereomers of urdamycins N6 (6) and N9 (9), respectively.
During the course of searching for novel anti-infective and antitumor agents from the marine environment, we found that the chemical profile of strain SCSIO GJ056 cultivated in AM2 medium revealed an array of secondary metabolites showing typical UV/VIS absorptions, which were similar to those of angucyclines/anthracyclines. Subsequent solvent extraction and isolation procedures led to the purification and structure elucidation of nine new angucycline glycosides, named urdamycins N1-N9 (1-9), together with two known urdamycins A (10) and B (11). Urdamycins N7 (7) and N8 (8) represent the first naturally occurring (5R, 6R)-angucycline glycosides. Herein, we report the fermentation, isolation, and structure elucidation of these compounds.
Results and Discussion
The strain SCSIO GJ056 was fermented (15 L) and the fermentation broth was extracted with butanone. The extract was subjected to repetitive silica gel column chromatography, followed by preparative HPLC purification to yield compounds 1-11 ( Figure 1). The known urdamycins A (10) Mar. Drugs 2018, 16 and B (11) were identified by comparisons of MS, 1 H, and 13 C NMR spectroscopic data with those previously reported [8]. Compound 1 was obtained as a yellowish powder. Its molecular formula was determined to be C 38 H 46 O 14 on the basis of HRESIMS peak at m/z 725.2834 [M − H] − , indicating 16 degrees of unsaturation. The 13 C and DEPT NMR spectra of 1 displayed 38 carbon resonances, including five methyls, six methylenes, 14 methines, and 13 nonprotonated carbons. The 1 H NMR spectrum showed one chelated hydroxy group signal at δ H 12.55 (1H, br s, 8-OH), a pair of ortho-coupled aromatic proton signals at δ H 7.81 (d, 7.8 Hz, H-10) and 7.59 (d,7.8 Hz,, and a singlet aromatic proton signal at δ H 7.65 (s, H-6). The HMBC correlations ( Figure 2) from H-6 to C-4a, C-5, C-7, and C-12a, from H-11 to C-7a, C-9, and C-12, and from H-10 to C-8, C-9, and C-11a confirmed the existence of the anthraquinone skeleton (rings B, C, and D). Further HMBC correlations of H 2 -2/C-1, C-12b, C-4; H 2 -4/C-2, C-4a, C-12b; and H 3 -13/C-2, C-3, C-4 allowed the assignment of the angular ring (ring A) with a methyl group (CH 3 -13) substitution at C-3. A methoxy group (OCH 3 -14) attached at C-5 in ring B was deduced by the HMBC correlation of H 3 -14/C-5. A hydroxy group linked at C-3 in ring A was inferred based on the 13 C NMR chemical shift at δ C 71.8. The absolute configuration of C-3 was tentatively deduced to be R, which was identical with that of urdamycinone B and N05WA963D in light of the similar 13 C NMR resonances of C-3 and CH 3 -13, as well as the similar biosynthetic pathway [8,9].
Compound 2, isolated as a dark red powder, has the molecular formula of C 38 H 44 O 13 on the basis of HRESIMS peak at m/z 707.2708 [M − H] − , showing 17 degrees of unsaturation and an 18 amu less than that of compound 1. An obvious red shift on the UV-VIS spectrum of 2 relative to that of 1 indicated an additional conjugated system in 2. The 13 C and DEPT NMR data of 2 displayed 38 carbon signals attributable to five methyls, four methylenes, 16 methines, and 13 nonprotonated carbons. The 1 H and HSQC NMR spectra suggested three singlet olefnic proton signals at δ H 7.58 (H-4), δ H 7.54 (H-6), and δ H 6.99 (H-2), and a pair of ortho-coupled aromatic proton signals at δ H 7.82 (d, 7.6 Hz, H-10) and 7.68 (d, 7.6 Hz, H-11). Comparing the 1 H and 13 C NMR spectroscopic data to those of 1 revealed that 2 possessed a similar core structure with that of 1. The difference between 2 and 1 was the aromatization of ring A in 2, which supported by the HMBC correlations from CH 3 -13 to C-2, C-3, and C-4, from H-2 to C-1, C-4, and C-12b, and from H-4 to C-2, C-4a, and C-12b. Compound 2 possessed the same trisaccharide moiety with 1 according to similar 1 H and 13 C NMR signals in aliphatic area. The structure of 2 was elucidated as shown in Figure 1 by detailed analysis of 2D NMR spectra data.
Compound 3, a dark green powder, was isolated as minor component from the extract. Its molecular formula of C 26 H 24 O 8 was determined by the HRESIMS peak at m/z 463.1409 [M − H] − , indicating 15 degrees of unsaturation. Comprehensive analysis of its 1 H and 13 C NMR spectroscopic data revealed that 3 had the same aglycone with that of 2. However, a set of 1 H and 13 C resonances ascribed to β-olivose-(1→4)-α-rhodinosyl moiety disappeared, indicating the absence of two sugar units in 3. This is consistent with the HRESIMS data, which showing a C 12 H 20 O 5 fragment loss relative to 2. Therefore, the structure of 3 was established and named urdamycin N3. Compound 4 was obtained as a dark green powder. Its molecular formula was determined to be C 37 H 42 O 13 by the HRESIMS peak at m/z 693.2554 [M − H] − . The 1 H and 13 C NMR data of 4 were closely similar to those of 2, except that the methoxy signals at δ H 4.14, δ C 56.6 in 2 were absent. The 13 C NMR signal of C-5 shifted from δ C 160.3 in 2 to δ C 163.6 in 4, indicating the OMe-5 in 2 was replaced by OH-5 in 4. Compound 4 was named urdamycin N4.
Compound 5 was obtained as a red powder. The molecular formula of C 37 H 42 O 12 , as determined by HRESIMS, which was one oxygen atom less than that of 4. The 1 H and 13 C NMR spectroscopic data were similar with those of 4, except that two pairs of ortho-coupled aromatic signals were observed. Additionally, the 13 C NMR signal at δ C 163.6 for the oxygen-bearing aromatic C-5 in 4 was replaced by an aromatic methine signal at δ C 135.4. Thus, the structure of 5 was determined as 5-demethoxy-urdamycin N2, designated as urdamycin N5.
The molecular formulae of compounds 6 and 7 were determined both to be C 27 H 28 O 9 by HRESIMS, indicating 14 degrees of unsaturation. The 1 H and 13 C NMR spectroscopic data of 6 were similar with those of 3, except that two aromatic carbon signals at δ C 160.5 (C-5) and 100.0 (C-6) in 3 were replaced by two oxygen-bearing methine carbon signals at δ C 78.1 (C-5) and 70.2 (C-6). Furthermore, two methoxys were attached at C-5 and C-6 based on the HMBC correlations of H 3 -14/C-5 and H 3 -15/C-6, respectively. Small coupling constants (2.8 Hz) between H-5 and H-6 revealed a trans configuration of H-5 and H-6, indicating an (5R, 6R) or (5S, 6S) configuration of 6.
To determine the absolute configurations of 6, comparisons of the experimental and ECD spectra using a time-dependent density functional theory (TDDFT) were employed. Comparison of the experimental and calculated CD spectra ( Figure 4) established the absolute configuration as (5S, 6S) for 6, which were the same as those of PMO70747, PD116740, and TAN-1085 [12][13][14][15][16][17]. Compound 7 possessed the same planar structure with that of 6, as deduced by the COSY and HMBC spectra ( Figure 2). However, the experimental and calculated CD spectra of 7 showed cotton effects totally opposite to those of 6, respectively, inferring the (5R, 6R) configuration for 7 ( Figure 4). Compounds 6 and 7 were named urdamycins N6 and N7, respectively. Compound 4 was obtained as a dark green powder. Its molecular formula was determined to be C37H42O13 by the HRESIMS peak at m/z 693.2554 [M − H] − . The 1 H and 13 C NMR data of 4 were closely similar to those of 2, except that the methoxy signals at δH 4.14, δC 56.6 in 2 were absent. The 13 C NMR signal of C-5 shifted from δC 160.3 in 2 to δC 163.6 in 4, indicating the OMe-5 in 2 was replaced by OH-5 in 4. Compound 4 was named urdamycin N4.
Compound 5 was obtained as a red powder. The molecular formula of C37H42O12, as determined by HRESIMS, which was one oxygen atom less than that of 4. The 1 H and 13 C NMR spectroscopic data were similar with those of 4, except that two pairs of ortho-coupled aromatic signals were observed. Additionally, the 13 C NMR signal at δC 163.6 for the oxygen-bearing aromatic C-5 in 4 was replaced by an aromatic methine signal at δC 135.5. Thus, the structure of 5 was determined as 5-demethoxy-urdamycin N2, designated as urdamycin N5.
Bacterial Materials
Strain SCSIO GJ056 was isolated from a mangrove-derived sediment sample collected in Yalong bay, China. It was identified as Streptomyces diastaticus subsp. on the basis of morphological characteristics and 16S rRNA sequence analysis by comparisons with other sequences in the GenBank database. The DNA sequence has been deposited in GenBank (accession no. MH368281). The strain was preserved at the RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences and also at the China General Microbiological Culture Collection Center (CGMCC, Beijing, China), CGMCC No. 13648. | v3-fos-license |
2017-11-21T12:56:33.624Z | 2017-05-04T00:00:00.000 | 273023517 | {
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} | pes2o/s2orc | A complex stereochemical relay approach to the antimalarial alkaloid ocimicide A1. Evidence for a structural revision
The core structure of the potent antimalarial alkaloid ocimicide A1 was prepared by a complex stereochemical relay. Computational studies suggest a structural revision of the metabolite is necessary.
SYBR Green-Based Parasite Growth Assay. This proliferation assay was adapted from the malaria SYBR Green I-based fluorescence assay. [7] The intermediates to be tested were added to a 96-well plate with final concentrations of 100 nM, 500 nM, 2 µM/5 µM. A highly synchronized early ring stage parasite culture was added to the plate containing conjugate. Controls were performed using non-infected erythrocytes, infected erythrocytes without conjugate, and infected erythrocytes treated with 2.5 μg/mL and 0.035 μg/mL of blasticidin. Plates were incubated for 72 and 96 h at 37 °C in a gas chamber. After 72 h and 96 h, erythrocytes were lysed with 20 mM Tris (pH 7.5), 5 mM EDTA, 0.008% saponin, 0.08% Triton-X 100, 1× SYBR Green I and incubated for 1 h in the dark at room temperature. Plates were read at 497/520 nm on a Synergy MX, Biotek fluorescent plate reader. The percent inhibition was calculated with no treatment as no inhibition and Blasticidin 2.5 μg/mL treatment as 100% inhibition. NMR Calculations. Following the protocol reported by Hoye and co-workers, [8] structures were generated in GaussView for all diastereomers of protonated ocimicide A2 (2) (at nitrogen 15 and carbons 12, 13, 14 and 17 based on patent numbering [1] ). This produced 32 initial structures, which were imported into BOSS [9] and subjected to a conformational search. Rotamers within 5.02 kcal/mol of the lowest energy structure (8-30 conformers for each diastereomer) were advanced to density functional theory geometry optimization [gas phase, B3LYP/6-31+G(d,p)] in Guassian 09 [10] . Geometry optimized conformers were confirmed as real local-minima by the absence of imaginary frequencies. The chemical shifts of the optimized conformers were calculated at the modified [3] WC04/6-31G(d) level of theory in methanol. Cartesian coordinates (numbering is unique for each conformer, and does not correspond to patent numbering), energy values (in A.U.), and NMR data [ 1 H and 13 C (ppm, δ scale); numbering is consistent with patent [1] ] are presented for each unique conformer (supplementary appendix).
General Experimental Procedures.
All reactions were performed in single-neck, flame-dried, round-bottomed flasks fitted with rubber septa under a positive pressure of argon, unless otherwise noted. Air and moisture-sensitive liquids were transferred via syringe or stainless steel cannula, or were handled in a nitrogen-filled drybox (working oxygen level <1 ppm). Organic solutions were concentrated by rotary evaporation at 30-33 °C. Flash-column chromatography was performed as described by Still et al, [11] employing silica gel (60 Å, 40-63 µm particle size) purchased from Sorbent Technologies (Atlanta, GA). Analytical thin-layered chromatography (TLC) was performed using glass plates precoated with silica gel (1.0 mm, 60 Å pore size) impregnated with a fluorescent indicator (254 nm). TLC plates were visualized by Instrumentation. Proton nuclear magnetic resonance spectra ( 1 H NMR) were recorded at 400 or 500 MHz at 24 °C, unless otherwise noted. Chemical shifts are expressed in parts per million (ppm, δ scale) downfield from tetramethylsilane and are referenced to residual protium in the NMR (CDCl3, δ 7.26; (CD3)2SO, δ 39.5). Data are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet and/or multiple resonances, br = broad, app = apparent), integration, coupling constant in Hertz, and assignment. Proton-decoupled carbon nuclear magnetic resonance spectra ( 13
S16
infrared spectra (ATR-FTIR) were obtained using a Thermo Electron Corporation Nicolet 6700 FTIR spectrometer referenced to a polystyrene standard. Data are represented as follows: frequency of absorption (cm -1 ), intensity of absorption (s = strong, m = medium, w = weak, br = broad). High-resolution mass spectrometry (HRMS) data were obtained using a Waters UPLC/HRMS instrument equipped with a dual API/ESI high-resolution mass spectrometry detector and photodiode array detector. Unless otherwise noted, samples were eluted over a reverse-phase C18 column (1.7 µm particle size, 2.1 × 50 mm) with a linear gradient of 5% acetonitrile-water containing 0.1% formic acid→95% acetonitrile-water containing 0.1% formic acid over 4 min, followed by 100% acetonitrile containing 0.1% formic acid for 1 min, at a flow rate of 800 µL/min. Nikolayevskiy et al. "A complex stereochemical relay approach to the antimalarial alkaloid ocimicide A1. Evidence for a structural revision." submitted to Chem. Sci.
Synthesis of the enaminone S2:
4-Methoxypyridine (10.0 mL, 98.5 mmol, 1 equiv) was added via syringe to a solution of 2-methyl-1-propenylmagnesium bromide (S1) in tetrahydrofuran (0.50 M, 197 mL, 98.5 mmol, 1.00 equiv) at 24 °C. The reaction mixture was cooled to -23 °C. Benzyl chloroformate (15.5 mL, 108 mmol, 1.10 equiv) was added via syringe to the cold reaction mixture. Upon completion of the addition, the mixture was stirred for 1 h at -23 °C. Aqueous hydrochloric acid solution (10% v/v, 100 mL) was added via syringe to the cold reaction mixture. The reaction mixture was allowed to warm over 30 min to 24 °C, with stirring. The warmed reaction mixture was stirred for 30 min at 24 °C. The product mixture was diluted with ethyl acetate (500 mL). The diluted product mixture was transferred to a separatory funnel that had been charged with saturated aqueous sodium chloride solution (300 mL) and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (2 × 300 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue obtained was dissolved in dichloromethane (20 mL). Silica gel (25.0 g) was added and the suspension was concentrated to afford a free-flowing powder. The dried powder was transferred to a column of silica gel. Purification by flash-column chromatography (eluting with 30% ethyl acetate-hexanes) afforded the enaminone S2 as a pale, yellow solid (20.9 g, 74%).
Synthesis of the vinylogous amide S3:
Sodium methoxide (1.90 g, 35.1 mmol, 10.0 equiv) was added to a solution of the enaminone S2 (1.00 g, 3.51 mmol, 1 equiv) in methanol (35 mL) in a 100-mL flask that had been fused to a Teflon-coated valve at 24 °C. The reaction vessel was sealed and the sealed vessel was placed in an oil bath that had been preheated to 65 °C. The reaction mixture was stirred and heated for 12 h at 65 °C. The reaction vessel was removed from the oil bath and the product mixture was allowed to cool over 10 min to 24 °C. The cooled product mixture was concentrated to dryness to afford a brown residue. The residue obtained was diluted with ethyl acetate (200 mL). The diluted product mixture was transferred to a separatory funnel that had been charged with water (100 mL) and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (3 × 150 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue obtained was purified by flash-column chromatography (eluting with 30% ethyl acetate-hexanes initially, grading to 20% methanol-ethyl acetate) to afford the vinylogous amide S3 as a yellow solid (529 mg, >99%).
Synthesis of the carbamate S4:
4-Dimethylaminopyridine (42.8 mg, 350 μmol, 0.10 equiv) was added to a solution of the enaminone S3 (529 mg, 3.50 mmol, 1 equiv) in tetrahydrofuran (7.0 mL) at 24 °C. The reaction mixture was cooled to 0 °C. Triethylamine (2.93 mL, 21.0 mmol, 6.00 equiv) was added dropwise via syringe to the reaction mixture. Upon completion of the addition, the reaction mixture was stirred for 10 min at 0 °C. Di-tert-butyl dicarbonate (965 μL, 4.20 mmol, 1.20 equiv) was added dropwise via syringe to the cold reaction mixture. Upon completion of the addition, the reaction mixture was stirred for 4 h at 0 °C. The product mixture was diluted with ethyl acetate (50 mL). The diluted product mixture was transferred to a separatory funnel that had been charged with water (100 mL) and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (3 × 50 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to afford the carbamate S4 was a pale, yellow solid (859 mg, 98%).
Synthesis of the vinyl triflate 9:
A solution of lithium tri-sec-butylborohydride in tetrahydrofuran (1.0 M, 67.0 mL, 67.0 mmol, 1.05 equiv) was added dropwise over 15 min via syringe pump to a solution of the carbamate S4 [16.0 g, 63.8 mmol, 1 equiv, dried by azeotropic distillation with benzene (10 mL)] and Comins' reagent (S5, 27.6 g, 70.2 mmol, 1.10 equiv) in tetrahydrofuran (320 mL) at −78 °C. Upon completion of the addition, the reaction mixture was stirred for 1 h at −78 °C. The cold reaction mixture was warmed over 30 min to 24 °C. The warmed reaction mixture was stirred for 2.5 h at 24 °C. The warmed product mixture was transferred to a separatory funnel that had been charged with aqueous sodium hydroxide solution (10%, 500 mL) and ethyl acetate (500 mL). The layers that formed were separated. The aqueous layer was extracted with ethyl acetate (2 × 400 mL). The organic layers were combined and the combined organic layers were washed sequentially with 1 N aqueous sulfuric acid solution (300 mL), saturated aqueous sodium bicarbonate solution (300 mL), and saturated aqueous sodium chloride solution (300 mL). The organic layer was dried over sodium sulfate and the dried solution was filtered. The filtrate was concentrated and the residue obtained was suspended in dichloromethane (50 mL). The heterogeneous mixture was filtered through a pad of celite. The filtrate was concentrated and the residue obtained was purified by flashcolumn chromatography (eluting with 10% acetone-pentane) to afford a sample of the vinyl triflate 9 contaminated with reagent-derived byproducts. Further purification of this sample by flash-column chromatography (eluting with 5% ethyl acetate-dichloromethane) provided the vinyl triflate 9 as a yellow solid (19.2 g, 78%).
Synthesis of the cyclohexenylquinoline 11:
A 100-mL round-bottomed flask fused to a Teflon-coated valve was charged with the vinyl triflate 9 (1.36 g, 3.90 mmol, 1 equiv) and 2-cyano-6-methoxy-3-(trimethylstannyl)quinoline (10, 1.50 g, 3.90 mmol, 1.00 equiv). Benzene (5.0 mL) was added and the solution was concentrated to dryness. This process was repeated twice. The vessel was sealed and the sealed vessel was transferred to a nitrogen-filled drybox. N,N-dimethylformamide (19.5 mL), tetrakis(triphenylphosphine)palladium (225 mg, 195 μmol, 0.05 equiv), cesium fluoride (1.18 g, 7.79 mmol, 2.00 equiv), and copper iodide (74.2 mg, 390 μmol, 0.10 equiv) were added in sequence. The vessel was sealed and the sealed vessel was removed from the drybox. The reaction mixture was stirred for 1 h at 24 °C. The product mixture was diluted with ethyl acetate (50 mL) and water (50 mL). The diluted product mixture was eluted through a pad of celite (length/diameter = 6/4 cm). The celite pad was washed sequentially with water (50 mL) and ethyl acetate (400 mL). The biphasic filtrate was transferred to a separatory funnel and the layers that formed were separated. The organic layer was washed sequentially with water (3 100 mL) and saturated aqueous sodium chloride solution (100 mL). The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue obtained was purified by flash-column chromatography (eluting with 2% acetone−dichloromethane) to afford the cyclohexenylquinoline 11 as a pale yellow solid (1.63 g, >99%). alkaloid ocimicide A1. Evidence for a structural revision." submitted to Chem. Sci.
Synthesis of the carboxylic acid 12:
2,6-Lutidine (363 μL, 3.13 mmol, 2.00 equiv) and N-methylmorpholine N-oxide (220 mg, 1.88 mmol, 1.20 equiv) were added in sequence to a solution of the cyclohexenylquinoline 11 (656 mg, 1.57 mmol, 1 equiv) in acetone (28 mL) and water (3.1 mL) at 24 °C. A solution of aqueous osmium tetroxide (2% w/w, 491 μL, 31.3 μmol, 0.025 equiv) was added to the reaction mixture at 24 °C. Upon completion of the addition, the resulting reaction mixture was stirred for 12 h at 24 °C. (Diacetoxyiodo)benzene (605 mg, 1.88 mmol, 1.20 equiv) was then added to the reaction mixture. The resulting reaction mixture was stirred for 3 h at 24 °C. An additional portion of (diacetoxyiodo)benzene (302 mg, 0.94 mmol, 0.60 equiv) was added to the reaction mixture and the reaction mixture was stirred for 1 h at 24 °C. The reaction mixture was concentrated to remove acetone (Caution: this operation should be performed in a well-ventilated fume hood). The oily residue in water was diluted with tetrahydrofuran (15.7 mL), tert-butyl alcohol (3.9 mL), and water (12.6 mL). 2-Methyl-2-butene (3.9 mL), monosodium phosphate (1.88 g, 15.7 mmol, 10.0 equiv), and sodium chlorite (991 mg, 11.0 mmol, 7.00 equiv) were added in sequence to the reaction mixture at 24 °C. The reaction mixture was stirred for 1 h at 24 °C. The product mixture was diluted with ether (300 mL). The diluted product mixture was transferred to a separatory funnel that had been charged with 5% aqueous sodium hydroxide solution (200 mL) and the layers that formed were separated. The organic layer was extracted with 5% aqueous sodium hydroxide solution (3 × 150 mL) and water (2 × 100 mL). The aqueous layers were combined and the combined aqueous layers were acidified to pH 4 with concentrated hydrochloric acid solution, to afford an aqueous suspension. The aqueous suspension was transferred to a separatory funnel that had been charged with ethyl acetate (300 mL) and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (3 × 300 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to afford the unpurified carboxylic acid 12 as a brown solid. 1 H NMR analysis (400 MHz, CDCl3) indicated >95% conversion to the carboxylic acid 12. The product so obtained was used directly in subsequent steps.
The carboxylic acid 12 was not amenable to purification by flash-column chromatography. Therefore, further characterization was not attempted.
Nikolayevskiy et al. "A complex stereochemical relay approach to the antimalarial alkaloid ocimicide A1. Evidence for a structural revision." submitted to Chem. Sci.
Note: The bromolactone 13 was found to be unstable towards prolonged exposure to silica gel (as indicated by two-dimensional TLC analysis). Therefore, the purification step should be performed as rapidly as possible.
S25
One-pot Synthesis of the epoxide 8: 4-Dimethylaminopyridine (35.5 mg, 291 μmol, 0.19 equiv) and Nbromosuccinimide (311 mg, 1.75 mmol, 1.12 equiv) were added in sequence to a solution of the unpurified acid 12 [1.56 mmol, 1 equiv; assuming quantitative yield in the saponification step, dried by azeotropic distillation with benzene (3 × 10 mL)] in dichloromethane (58 mL) at 24 °C. The reaction mixture was stirred for 1 h at 24 °C. The reaction mixture was concentrated to dryness. The residue so obtained was dissolved in tetrahydrofuran (24 ml) and methanol (48 mL). Potassium carbonate (1.00 g, 7.27 mmol, 4.65 equiv) was added to the reaction mixture at 24 °C. The resulting suspension was vigorously stirred for 1 h at 24 °C. The product mixture was diluted with ethyl acetate (250 mL). The diluted product mixture was transferred to a separatory funnel that had been charged with 100 mM aqueous sodium phosphate buffer solution (pH 7, 200 mL) and the layers that formed were separated. The aqueous layer was extracted with ethyl acetate (3 × 100 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried organic solution was filtered and the filtrate was concentrated. The residue obtained was purified by flash-column chromatography (eluting with 10% acetone-dichloromethane) to afford the epoxide 8 as an off-white solid (308 mg, 45% from 11).
Synthesis of the alcohol 7:
In a nitrogen-filled drybox, toluene (11 mL) and lithium bis(trimethylsilyl)amide (20.6 mg, 123 μmol, 1.10 equiv) were added in sequence to a 25-mL round-bottomed flask fused to a Teflon-coated valve that had been charged with the epoxide 8 [49.1 mg, 112 μmol, 1 equiv, dried by azeotropic distillation with benzene (4 × 1.0 mL)]. The vessel was sealed and the sealed vessel was removed from the drybox. The reaction vessel was placed in an oil bath that had been preheated to 103 °C. The reaction mixture was stirred and heated for 2 h at 103 °C. The reaction vessel was removed from the oil bath and the product mixture was allowed to cool over 1 min to 24 °C. The cooled product mixture was transferred to a separatory funnel that had been charged with saturated aqueous ammonium chloride solution (20 mL) and ethyl acetate (30 mL). The layers that formed were separated. The aqueous layer was extracted with ethyl acetate (4 × 30 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to afford a red viscous oil. 1 H NMR analysis (400 MHz, CDCl3) indicated 75% conversion to the alcohol 7. Purification by flash-column chromatography (eluting with 30% ethyl acetate−dichloromethane, grading to 50% ethyl acetate−dichloromethane, one step) afforded the alcohol 7 as an off-white solid (21.4 mg, 44%) and the recovered epoxide 8 as a pale, yellow solid (9.2 mg, 19%).
Synthesis of the ester 6:
Triethylamine (1.06 mL, 7.59 mmol, 10.0 equiv) and methanesulfonyl chloride (70.1 μL, 910 μmol, 1.20 equiv) were added in sequence to a solution of the alcohol 7 [333 mg, 759 μmol, 1 equiv, dried by azeotropic distillation with benzene (10 mL)] in dichloromethane (10 mL) in a 50-mL round-bottomed flask fused to a Teflon-coated valve at 24 °C. Upon completion of the addition, the reaction mixture was stirred for 1 h at 24 °C. An additional portion of methanesulfonyl chloride (10.0 μL, 130 μmol, 0.17 equiv) was then added at 24 °C. Upon completion of the addition, the reaction mixture was stirred for 30 min at 24 °C. Sodium methoxide (150 mg, 2.78 mmol, 3.67 equiv) and methanol (5.0 mL) were added in sequence to the reaction mixture at 24 °C. The resulting mixture was stirred for 1 min at 24 °C, and then the reaction mixture was concentrated to dryness. Sodium methoxide (328 mg, 6.07 mmol, 8.00 equiv) and methanol (15 mL) were added in sequence to the reaction vessel at 24 °C. The reaction vessel was sealed and the sealed vessel was placed in an oil bath that had been preheated to 65 °C. The reaction mixture was stirred and heated for 12 h at 65 °C. The reaction mixture was concentrated to dryness and the residue obtained was dissolved in tetrahydrofuran (76 mL). Aqueous sulfuric acid solution (2 N, 15 mL) was added to the reaction mixture at 24 °C. The reaction mixture was stirred for 2 h at 24 °C. The product mixture was diluted with dichloromethane (300 mL). The diluted product mixture was poured slowly into a separatory funnel that had been charged with saturated aqueous sodium bicarbonate solution (300 mL) and the layers that formed were separated. The aqueous layer was extracted with dichloromethane (3 × 250 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to afford the ester 6 as an offwhite solid (321 mg, 96%).
Synthesis of the acid 17:
Aqueous sodium hydroxide solution (1.25 M, 2.3 mL) was added to a solution of the ester 6 (20.4 mg, 46.4 μmol, 1 equiv) in methanol (4.6 mL) in a 10-mL roundbottomed flask fused to a Teflon-coated valve at 24 °C. The reaction vessel was sealed and the sealed vessel was placed in an oil bath that had been preheated to 80 °C. The reaction mixture was stirred and heated for 14 h at 80 °C. The product mixture was allowed to cool over 10 minutes to 24 °C. The cooled product mixture was concentrated to remove methanol. The residue was diluted with water (8.0 mL) and the diluted mixture was acidified to pH 6 with concentrated hydrochloric acid solution. The resulting mixture was loaded onto a column of reverse-phase silica (length/diameter = 15/1 cm). The column was washed with water (20 mL) to remove inorganic salts and the washed column was dried under a stream of nitrogen. The product was then eluted from the dried reverse-phase silica column with methanol (50 mL). The methanol collected and concentrated to afford unpurified acid 17. The product so obtained was used directly in the following step.
Nikolayevskiy et al. "A complex stereochemical relay approach to the antimalarial alkaloid ocimicide A1. Evidence for a structural revision." submitted to Chem. Sci.
Synthesis of the Weinreb amide 18:
Methanesulfonyl chloride (165 μL, 2.13 mmol, 10.0 equiv) was added dropwise to a solution of the unpurified acid 17 [213 μmol, 1 equiv; assuming quantitative yield in the preceding step, dried by azeotropic distillation with benzene (3 × 4.0 mL)] and N,Ndi-iso-propylethylamine (743 μL, 4.26 mmol, 20.0 equiv) in tetrahydrofuran (18 mL) at 0 °C. The reaction mixture was stirred for 30 min at 0 °C. N,O-Dimethylhydroxylamine (385 μL, 5.02 mmol, 23.6 equiv) was added to the reaction mixture at 0 °C. The reaction mixture was stirred for 3 h at 0 °C. The product mixture was diluted with water (5.0 mL). The diluted product mixture was transferred to a separatory funnel that had been charged with saturated aqueous sodium bicarbonate solution (10 mL) and dichloromethane (30 mL). The layers that formed were separated. The aqueous layer was extracted with dichloromethane (4 × 30 mL). The organic layers were combined and the combined organic layers were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue obtained was dissolved in dichloromethane (6.0 mL), and silica gel (800 mg) was added to the diluted product mixture. The resulting suspension was concentrated to afford a free-flowing powder. The dried powder was loaded onto a column of silica gel. Flash-column chromatography (eluting with 10% methanol−ethyl acetate) afforded the Weinreb amide 18 as a white solid (82.9 mg, 83% from 6).
Synthesis of the amine 19:
Trifluoroacetic acid (209 μL) was added to a solution of the ester 6 (6.0 mg, 13.7 μmol, 1 equiv) in dichloromethane (1.4 mL) at 24 °C. Upon completion of the addition, the reaction mixture was stirred for 12 h at 24 °C. The product mixture was concentrated to provide the amine 19 as an off white solid. 1 H NMR analysis (400 MHz, CDCl3) indicated >95% conversion to the secondary amine 19.
The secondary amine 19 was found to be unstable towards neutralization and purification by flash-column chromatography. Therefore, further characterization was not attempted.
Nikolayevskiy et al. "A complex stereochemical relay approach to the antimalarial alkaloid ocimicide A1. Evidence for a structural revision." submitted to Chem. Sci.
Synthesis of the amine 21:
Trifluoroacetic acid (52.4 μL, 68.5 μmol, 30.0 equiv) was added to a solution of the Weinreb amide 18 (10.7 mg, 22.8 μmol, 1 equiv) in dichloromethane (200 μL) at 0 °C. Upon completion of the addition, the reaction mixture was stirred for 12 h at 0 °C. The product mixture was concentrated to provide the secondary amine 21 as an off white solid. 1 H NMR analysis (500 MHz, CD3OD) indicated >95% conversion to the secondary amine 21.
The secondary amine 21 was found to be unstable towards neutralization and purification by flash-column chromatography. Therefore, further characterization was not attempted. = 0.192). Low-temperature diffraction data (ω-scans) were collected on a Rigaku MicroMax-007HF diffractometer coupled to a Saturn994+ CCD detector with Cu Kα (λ = 1.54178 Å) for the structure of 007-14186. The diffraction images were processed and scaled using Rigaku Oxford Diffraction software (CrysAlisPro; Rigaku OD: The Woodlands, TX, 2015). The structure was solved with SHELXT and was refined against F 2 on all data by full-matrix least squares with SHELXL (Sheldrick, G. M. Acta Cryst. 2008, A64, 112-122). All nonhydrogen atoms were refined anisotropically. Hydrogen atoms were included in the model at geometrically calculated positions and refined using a riding model. The isotropic displacement parameters of all hydrogen atoms were fixed to 1.2 times the U value of the atoms to which they are linked (1.5 times for methyl groups). The only exception is H3, which is freely refining and a part of a hydrogen bond between N3 and O3. The crystal structure reported here contains solvent accessible voids in the unit cell. In spite of numerous attempts, no sensible solvent model could be established, and the solvent is assumed to be disordered within these voids. The crystals had been obtained from a ethyl acetate. The program SQUEEZE was used to compensate for the contribution of disordered solvents contained in voids within the crystal lattice from the diffraction intensities. This procedure was applied to the data file and the submitted model is based on the solvent removed data. Based on the total electron density found in the voids, it is likely that 2 molecules of ethyl acetate molecules are present in the unit cell. See "_platon_squeeze_details" in the .cif for more information. The full numbering scheme of compound 007-14186 can be found in the full details of the X-ray structure determination (CIF), which is included as Supporting Information. CCDC number 1536046 (007-14186) contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Center via www.ccdc.cam.ac.uk/data_request/cif. [3] The modified functional was invoked using WC04 internal option (IOp) statements and the B3LYP functional.
Crystallographic data for the Weinreb amide 18 (Rint
[4] The modified functional was invoked using WC04 internal option (IOp) statements and the B3LYP functional.
[5] The modified functional was invoked using WC04 internal option (IOp) statements and the B3LYP functional.
[6] The modified functional was invoked using WC04 internal option (IOp) statements and the B3LYP functional. | v3-fos-license |
2014-10-01T00:00:00.000Z | 1998-03-09T00:00:00.000 | 2932618 | {
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} | pes2o/s2orc | Elemental and Molecular Heritage: An Internet-based Display
The background to a Web page describing elemental and molecular heritage at Imperial College chemistry department is described. Photographs are shown of the original samples of elemental bromine and crystalline silicon, and molecular ferrocene and mauveine. 3D "Hyperactive" models of these systems are shown, together with a recently discovered heterocyclic systems scorpionine, which like mauveine is made by a deceptively simple chemical synthesis.
Introduction
Much like musical heritage, molecular heritage comes in two parts; a set of instructions on how molecules can be prepared, and an actual sample created by an individual or group.The analogy may also extend in another sense; most of the new molecules described in the scientific literature have probably only been prepared once, by their original discoverers.Of the 18 million or so molecules whose preparation has been described, relatively few probably exist as physical specimens at any given moment in time.Curiously however, unlike artistic heritage, the chemical community has often placed relatively little importance on the preservation of original chemical specimens and the actual samples prepared by their discoverers.Often, any specimens that have been preserved remain un-indexed and probably obscurely stored.It was of some interest therefore to discover what the preserved chemical heritage was of Imperial College, the origins of which date back to the middle of the 19th century.
My own personal interest in the departmental chemical and molecular heritage dates back some 30 years, when as a prospective entrant to the undergraduate course at Imperial College, I was invited for an interview.During the time waiting for my interview, I was left to enjoy the pleasures of the main lecture theatre, a magnificent building constructed around 1906 and demolished in 1996.At the rear, I discovered a display case containing specimens that dated back to the early 1800s.That memory has stayed with me, despite the fact that by the time I actually become a student at Imperial College, the specimens I recollected seeing were no longer on display.As it turned out, I was not to find them again for 30 years.My re-discovery of these recently in turn triggered an urge to share their background with others.Thus was born, http://www.ch.ic.ac.uk/heritage/Along with this Web page came the realisation that the Internet could form an ideal mechanism for linking institutions with interesting chemical heritages.
Organisations such as the Chemical Heritage Foundation [1] are also beginning to assume such a role on a global scale.
The Internet also offers the possibility of enhancing molecular information in a "hyperactive" manner [2].This is very much in the style of the "molecules of the month" [3].For the molecular heritage collection below, three dimensional molecule (or crystallographic unit cell) information has been hyper linked to the photographs in a form which can be displayed by e.g. the Chime browser plug-in from MDL Information Systems [4].The entries that follow represent a February 1998 snapshot of a collection that continues to grow.The author expresses the hope that others may be persuaded to similarly document their own institutional molecular heritage.
Elemental Heritage
Whilst the vast majority of the 18 million or so chemical compounds known to science in 1998 were discovered in the 20th century, most of the natural elements themselves were in fact isolated in their elemental form in the 19th century.It is not known exactly how many original samples of the 80 or so nonradioactive elements that can be placed in a sample tube are still extant.The entries below relate to those elements that exist either at Imperial College, or at the nearby Royal Institution of Great Britain [5].The author welcomes information on the existence of any other original elemental samples prepared by their discoverer.
The Imperial College archives contain a sample of elemental potassium which is still in a beautifully lustrous condition (Figure 1).The date of this sample has not been firmly established, although the label dates from the period around 1881.Potassium was originally isolated as an element by Sir Humphry Davy in 1807, and Davy's original samples of metallic sodium (1807), magnesium (1808), barium (1808) and calcium (1808) still exist on display at the Royal Institution of Great Britain.Davy also first isolated metallic lithium, but it is not known if a sample still exists.Samples of the other alkali metals (Rb, Cs) are suspected to exist in Heidelberg (Germany) where the discoverers Robert Bunsen and Gustav Kirchhoff worked.The Science Museum, London, located just to the south of the modern chemistry department at Imperial College, also has various samples made by Bunsen, inert gases isolated by William Ramsay in 1894 (probably diffused out by now!), platinum made by Wollaston, and a large collection of molecular samples.
Sir Humphry Davy and his assistant and then successor Michael Faraday worked at the Royal Institution during the first half of the 19th century.During this period many interesting chemical samples were prepared, or acquired from other scientists.Davy was the first to show that chlorine was elemental in 1810, and a sample of his is on display at the Royal Institution.Some of the specimens acquired by Faraday are now part of the Royal College of Science collection at Imperial College.These include a sample of Bromine (Figure 2), isolated and identified as a new element in 1826 by Antoine Balard.One of the odder compounds formed by bromine is the complex resulting from reacting it with the simple tertiary base DABCO in the presence of dichloromethane and atmospheric oxygen [6].The bromine takes the form of linear chains of Br 7 3-ions, the mystery being why a species with three negative charges does not simply dissociate to Br -, and 2 Br 3 -ions.Complex products formed by reactions of simple bases are characteristic of Mauveine and Sscorpionine as well (see below).
The author would like to hear whether any original sample of Iodine made by Bernard Courtois in 1811 still exists.Given the reactivity of fluorine, it seems improbable that any sample deriving from Henri Moisson (1886) has survived.
The Faraday collection also includes an original sample of crystalline silicon prepared for the first time by H.St. C. Deville in 1854 by the action of SiCl 4 over molten aluminium (Figure 3).Crystalline silicon of course underpins the world's computer industry, and is arguably the most industrially important element, more so than even gold and diamond, and much more useful!
Molecular Heritage
Another famous student of the Royal College of Chemistry was William Perkin, who in 1856 discovered a brilliantly coloured substance which became known as mauveine, and which belongs to a class of substances known as heterocyclic molecules.Mauveine was the subject of the first "molecule-of-the-month" article in December 1995 [7].A piece of silk dyed with an original batch of mauveine prepared by Perkin himself is attached to the bottom of a letter written in 1922 by William Perkin's son to Henry Armstrong, a Professor at the by then named Royal College of Science (Figure 4).This silk is part of a batch that originally was made into a dress for Queen Victoria.Perkin went on to found a factory in Greenford, West London, to manufacture mauveine.Arguably, this site represents one of the first sources of the modern organic fine-chemical manufacturing and pharmaceutical industry, and which therefore can be argued to rank in importance with the original industrial revolution, which had occurred at Ironbridge in the midlands of England in the early 1700s.Perkin was also associated with the early Chemical Society of Great Britain, now known as The Royal Society of Chemistry.A past president of the Royal Society of Chemistry is Charles Rees, who is also the current Hofmann Professor of Chemistry at Imperial College Chemistry Department (the direct successor to the founder August von Hofmann).A characteristic bow tie dyed with an original sample of mauveine was presented to Professor Rees by Professor Otto Meth-Cohn, who had recently corrected the molecular structure of mauveine (appropriately enough reporting the result in the journal named after Perkin) [8].The photograph (Figure 5) shows Professor Rees holding the February 1998 issue of this journal.The molecule linked to the photograph is Scorpionine, which like mauveine is another complex and deeply coloured heterocyclic system made in a single step from a simple base, this time recently discovered by Professor Rees himself [9].
Conclusions
A surprising number of elemental and molecular chemical samples from the 19th century still exist, although their existence is not always widely known.This article (at least in its active Web-based form rather than its printed version) attempts to show how this heritage can be documented in a novel and global manner.Perhaps by this mechanism, other hitherto unknown samples might be brought to light, in a celebration of more than two hundred years of chemical heritage.
Figure 5 .
Figure 5. Professor C. W. Rees wearing the "mauveine" bow tie.© H. S. Rzepa, 1998.All rights reserved.Hofmann's successor at the Royal College of Chemistry was Sir Edward Frankland, who was a pioneer of the then new field of organometallic chemistry.Continuing the tradition at the Royal College of Science was Nobel Laureate Geoffrey Wilkinson, who discovered | v3-fos-license |
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} | pes2o/s2orc | Novel Magnetic Domain Structure in Iron Meteorite Induced by the Presence of L10-FeNi
Photoelectron emission microscopy has been carried out to study the magnetic properties of iron meteorite associated with the Widmanstätten structure for the first time. A magnetic circular dichroic image reveals a unique magnetic domain structure, resulting in the “head-on” magnetic coupling over the interface between the α and γ lamellae. Such a magnetic domain structure is unfavorable in any synthetic Fe–Ni alloys. Micromagnetics simulation reasonably explains that the formation of magnetic domains is induced by the L10-type FeNi (tetrataenite) phase segregated at the boundary in the Widmanstätten structure.
I ron meteorite shows an extraterrestrial pattern termed as the Widmanstätten structure [ Fig. 1(a)]. Its metallographic feature has been of great benefit to planetary scientists for studying the history of the solar system. [1][2][3][4] The scientists have believed that the Widmanstätten structure was formed by the long-range thermal diffusion of Fe and Ni in asteroid's core over a period of 4.6 billion years. 2) Meanwhile, the iron meteorite is also characterized by remarkable magnetic properties, namely large magnetic anisotropy and strong coercivity, differing from those of synthetic Fe-Ni alloys. 5) However, there is no explanation how the magnetic properties are associated with the Widmanstätten structure. From the viewpoint of materials science, the Widmanstätten structure is regarded as Fe-Ni alloy segregated (bcc-FeNi, Kamacite) and (fcc-FeNi, Taenite) lamellae on the micrometer scale [ Fig. 1(b)]. 1) The Ni concentration in the lamella rapidly increases toward the interface, 2) resulting in several laminated Fe-Ni alloys, namely invar alloy (Fe 65 Ni 35 ), 7) tetrataenite (Fe 50 Ni 50 ) [8][9][10][11] and permalloy (Fe 25 Ni 75 ). 12,13) The f110g bcc axis maintains its parallel orientation to f111g fcc , and either h001i bcc or h1 11i bcc orients parallel to h1 10i fcc known as the Nishiyama-Wassermann (NW) or Kurdjumov-Sachs (KS) orientation, respectively. 6) Such a heterogeneous structure near boundary can be considered as same sort of magnetic multilayer system. In this study, we investigate the magnetic properties of iron meteorite resulting from the Widmanstätten structure for the first time.
Among the Fe-Ni alloys, we pay particular attention to the tetrataenite phase, which is described as a chemically ordered FeNi alloy with L1 0 -type superstructure. 9,10) It is also well known that tetrataenite exhibits significantly different magnetic properties from synthetic Fe-Ni alloys. Néel et al. reported the magnetic anisotropy energy of tetrataenite as 3:2 Â 10 5 J/m 3 , 8) which is more than one order of magnitude larger than those of Fe, invar alloy, ordered permalloy and pure Ni (4:8 Â 10 4 , 5 Â 10 2 , À2:5 Â 10 3 , and À6 Â 10 3 J/m 3 , respectively 7,8,12,13) ). Our pilot magnetic hysteresis measurement for tetrataenite flake shows coercive force of more than 1:0 Â 10 5 A/m, which is entirely larger than common Fe-Ni alloys. 8,12) In other words, tetrataenite is characterized as a hard ferromagnet with a strong anisotropy despite the fact that common Fe-Ni alloys are classified as softmagnetic materials.
To elucidate the relationship between the Widmanstätten structure and the magnetic property, we used Gibeon iron meteorite, which is one of the typical iron meteorites showing a clear Widmanstätten structure. 1) Figure 1(c) shows the image of the Widmanstätten structure; a thin lamella of 4 m width is separated from a thick lamella of 40 m width by lamellae. Recent progress in photoelectron emission microscopy (PEEM) using synchrotron radiation enables us to obtain the spatial information on composition, electronic state, 14) crystallographic structure, 15,16) and magnetic domain structure [17][18][19] in the combination with X-ray absorption fine structure (XAFS) or magnetic circular dichroism (MCD). The spatial resolution of apparatus reaches well below 100 nm.
Specimen was sliced nearly parallel to the ð001Þ bcc plane of the lamella. The surface was carefully prepared using automatic mechanical polisher with a 6 m diamond slurry for rough treatment and finished by buff polishing with a 1 m diamond slurry. AC demagnetization field is applied to the specimen to cancel residual magnetization or improper magnetic treatment since 19th century. We examine the surface irregularity using AFM that shows the scratches with a typical width of 100 nm and depth of 10 nm. We adopted even deeper scratch in the numerical calculation, but the scratch does not influence observed magnetic domains. The influence of shape anisotropy energy is negligibly smaller than exchange energy to produce the scratch induced magnetic domain structure. PEEM measurement was performed in the region with the lowest density of scratches.
The spatial distribution of Ni is shown in Figs. 2(a) and 2(b) for the boundary regions indicated by circles in The typical exposure time per image was 10 s. Ni composition was estimated by edge-jump of the XAFS spectrum. The Ni composition in lamella in Fig. 2(a) shows spatially homogeneous profile of 6.6 at. %. On the other hand, that in lamella is highly condensed of 28 at. %. Figure 2(c) is typical XAFS spectra obtained for and lamella. As denoted by arrows, the spectral change from a single to a double peak on the crest is ascribed to the structural alternation from bcc to fcc over the transition threshold at 25 at. % Ni. 21) Figure 2(d) shows the Ni line profile at the boundary region in Fig. 2(b), indicating that local Ni composition is rapidly increasing from 20 to 35 at. % toward the interface. L1 0 structure was not directly recognized here because of the limit of resolution, however such structural alternation associated with chemical composition suggests that tetrataenite is segregated at the boundary from the metallurgical viewpoint. 2) To confirm the presence of tetrataenite, chemical etching by 5% HCl for several minutes reveals the tetrataenite at this region. Scanning electron microscopy and electron probe microanalyzer (SEM-EPMA) estimates the chemical composition as to be Fe 50 Ni 50 . Next, the surface magnetic domain structure was probed by MCD-PEEM for the same area. The circularly polarized light from BL25SU 22) was used to illuminate the specimen along ½110 bcc of lamella as shown by a thick arrow in Figs. 2(e) and 2(f). The exposure time of PEEM was set to be about 20 min to accumulate the image at the Fe L 3 edge (708.4 eV). The red-to-blue color scale indicates the MCD intensity. As shown in Fig. 2(e), a transversal domain lying parallel to the interface is observed clearly at 6 and 12 m from the interface. In common bcc-Fe, such as whiskers, the magnetic domain shows a wide rectangular structure with a sharp domain wall; thus the transversal stripe in meteoritic iron shows a behavior different from that of the bcc Fe-Ni alloy. 12,13) By considering its direction, the striped domain may be associated with the boundary.
Over the = interface, as shown in Figs. 2(e) and 2(f), a fine structure of about 2 m width is also observed; this structure is characterized by an elongated shape oriented parallel to the ½110 bcc direction, and the direction of magnetization orients parallel or antiparallel to the ½110 bcc direction, as indicated by arrows in Fig. 2(f). The magnetizations on both sides of the interface align opposite to each other and orthogonal to the domain wall, and then this magnetic domain eventually forms a ''head-on'' structure, which requires a large amount of magnetostatic energy for demagnetizing field. 12,23) For a typical 180 domain structure, the magnetization orients parallel to the domain wall so as to reduce the static magnetic energy. For the polycrystalline Fe-Ni alloy, the magnetization over the grain boundary aligns in the same continuous direction; 23,24) thus the head-on configuration in the iron meteorite is completely different from the case of Fe-Ni alloy. In epitaxially grown Fe ultrathin films on Ni(111) system, Fe moment align perpendicularly or parallel to Ni moments. 25,26) However, this is also not the case of iron meteorite. The head-on configuration is not simply explained by interface mismatch or atomic relaxation. It is concluded, therefore, that the striped magnetic domain and head-on magnetic coupling are unique properties of the magnetic domain in iron meteorite.
To verify such a magnetic domain, we performed micromagnetics simulation solving the Landau-Lifshits-Gilbert (LLG) equation. 27,28) Numerical study is achieved fully three dimensional with a functional form of boundary condition. We used two simple theoretical models, namely Fe/Ni [ Fig. 3(a)] and Fe/tetrataenite/Ni [ Fig. 3(b)] interface. We assumed a spatially uniform composition for the Ni lamella here. Downward (Àz) of the specimen uses a continuous boundary, and upward (þz) free. Longitudinal direction (x) uses a continuous boundary, and transversal (y) periodic. 1:6 Â 6:4 Â 1:6 mm 3 with 100 nm grid is adopted for simulating boundary region. Magnetic moment is referred to 2.2 and 0.6 B /atom for Fe and Ni, respectively, and 1.33 B /atom evaluated by superconducting quantum interference device (SQUID) is used for tetrataenite. Exchange stiffness is adopted as 1:3 Â 10 À11 , 1:0 Â 10 À11 , and 0:8 Â 10 À11 J/m for Fe, tetrataenite and Ni respectively. Averaged value is used for the interface exchange stiffness here. Magnetic anisotropy energy is referred to the values as described above. 7,8,12,13) Calculation runs from random magnetization to a cooled equilibrium state under zero magnetic field. Numerical simulation was performed for both NW and KS configurations and for 1.4-, 1.2-, 1-, 0.8-, 0.6-, 0.4-, and 0.2-m-thick tetrataenite films to examine the dependences of orientation and thickness. Here, we present the NW configuration of the 1-m-thick tetrataenite film as a representative result. Top layer is responsible to experimental results. To confirm the entire region, we also performed the calculation for large area of 16 Â 72 Â 8 m 3 with 1 m gird, and the result was consistent with that for 100 nm grid and experimental result.
As indicated in Fig. 3(a), the Fe/Ni interface shows a simple magnetic domain, and no head-on magnetic domain is formed. Most magnetic moments in both Fe and Ni lamellae align to the bulk-like easy axis as h100i bcc and h111i fcc . The Fe moment gradually cants while approaching the interface, because of the requirement for continuity of the magnetization in x-direction. On the other hand, magnetic domain is disarranged in the Fe/tetrataenite/Ni system [ Fig. 3(b)], and head-on structure definitely reveals at nearby interface. Such head-on domains are commonly formed at any tetrataenite film thickness and in both the KS and NW configurations.
According to technical magnetization, magnetic domain structure is determined so as to minimize the total energy. The magnetic anisotropy of tetrataenite is extremely larger than that of surrounding soft magnetic Fe and Ni. Thus, magnetization in tetrataenite remains in the direction of an easy axis. As shown in the inset, the magnetic pole with z component is produced at bare surface of tetrataenite, resulting in the increase of magnetostatic energy. In order to cancel the surface pole at tetrataenite (N-pole in Fig. 3), the S-pole is created at the surface of Ni. These upward and downward configurations of magnetization increase the exchange energy at the interface between Ni/tetrataenite. However, the exchange energy between Fe/tetrataenite is larger than that of Ni/tetrataenite. Accordingly, the configuration in Fig. 3 produces the lower energy. On the other hand, in x-direction, the S-pole is created at the interface between Ni/tetrataenite, because the magnetization of tetrataenite is larger than that of Ni. In order to cancel the influence of that pole, the generation of N-pole is required at the interface between tetrataenite/Fe. Therefore, the magnetization in Fe shows the opposite direction against the magnetization in tetrataenite. This configuration causes the head-on domain, and increases the exchange energy. However, the cost of the exchange energy in head-on domain wall is equivalent to that in 180 domain wall, assuming the same wall width. Thus, head-on domain is agreeable to reduce the magnetostatic energy in the system. Consequently, we can conclude that the observed magnetic domains in iron meteorite are induced by the large magnetic anisotropy of the tetrataenite phase at the boundary. Tetrataenite will play a key role in the magnetic anisotropy of iron meteorite.
Synthesis of tetrataenite phase is currently attracting new attention because of inexpensive and abundant resource of Fe and Ni, 29) thus tetrataenite phase will offer potential applications in magneto-electronic devices. The tetrataenite thin film located at the boundary exhibits a high magnetic anisotropy, and behaves similarly to a permanent magnet against adjacent soft-magnetic FeNi alloys, resulting in the formation of the head-on and striped magnetic domains. | v3-fos-license |
2019-06-07T21:13:18.795Z | 2019-06-07T00:00:00.000 | 182409592 | {
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} | pes2o/s2orc | Target Identification of Active Constituents of Shen Qi Wan to Treat Kidney Yang Deficiency Using Computational Target Fishing and Network Pharmacology
Background: Kidney yang deficiency syndrome (KYDS) is one of the most common syndromes treated with traditional Chinese medicine (TCM) among elderly patients. Shen Qi Wan (SQW) has been effectively used in treating various diseases associated with KYDS for hundreds of years. However, due to the complex composition of SQW, the mechanism of action remains unknown. Purpose: To identify the mechanism of the SQW in the treatment of KYDS and determine the molecular targets of SQW. Methods: The potential targets of active ingredients in SQW were predicted using PharmMapper. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out using the Molecule Annotation System (MAS3.0). The protein–protein interaction (PPI) network of these potential targets and “components-targets-pathways” interaction networks were constructed using Cytoscape. We also established a KYDS rat model induced by adenine to investigate the therapeutic effects of SQW. Body weight, rectal temperature, holding power, water intake, urinary output, blood urea nitrogen (BUN), serum creatinine (Scr), adrenocorticotrophic hormone (ACTH), cortisol (CORT), urine total protein (U-TP), and 17-hydroxy-corticosteroid (17-OHCS) were measured. Additionally, the mRNA expression levels of candidates were detected by qPCR. Results: KYDS-caused changes in body weight, rectal temperature, holding power, water intake, urinary output, BUN, Scr, ACTH, CORT, U-TP, and 17-OHCS were corrected to the baseline values after SQW treatment. We selected the top 10 targets of each component and obtained 79 potential targets, which were mainly enriched in the proteolysis, protein binding, transferase activity, T cell receptor signaling pathway, and focal adhesion. SRC, MAPK14, HRAS, HSP90AA1, F2, LCK, CDK2, and MMP9 were identified as targets of SQW in the treatment of KYDS. The administration of SQW significantly suppressed the expression of SRC, HSP90AA1, LCK, and CDK2 and markedly increased the expression of MAPK14, MMP9, and F2. However, HRAS levels remained unchanged. Conclusion: These findings demonstrated that SQW corrected hypothalamic–pituitary–target gland axis disorder in rats caused by KYDS. SRC, MAPK14, HRAS, HSP90AA1, F2, LCK, CDK2, and MMP9 were determined to the therapeutic target for the further investigation of SQW to ameliorate KYDS.
INTRODUCTION
Kidney yang deficiency syndrome (KYDS) is a diagnostic pattern in traditional Chinese medicine (TCM) and was first documented in Huang Di Nei Jing, one of the four great classical textbooks of TCM . KYDS is characterized by warm dysfunction and a metabolic disorder of the body fluid, causing aversion to cold, cold limbs, cold of waist and back, soreness and weakness of waist and knee, tinnitus, fatigue, impairment of hearing, and looseness of teeth (Lu et al., 2011;Tan et al., 2014;Rong et al., 2016;Xiong et al., 2019). Modern studies have indicated that functional disorders with different degrees of hypothalamic-pituitary-target gland axis, including adrenal glands, thyroids, and gonads, are the crucial pathological mechanism leading to KYDS (Lu et al., 2011;Tan et al., 2014;Nan et al., 2016;Zhang et al., 2017;Tang et al., 2018). KYDS can be present in chronic diseases such as rheumatoid arthritis, hypertension, and diabetes, posing a considerable challenge to the medical system. A valid and classic rat model of KYDS has been developed via the administration of a high dose of adenine by oral gavage, which precipitates in renal tubules, leading chronic renal failure, and the animals exhibit the clinical characteristics of KYDS.
Shen Qi Wan (SQW) is a frequently used Chinese formula described by Zhang Zhongjing in Synopsis of Prescriptions of the Golden Chamber (also named Jin Kui Yao Lue in Mandarin). It can be traced back to nearly 2,000 years ago in ancient China (Xiong et al., 2015). The SQW formula is based on the combinatorial principle of "emperor-ministeradjuvant-courier" (jun-chen-zuo-shi in Chinese) to combine multiple herbs. The jun herb of SQW contains Cinnamomum cassia (L). J.Presl and Aconitum carmichaelii Debeaux to treat the main cause or primary symptoms of KYDS. The chen herb of SQW is Rehmannia glutinosa (Gaertn). DC., Cornus officinalis Siebold & Zucc., and Dioscorea oppositifolia L. assist the jun herb to enhance its therapeutic effects and relieve the accompanying symptoms. The zuo shi herb includes Poria cocos (Schw). Wolf, Alisma plantago-aquatica L., and Paeonia × suffruticosa Andrews to counteract the possible toxicity or side effects of other herbs and to ensure the absorption of the formula components and help deliver or guide them to the target organs (Qiu, 2007;Yao et al., 2013). For centuries, SQW has been effectively used in treating various diseases associated with KYDS. However, the therapeutic mechanism remains unknown, the complexity of multiple components, multiple targets, and multiple pathways involved in KYDS make it difficult to elucidate using classical pharmacological approaches.
Network pharmacology is a distinctive new approach based on advances in polypharmacology and network biology to shift away from the traditional "one drug, one target" strategy and move toward sub-network targets and systems, providing a more comprehensive understanding of the mechanism, targets, and pathways behind drug action (Tang and Aittokallio, 2014;Poornima et al., 2016). With the combination of the "medicines-targets" network and biological system network, network pharmacology is becoming more widely known and more frequently used in the field of drug research. PharmMapper (http://www.lilab-ecust. cn/pharmmapper/) is a freely accessed web server designed to identify potential target candidates for probe small molecules of interest using pharmacophore mapping approach (Liu et al., 2010). PharmMapper provides deeper insights and scientific evidence for TCM and helps identify potential targets of Chinese herbs and their underlying mechanisms.
In the present study, investigations based on the pharmacology database and previous studies were conducted to investigate the warm yang and the involvement of several compounds of interest. Potential targets of SQW were predicted by reverse docking to analyze the biological information of potential targets and associated pathways using the network pharmacology method. Moreover, we aimed to identify the potential therapeutic target genes and explore the effects of SQW on the mRNA expression levels of the candidate targets to preliminarily discuss the involvement of the candidate targets in KYDS.
Compound Preparation
To collect the compounds of SQW, we combined the Traditional Chinese Medicine Systems Pharmacology Database (TcmSP ™ , http://lsp.nwu.edu.cn), a unique system pharmacology platform designed for Chinese herbal medicines and the review of previous studies . In addition, we used China National Knowledge Infrastructure (CNKI) and PubMed to obtain information on the modern pharmacology of the compounds in SQW. CAS No. comes from the Chemical Abstract Service (http://www.cas.org/). We finally selected several compounds of each herb in SQW, every compound we chose has various pharmacological effects such as vascular and tracheal relaxation effect; anti-thrombotic, anti-apoptotic, anti-oxidative effects; and anti-inflammatory and immunomodulatory effects.
Preparation of Mol2 Format Files
Using the software ChemBioDraw Ultra 14.0 (Version 14, PerkinElmer Inc), we transformed the structures of active components into the sdf structure format. Then, we transformed the sdf structure format into mol2 format files using ChemBio3D Ultra 14.0 (Version 14, PerkinElmer Inc) to obtain the corresponding three-dimensional molecular ball-and-stick model.
Prediction and Screening of Targets
To predict the potential target candidates, we imported the mol2 format files into the freely accessed web server of the target database of pharmacophore PharmaMapper website (http:// www.lilab-ecust.cn/pharmmapper/) to perform reverse docking. Subsequently, we employed UniProtKB (http://www.uniprot. org/), which is the central hub for the collection of functional information on proteins, to correct the unstandardized drug target naming by converting the protein names with the species limited to "Homo sapiens" to its official symbol. We selected the top 10 targets of each active component for the subsequent study.
Construction of Protein-Protein Interaction Network for Potential Targets
By using the STRING (Version 10.0) database (http://version10. string-db.org//), we identified the direct physical interactions of proteins and their functional interactions (Wu et al., 2016). We uploaded the gene symbols of potential targets and drew a protein-protein interaction (PPI) network graph online to evaluate the interactions among the potential targets. Then, we imported the PPI data in text format into Cytoscape (http://www. cytoscape.org/) to visualize relationships and used its network analyzer plugin to calculate the degree of PPI network.
Investigation of Biological Information for Potential Targets of SQW
We imported the potential targets into the Bio database (http:// bioinfo.capitalbio.com/mas3/ Version 3.37) to perform the analysis for GO and KEGG pathway enrichment and then screened for pathways with a cut-off p < 0.05 (Wu et al., 2016).
Construction of the Component-Target-Pathway Network
Based on the screening of pathways with their corresponding targets and components, we created a component-target-pathway illustration using Cytoscape, which not only applies to visualizing biological pathways and intermolecular interaction networks but also supplies a basic set of features for data integration, analysis, and visualization for complicated network analysis . In the network, the node stands for the constituents of SQW, chemical components, component targets, and component pathways. These constituents are connected by an edge when a target is a potential target of a compound. With this network, we studied the effects of multiple components, multiple targets, and multiple pathways of SQW, which ameliorates KYDS.
Animal Study and Sample Collection
The animal study was approved by the Ethics of Committee of Zhejiang Chinese Medical University. Thirty male Wistar rats (250 ± 30 g, animal license no. SCXK-2013-0033) were obtained from the Animal Center of Zhejiang Chinese Medicine University [Laboratory rearing room Permit No. SYXK (Zhejiang) 2013-0184]. All of the animals were housed at 22 ± 2°C with 50-60% relative humidity. A 12 h light/12 h dark cycle was set, and the animals had free access to standard diet and water. All animals were randomly divided into the control group (n = 10), KYDS model group (n = 10), and SQW group (n = 10). In the first 21 consecutive days, the control group was administered normal saline, the model group and the SQW group were administered adenine (Lot: 131203. Shanghai Bo'ao Biological Technology Co., Ltd) at 200 mg/kg per day. From the 22 nd day, the control group was administered normal saline, the model group was administered adenine at 200 mg/kg and normal saline via gavage after 1 h per day in the next 21 consecutive days, and the SQW group was administered adenine at 200 mg/kg per day and SQW (Lot: 130904. Henan WanXi Pharmaceuticals Co., Ltd) at 3g/ kg via gavage after 1 h per day for the next 21 consecutive days. The body weight, rectal temperature, and holding power were detected every 4 days. Water intake and urinary output were observed every 7 days. The urine was obtained from metabolism cages and was used to detect the urine total protein (U-TP) by an automatic biochemical analyzer; 17-hydroxy-corticosteroids (17-OHCSs) were measured using ELISA kits (CAS: 14020809, Biovol Technologies Co. Ltd. Shanghai) after adenine administration at day 21. Blood samples were collected from the heart after pentobarbital sodium (45 mg/ kg, i.p.) anesthesia, and then the kidney tissues were rapidly excised, quickly frozen in liquid nitrogen, and stored at −80°C to perform the quantitative real-time PCR assays. Serum was separated by centrifugation at 3,000 rpm for 15 min at 4°C after standing for 30 min to detect blood urea nitrogen (BUN) and serum creatinine (Scr) with an automatic biochemical analyzer (Hitachi, Japan); adrenocorticotrophic hormone (ACTH) and cortisol (CORT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (CAS: 140208, 14020807. Biovol Technologies Co. Ltd., Shanghai). Throughout the experimental period, no animals died before the experimental endpoint. Euthanasia was performed under sodium pentobarbital anesthesia followed by cardiac puncture/kidney removal for all animals.
Quantitative Real-Time PCR Analysis
Total RNA separation and extraction methods were performed according to the instructions of the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Clontech). Spectrophotometric measurements at 260/280 nm (Thermo Scientific, USA) were used to determine the purities and concentrations of the total RNA samples. Reverse transcription reactions were performed using 300 ng of RNA with PrimerScript ™ RT Master Mix (Perfect Real Time) for cDNA. Table 1 lists the primer sequences. SRC, MAPK14, HRAS, HSP90AA1, F2, LCK, CDK2, and MMP9 gene expression was investigated. The samples were exposed to pre-denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s, and annealing at 60°C for 30 s. The dissolution curve conditions were 65°C for 0.05 s and 95°C for 0.5 s using 5 µL 5× SYBR Green qPCR Mix, 0.4 µL 20 µmol/L forward primer, 0.4 µL 20 µmol/L reverse primer, and 1 µL cDNA. Water was added to achieve a total volume of 10 µL. β-Actin was used as the internal control, and the data were analyzed using the 2 -ΔΔCt method. The experiment was repeated three times.
Preparation of Reference and Sample Solutions
A mixed standard solution was obtained by dissolving the five standards (o-anisaldehyde, higenamine, coryneine chloride, salsolinol, and cinnamic acid) in methanol. The final concentration is 2 mg/ml. SQW was ultrasonically extracted by 10-fold volume pure water twice for 30 min each time. The solution was concentrated to 40 mg/ml then filtered by a 0.22-μm Millipore filter. The injection volume was 10 μl in the same.
Statistical Analysis
The SPSS 22.0 statistical software package (SPSS, Chicago, IL, USA) was used for the analysis of variance followed by one-way ANOVA. Data were presented as the mean values ± standard deviation. Statistical significance was considered if p < 0.05 was observed.
Compound Information
A search of the TcmSP ™ identified 1,345 items, including 130 in A. carmichaelii Debeaux, 200 in C. cassia (L.) J.Presl, 151 in R. glutinosa (Gaertn.) DC., 452 in C. officinalis Siebold & Zucc., 142 in D. oppositifolia L., 68 in P. cocos (Schw). Wolf, 92 in A. plantagoaquatica L., and 110 in Paeonia × suffruticosa Andrews. Wang et al. (2016), in "an integrated chinmedomics strategy for discovery of effective constituents from traditional herbal medicine, " reported 84 compounds, among which 51 compounds in negative ion mode and 33 compounds in positive ion mode were identified from SQW. Moreover, 20 compounds absorbed into the blood, such as azelaic acid-O-glucuronide, jionoside D, azelaic acid, and poricoic acid, had a strong relationship with the therapeutic effect of SQW on KYDS. Database search and current studies were used to select the chemical components of SQW according to its pharmacological activities, such as a cardiac-stimulating effect, heightened adrenal cortex function, promoting diuresis and detumescence, invigorating spleen and dampness removal, as shown in Table 2. Higenamine, coryneine chloride, salsolinol, o-anisaldehyde, cinnamic acid, catalpol, acteoside, loganin, diosgenin, morroniside, pachymic acid, acetophenone, paeoniflorin, alisol A, and alisol B were probably associated with warming yang attributes of SQW, which were used for further study.
Construction of the Interaction Network and Network Analysis
Ranked by fit score in descending order, three hundred potential targets were predicted by PharmMapper. We selected the top 10 targets of each component, if one gene symbol of a component with a different subunit remained. Subsequently, 79 potential targets were selected for further investigation. In the present study, the components of SQW could dock the same or different targets, implying that SQW had a therapeutic effect on the treatment of KYDS through a "multiple components-multiple targets" mechanism. We evaluated 79 potential targets by using the STRING version 10.0 database to identify the interactions between identified 68 proteins. Then, we constructed a PPI network (Figure 1) by using Cytoscape. We deleted the isolated pairs of linked nodes, which were not meaningful. The resulting network was composed of 68 nodes and 229 edges, with 27 as the maximum degree of connectivity of a node and 1 as the minimum. We evaluated a node with a degree, which denotes the number of edges between a node and other nodes in a network. A high-degree node was the most influential node in the network, and a hub node was a component of a network with a high-degree node. The average degree of connectivity of the nodes in the network was 6.74, and the standard deviation was 5.84. In this study, we selected the hub nodes with a degree of connectivity set as ≥ the mean value + standard deviation.
GO Enrichment and Pathway Analysis for Potential Targets of SQW
We imported the selected potential 79 target genes into the Molecule Annotation System for GO enrichment and pathway analysis. GO analysis results revealed that the functions of these potential targets are related to many biological processes that may be important for the occurrence and development of KYDS, such as proteolysis, FIGURE 1 | Candidate target genes identified in the protein-protein interaction network constructed using Cytoscape software.
oxidation reduction, signal transduction, and metabolism. Binding (protein, nucleotide, zinc ion, metal ion) and activity (transferase, peptidase) are closely related in molecular function and the cellular components, including cytoplasm, nucleus, and cytosol, these proteins were ranked highly as potential targets (Figure 2). A total of 105 pathways were obtained by GO analysis, from which we selected the top 76 pathways that met the criterion of p < 0.05. Numerous pathways for potential target genes were identified. Our study found that the ErbB signaling pathway, VEGF signaling pathway, and MAPK signaling pathway are associated with signal transduction, the insulin signaling pathway, metabolism of xenobiotics by cytochrome P450, drug metabolism-cytochrome P450, and the PPAR signaling pathway. Androgen and estrogen metabolism are associated with the endocrine system. The focal adhesion and the T cell receptor signaling pathway are also closely related to immunological stress or inflammation. Moreover, we found some disease-related pathways such as prostate cancer, non-small cell lung cancer, endometrial cancer, and thyroid cancer, which indicate that SQW has a potential application in other diseases (Figure 3). The results prompted that SQW ameliorated the imbalance of body by regulating the neurological, endocrine, and immune processes.
Pharmacology Network of SQW
We constructed a pharmacology network of SQW (Figure 4) using the Cytoscape software, which showed the relationships among the constituents, chemical components, and potential targets of SQW and the selected 76 pathways (p < 0.05). We obtained a preliminary understanding of the mechanism of SQW through this network. The potential targets of the effective components are distributed in different metabolic pathways to jointly affect the occurrence and development of KYDS.
Adenine-Induced KYDS
To validate the establishment of the animal model, the body weight, rectal temperature, and the holding power were measured on days 0, 4, 8, 12, 16, and 20. The results (Figure 5A, B, and C) demonstrated that the body weight, temperature, and the holding power values of the KYDS model rats gradually decreased as the time increased compared to those of the rats in the control groups (p < 0.01), whereas the water intake and urinary output of KYDS model rats were higher than those of the rats in the control groups (p < 0.01) on the 7th, 14th, and 20th days (Figure 5D and E). As shown in Table 3, the contents of BUN, Scr, ACTH, and CORT were determined in rat serum on the 21 st day. The BUN and Scr of the KYDS model were significantly increased (p < 0.01), whereas the ACTH and CORT of the KYDS model rats were significantly lower than those of the rats in the control group (p < 0.01). Moreover, the concentration of 17-OHCS in the KYDS rats was decreased compared with that in the control group rats (p < 0.01), but the U-TP in the urine of KYDS model rats was significantly increased (p < 0.01). These results indicated that the rats presented symptoms such as sluggishness, languorousness, and a crouched posture, which are the typical pathological features of KYDS. The biochemical results indicated that the KYDS model was successfully established for subsequent experiments.
Treatment of KYDS With SQW
Treatment group rats recuperated after intra-gastric administration of SQW. The body weight improved significantly in the SQWtreated rats compared to that in model group rats (p < 0.01) on the 12 th day of intra-gastric administration of SQW ( Figure 6A). The rectal temperature and holding power of the SQW-treated rats were ameliorated compared with that in the model groups (p < 0.01) at the beginning of the 8 th day of intra-gastric administration of SQW (Figure 6B and C). The levels of water intake and urinary output in the SQW-treated rats gradually returned to the baseline levels (p < 0.01) of the control group (Figure 6D and E). The body weight, rectal temperature, holding power, water intake, and urinary output of the model groups showed significant differences compared with those in the control groups (p < 0.01) throughout the treatment period. As shown in Table 4, the SQW treatment obviously improved the numeral values of ACTH, CORT, and 17-OHCS (p < 0.01), while BUN, Scr, and U-TP showed a significant decrease compared with the model groups (p < 0.01), demonstrating that SQW could effectively ameliorate KYDS and had a therapeutic effect on the rat KYDS models.
Results of qPCR for Candidate Target Genes
The hub genes were identified in the PPI network with high degree of connectivity. Among them, SRC, MAPK14, HRAS, HSP90AA1, F2, LCK, CDK2, and MMP9 were closely related to the emperor's constituents. Then, we explored the effect of SQW on mRNA expression in the kidney using qPCR, as shown in Figure 7. The mRNA expression levels of SRC, HSP90AA1, LCK, and CDK2 in the SQW-treated group were significantly decreased compared to those in the model group (Figure 7A-D), whereas MAPK14, MMP9, and F2 expression levels were significantly higher in the SQW-treated group than those in the model group (Figure 7E-G). Although the mRNA expression levels of HRAS showed no significant difference compared with the model group, there was a weak trend ( Figure 7H). These results indicate that SQW treatment could effectively ameliorate KYDS via the synergy of multiple targets.
DISCUSSION
KYDS is most prevalent in older men and women and increases with age (Chen et al., 2010;Rong et al., 2016). TCM considers KYDS to be a complex kidney disorder, and "kidney yang" activates the power of human vitality (Lu et al., 2011;Huang et al., 2013;Zhao et al., 2013). The primary cause of KYDS is a decline Frontiers in Pharmacology | www.frontiersin.org in kidney-yang and transformative action, which is similar to a debilitating disease, such as chronic prostatitis, nephrotic syndrome, adrenocortical insufficiency, chronic nephritis, and diabetes mellitus in Western medicine. SQW is a typical TCM formula widely used for the treatment of chronic diseases associated with KYDS in China. Nevertheless, due to the complex pathological properties and multiple targets in KYDS, it is not easy to explore the mechanism of action of SQW using traditional methods. In this study, reverse pharmacophore docking and network pharmacology strategies were used to study the characteristics of "multiple components-multiple targets-multiple pathways" associated with SQW in the treatment of KYDS.
PharmMapper was designed to identify potential target candidates for given small molecules (drugs, natural products, or other newly discovered compounds with unidentified binding targets) by the mutual recognition of space and the ability to find the best mapping configurations (Liu et al., 2010). Ma et al. (2016) showed the "multiple components-multiple targetsmultiple pathways" mechanism of Naoxintong capsule with the PharmMapper database and network pharmacology. Tao et al. (2016) used PharmMapper and the KEGG bioinformatics websites to predict the target proteins and related pathways of Chuanbei Pipa dropping pills to clarify the anti-inflammatory and cough-suppressing mechanisms. Using a network pharmacology method provides a basis for understanding the mechanism of action of SQW and is indispensable in the study of complex drugs.
We successfully predicted the drug targets of 15 compounds in SQW. The results of PPI network suggested 13 hub genes, which play important roles in the PPI. Among them, SRC, MAPK14, HRAS, HSP90AA1, F2, LCK, CDK2, and MMP9 were closely associated with higenamine, coryneine chloride, salsolinol, o-anisaldehyde, and cinnamic acid. These compounds were found in R. Cinnamomi and Radix aconiti lateralis preparata, the jun herb of SQW treating the main cause or primary symptoms of KYDS.
Proto-oncogene tyrosine-protein kinase Src (SRC) is a nonreceptor tyrosine kinase. Once SRC is activated, the intracellular signal transduction cascades are triggered and subsequently multiple cellular functions such as cell proliferation, differentiation, and metabolism are modulated. Further investigations revealed that SRC activation is critically involved in the development of chronic kidney disease. Yan et al. (2016) observed that SRC kinase is activated in cultured kidney fibroblasts, and the inhibition of SRC by PP1, a selective small-molecule inhibitor of SRC kinase, appeared to disrupt TGFβ1/Smad3 and epidermal growth factor receptor (EGFR) signaling. Another study demonstrated that the inhibitor of SRC kinase effectively blocked the expression of α-SMA, which is associated with the progression of renal fibrogenesis (Hu et al., 2014). Even more, SRC can be activated by autophosphorylation of Tyr416, which is induced in response to a wide variety of cytokines/growth factors/transmembrane receptor proteins, including receptor tyrosine kinases, cytokine receptors, TGF-β1, and EGF (Yan et al., 2016;Zhou and Liu, 2016). Thus, SRC may be a potential therapeutic target for the treatment of chronic renal fibrosis with KYDS.
Mitogen-activated protein kinase 14 (MAPK14) encodes P38 mitogen-activated protein kinase and can be activated by various environmental stressors and pro-inflammatory cytokines (Han et al., 2015). MAPK14 regulates the activation of several transcription factors responses, including gene expression, growth, inflammation, metabolism, and apoptosis (Umasuthan et al., 2015). MAPK14 activity-deficient mice had less kidney dysfunction, inflammation, and apoptosis in acute folate nephropathy, while MAPK14 siRNA targeting decreased inflammation and cell death in cultured tubular cells (Ortiz et al., 2017). We conclude that MAPK14 promoted kidney injury through the promotion of inflammation and cell death and that it is a putative novel therapeutic target of SQW to ameliorate KYDS.
HRAS, a small GTPase from the Ras family, encodes the GTPase HRas, which is also known as the transforming protein p21 (Sugita et al., 2018). HRAS plays a role in regulating the growth, differentiation, and death of endothelial cells while enhancing the effects of the growth factor (Burgoyne et al., 2012). Moreover, HRAS participates in focal adhesion and the MAPK pathway by relieving inflammation (Tao et al., 2016).
Heat shock protein (HSP) is a highly conserved protein that is synthesized in response to physical, chemical, biological, and/or mental stimulation. Heat shock protein HSP 90α (HSP90AA1) belongs to the HSP90 protein superfamily, which is a molecular chaperone of numerous oncoproteins and a mediator of cellular homeostasis to maintain cell survival under stimulation (Trepel et al., 2010). Hsp 90 inhibition represses the TLR4-mediated NF-κB activity primarily through IKK to reduce renal ischemiareperfusion acute injury (O'Neill et al., 2015). Moreover, inhibiting HSP90 activation prevents the development of renal fibrosis through the degradation of TβRII depending on Smurf2mediation (Noh et al., 2012). These intriguing findings suggest that the kidney-protective functions of SQW may occur by regulating the expression of HSP90AA1.
Coagulation factor II (F2) encodes the prothrombin protein, which functions in blood homeostasis, inflammation, and wound healing. Qi deficiency and blood stasis are the key factors of KYDS, which is characterized by decreased gasification, a disorder of vital energy and blood, and cold limbs. We speculate that blood rheology abnormalities cause the deficiency of heat production and the ability of the kidney-yang to transfer body fluid into energy. However, the function of F2 in KYDS should be further studied.
Tubular epithelial cells (TECs) play an important role in renal diseases, especially in tubulointerstitial inflammation and fibrosis, which is a pathological process involved in a variety of cytokines and inflammatory mediators. Lymphocyte-specific protein-tyrosine kinase (LCK) and a SRC family protein-tyrosine kinase are located in the cytoplasm of TECs and form the key signal transduction molecule in the process of intracellular signal transduction (Singh et al., 2017). Li et al. have investigated the effect of the LCK pathway activation on the IL-12 signal transduction of TECs and found that LCK may regulate the LCK c-Jun signaling pathways in TEC, while the inflammation of TEC mediated by the activation of the LCK pathway is related to the expression of c-Jun promoted by IL-12 (Li et al., 2001). Glomerular mesangial cell proliferation is a common pathological feature of many glomerular diseases (Lin et al., 2017). Cyclin-dependent kinase 2 (CDK2) is a serine/threonineprotein kinase involved in the control of the cell cycle. Yu et al. (2007) observed that the proliferation of mesangial cells is directly related to the high expression of CDK2, which indicates that SQW probably improves KYDS by depressing the expression of CDK2.
Renal fibrosis is a common disease with pathological characteristics of the accumulation of extracellular matrix (ECM) and also strongly associated with the progression of chronic kidney disease to end-stage renal disease. Matrix metalloproteinases (MMPs) are renal, physiological regulators of ECM degradation. Matrix metalloproteinase 9 (MMP9), a 92 kDa type IV collagenase, can specifically degrade type IV and V collagens and gelatin to maintain homeostasis of the ECM in the kidney (Lenz et al., 2000). ECM components accumulate due to an imbalance in ECM production and defective ECM degradation by proteolytic enzymes during renal fibrosis (Tsai et al., 2012). The results of GO enrichment showed that protein hydrolysis has an important role, which is consistent with the function of MMP9. The preliminary results in our research demonstrated that the medicated serum of 3.0 and 6.0 g/kg SQW significantly increased the expression of MMP9 protein in NRK-52E cells. Target prediction also showed that salsolinol is associated with MMP9, MMP3, and MMP8, suggesting an interaction relationship between SQW and the matrix metalloproteinases (MMPs) family. These findings suggest FIGURE 6 | Effects of SQW on body weight (A), rectal temperature (B), holding power (C), water intake (D), and urinary output (E) in rats. **p < 0.01, the model group (n = 10) versus the control group (n = 10). ▲▲ p < 0.01, the SQW group (n = 10) versus the model group (n = 10). Values are presented as the means ± SD. The p-values were calculated using a one-way ANOVA. that SQW reduces the accumulation of EMC in renal epithelial cells via the metalloproteinases. Moreover, the effect of SQW on AQPs, the aquaporin channel family, and on the relation between AQP1 and MMP9 showed a trend of enhancement to promote the migration of renal TECs for renal injury repair. Moreover, SQW has a therapeutic effect on water metabolism disorder by promoting the mRNA and protein expression levels of AQP2. Furthermore, SQW significantly increased the ACTH, while CORT regulated the hypothalamicpituitary-adrenal axis to exploit the R. Cinnamomi and Radix aconiti lateralis preparata role in tonifying the kidney yang (Xu et al., 2014). These results are consistent with the therapeutic effect of SQW observed in the present paper.
Our study showed that SQW treatment dramatically improved the common physiological symptoms of KYDS and had protective effects on the hypothalamic-pituitary-adrenal axis in KYDS model rats. The potential targets of SQW were identified using PharmMapper, bioinformatics, and PPI network analysis. We found 79 potential target genes and identified SRC, MAPK14, HRAS, HSP90AA1, F2, LCK, CDK2, and MMP9 as the key potential therapeutic targets of SQW. The 79 target genes mainly related to the metabolism of xenobiotics by cytochrome P450, prostate cancer, and the T cell receptor signaling pathway. Wang et al. (2016) also reported 14 important potential targets associated with the aldosterone-regulated sodium reabsorption and adrenergic signaling pathways. However, further studies are required to confirm the results of this study. We explored the potential targets and pathways of SQW from a different perspective and using novel methods, and we conclude that multiple components, multiple targets, and multiple pathways of SQW led to a therapeutic effect on KYDS. This study shows that cell proliferation, differentiation, apoptosis, migration (SRC, HRAS, HSP90AA1, CDK2), and ameliorating chronic kidney disease (MAPK14, F2, LCK, MMP9) appear to play important roles in the therapeutic effect of SQW.
In this study, SQW ameliorated KYDS characteristics in rats presumably by eight target genes. Further studies are needed to analyze the protein levels of these targets. Moreover, other species should be considered for further verification.
CONCLUSION
In summary, SQW has a therapeutic effect on the treatment of KYDS through the "multiple components-multiple targets-multiple pathways" mechanism. We found that SRC, MAPK14, HRAS, HSP90AA1, F2, LCK, CDK2, and MMP9 genes were highly involved and may be potential targets in the treatment of KYDS.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.
ETHICS STATEMENT
This study was carried out in accordance with the recommendations of the Ethics of Committee of Zhejiang Chinese Medical University (permit number: SYXK 2013-0115). The protocol was approved by the Ethics of Committee of Zhejiang Chinese Medical University (permit number: SYXK 2013-0115). All procedures were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
AUTHOR CONTRIBUTIONS
JZ, CH, YY and CL conceived and designed the experiments. JZ and CH performed the experiments. HC, XZ and YZ analyzed the data. JZ, HC, TE, YY and CL contributed reagents/materials/ analysis tools. JZ and TE wrote and edited the paper. JZ and CH contributed equally to this work. TE, YY and CL contributed equally to this work. | v3-fos-license |
2019-07-12T13:14:36.531Z | 2019-06-01T00:00:00.000 | 195856101 | {
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} | pes2o/s2orc | Corn starch-based coating enriched with natamycin as an active compound to control mold contamination on semi-hard cheese during ripening
The effectiveness of natamycin supported in corn starch-based films to control environmental molds (mainly Penicillium spp) activity that could colonize the surface of semi-hard cheese during ripening, was evaluated. The starch amount was maximized, and this was achieved by adding polyvinyl alcohol (PVA) and also polyurethane (PU) to the formulation. The PU acted as plasticizer and also provided functional groups that interacted with the natamycin and affected its diffusion. When 5 % PU was added, the natamycin migration of the coating doped with 1% natamycin was reduced by half. The natamycin distribution on both sides of the film was also evaluated, concluding that, in line with the reduced migration, when polyurethane is included, the formulation presents high hydrophobicity and natamycin is left with a preferential distribution towards the air face (exterior). For microbiological tests, microorganisms were isolated from cheese factories. Natamycin solutions showed inhibitory effect against environmental molds including Penicillium spp. Accordingly, films loaded with 0.1 % natamycin showed a significant inhibitory effect against Penicillium spp. The polymer combination in this work was optimized to obtain an active coating with good physicochemical properties and enriched with natamycin that has proven to be available for acting against molds and preferentially on the surface exposed to potential mold attack during ripening.
Introduction
Food safety is a global priority and one of the central issues of the current food legislation [1]. Natamycin is a polyene antifungal antibiotic produced by Streptomyces natalensis and plays a major part in the food industry to prevent yeasts and molds contamination of cheese and other non-sterile foods such as meat products (dried sausage or salami, for example) [2]. It is approved as a food additive in more than 40 countries and the FDA consider it as a GRAS (generally recognized as safe) product [3]. Natamycin inhibits vacuolar fusion [4,5], not permeating but binding to ergosterol in the cytoplasmic membrane. As a result, natamycin is highly effective against molds and yeasts but not against bacteria and other microorganisms. Regarding application methods, it can be incorporated directly in a liquid matrix [6], or applied on solid surfaces by spraying, brushing or dipping. It is known that direct use of additives has limited benefits: the interaction with other components or additives in the food matrix could lead to a reduction in the active concentration of the antimicrobial component [7].
The two leading causes of food deterioration are microbial growth and oxidation reactions that take place on the product surface [8,9]. Uncontrolled opportunistic microorganisms generate unwanted taste or odor, discoloration and can sometimes produce toxic secondary metabolites. This could be risky to most consumers, and it is also associated with a negative impact on product acceptance [10].
Incorporation of antimicrobials in film formulations can regulate the diffusion rate into the product, providing a way of maintaining high concentrations of the active ingredient on the surface [11]. The fact of restraining the additive to the surface also diminishes the interaction with food components and/or other additives [12]. As a consequence, films or coatings with antimicrobial activity are an auspicious form of antimicrobial delivery regarding food preservation [7,13,14,15,16].
Much research in recent years has focused on starch because of its low cost and easy availability from renewable sources. Unfortunately, pure starch films have limited application on account of the low water resistance and high brittleness of this material. Starch/polyvinyl alcohol (PVA) polymer combination is one of the most studied blends due to PVA's advantages such as high film forming capacity, good adhesive properties and high thermal stability. Nevertheless, numerous experiments have established that great amounts of PVA are required for the purpose of obtaining the desired starch/PVA film properties. In this work, a polyurethane (PU) of low glass transition temperature was incorporated as a plasticizer, since the purpose was to obtain films containing starch as the major component. Starch/PVA/PU films were obtained and studied in previous works, demonstrating that incorporating PU to the starch/PVA blends at around 15 wt. % represents a good method for improving the properties of the films [17] and that natamycin incorporation at 0.1 % causes minimal variations in the global characteristics and properties of films [18]. This paper focuses on the evaluation of the effectiveness of natamycin in starch-based films principally against Penicillium spp and typical mold that could colonize the surface of semi-hard cheese during ripening. To achieve this, microorganisms were isolated from two cheese productive establishments and microbiological tests were carried out.
Preparation of film dispersion
The current investigation involved preparing the dispersions corresponding to the films starch/PVA/PU 70:30:0, 70:25:5 and 70:15:15. These were obtained following the procedure described by Gonz alez Forte et al. [17]. Briefly, starch and PVA suspensions were prepared (starch-gelatinized dispersion by heating 3 wt.% of starch in water at 90 C for 1 h and PVA by dissolving in water at 90 C for 24 h, both with magnetic stirring) and a polyurethane previously synthetized in the laboratory was added to the mixture. For the polyurethane synthesis, PPG2000 and dimethylol propionic acid (DMPA, Sigma Aldrich, USA) were charged into a dried 1000 mL flask with a mechanical stirrer, thermocouple, condenser, sampling tube, inlet system for gases and pump feed inlet. While stirring, the mixture was heated to 90 C, homogenized and bubbled dried N 2 for approximately 60 min, followed by increasing the temperature to 98 C and adding a mixture of H12MDI and dibutyltindilaurate catalyst (DBTDL, Sigma Aldrich, USA). After 2 h, the prepolymer was cooled to 45 C and 2-hydroxy ethylmethacrylate (HEMA, Sigma Aldrich, USA) dissolved in acetone was added slowly and allowed to react for approximately 90 min. Then, the temperature was raised to 60 C and kept constant until the isocyanate (NCO) content reached the desired value, determined using the conventional dibutylamine back-titration method [20]. The mixture was cooled to 55 C and triethylamine (TEA, ADELFA S.A.) (in acetone) was fed in slowly over 50 min to reach the theoretical NCO value (ca. 4.7 %). After neutralization, the temperature was lowered to room temperature. An aqueous dispersion of PU was obtained by adding the PU prepolymer to water containing the appropriate amount of ethylene diamine (EDA, Sigma Aldrich, USA) to perform the chain extension reaction. The dispersion was performed at about 300 rpm in an ordinary glass reactor at 30 C for 45 min. The resulting product was a stable dispersion with solid content of about 30 wt. % [21].
For the active films or coatings, natamycin water solution was prepared at 40 C for 2 h in darkness and subsequently added to each mixture to obtain a final concentration of 1 % or 0.1 wt. %.
Study of the natamycin distribution in the film
The distribution of natamycin on both sides of films (air and substrate) containing 1 wt. % natamycin was determined with infrared spectroscopy using an ATR accessory (Nicolet 380 spectrometer FTIR, Thermo Scientific, USA; ATR ZnSe IRE). The FTIR spectra were obtained by recording 64 scans between 4000-650 cm À1 with a spectral resolution of 4 cm À1 . To achieve this, a comparison of the absorption spectra by FT-IR with the ATR accessory on both sides was made, particularly studying the contribution of the stretching band of the carbonyls from the conjugated esters of the lactone ring of natamycin at 1715 cm À1 [22,23]. Spectral data were acquired with EZ OMNIC software (Thermo Electron Corporation, USA), and applying baseline correction, ATR correction and noise reduction.
Coating application on a model food
A commercial Tybo type semi hard cheese was chosen as a model system to evaluate natamycin effectiveness and behavior since cheese is one of the most popular products in which natamycin is applied worldwide. The choice of this type of cheese was made taking into account that this is a cheese with a homogeneously distributed humidity degree throughout its mass [24], allowing performing the tests with pieces simulating hole cheeses prior to ripening.
Coating application and thickness measurement
. Small pieces of Tybo cheese (3 Â 3 Â 1.5 cm 3 ) were covered with the coatings to measure the final thickness obtained. In order to establish a clear difference between the film and the cheese surface, a concentrated aqueous solution of violet crystal dye was prepared and added to a starch/PVA/PU dispersion. Successive layers were made by immersing the cheese pieces in the dispersions for 5 s and immediately lifting them up to drain the excess. For drying, each piece was placed on a silicon plate that was put into a convection oven at 30 C straightaway; flips were made every 10-15 min. This process was repeated until all the pieces were covered with three layers. Subsequently, a cut was made to each piece, and photographs were taken with a magnifying glass (10X magnification). The thickness was determined with Image-Pro Plus software. Triplicates were made for each sample.
Natamycin diffusion assay.
In the particular case of cheeses, the Argentine Food Code (AFC) establishes that natamycin is a permitted preservative on the surface that should not be detectable at 2 mm depth from the rind and, consequently, has to be absent in the mass [25,26].
For this experiment, Tybo cheese was cut into cubes of 3 Â 3 Â 3 cm 3 . The cubes were immersed in the corresponding mixtures for 5 s and immediately lifted up to drain the excess, completing the process until covering the cubes with 3 layers. Triplicates were made for each sample.
The coated cubes were individually placed in plastic cheese bags, vacuum sealed with a Vacuum Saver kit (Food Saver, Tila, Italy) and then placed in a chamber at 10 C for 75 days to simulate a ripening process.
To evaluate if natamycin diffused and penetrated more than 2 mm into the cheese mass, the technique of de Ruig & van Oostrom [27], which partially corresponds to ISO 9233 [28], was followed. Briefly, after the exterior first 2 mm of each coated piece were discarded, the cheese was homogenized and a methanol:water (MeOH: H 2 O) extract was obtained. UV spectra were recorded for each sample, and analyzed particularly in the natamycin absorption wavelengths. Calibration curves corresponding to the minimum absorbance at 311.5 nm, and also for the maxima at 318 and 329 nm were conducted, choosing 311.5 and 329 nm wavelengths because of their best fitting. A solution of MeOH:H 2 O in 2:1 ratio was used as blank. This procedure was also used by Vierikova, Hrnciarikova, & Lehotay [29]. It has been reported that the application method has a great influence on diffusion: the interaction between cheese surface and coating has a direct influence on the migration efficiency of an active component [30,31].
Microbiological tests
2.2.4.1. Isolation of most representative colonies from cheeses. Three different strains were isolated from the surface of molded chesses from the "C atedra de Agroindustrias, Facultad de Ciencias Agrarias y Forestales de la Universidad Nacional de La Plata" and from the "Colegio Agrot ecnico Don Bosco Uribelarrea" of Buenos Aires Province. The surface of the cheese was scraped with a sterile swab, and then strains were isolated and grown in Sabouraud slanted agar at 28 C for ten days [32]. The most representative strains were replicated on glucose-potato agar and Czapek-Dox agar. Macro and micromorphological studies were performed to the biggest colonies and microcultures. in Petri plates with a paper soaked in sterile distilled water; 3) Films exposed for 1 h to the environment, without inoculation, in Petri plates with a paper soaked in sterile water.
Subsequently, the films were incubated at 10 C for 45 days. All tests were performed in duplicate.
Agar diffusion method
2.2.4.3.1. Inhibitory activity of natamycin against isolated molds. For the purpose of evaluating the inhibition capacity of natamycin, three solutions were exposed to the different strains previously isolated and identified: Penicillium sp. 1, sp. 2 and sp. 3, Alternaria sp., Fusarium sp., Aspergillus Niger sp., Mucor sp. and Cladosporium sp. All fungi isolates were cultured on petri plates prior to microbiological essays.
To perform the Kirby-Bauer method Petri plates with Mueller-Hinton agar were used, and an inoculum containing 1 Â 10 9 CFU/mL of the strain was placed and spread homogeneously on the surface. Then, with the aid of a punch, 3 wells were made in the agar, in which natamycin solutions at 0.1, 0.2 and 0.3 % were placed. Finally, plates were incubated at 25-28 C for 96 h and after that the inhibition zones were measured. Determinations were made in duplicate.
2.2.4.3.2. Antimicrobial capacity of natamycin of loaded films. The agar diffusion test was also used to determine the antimicrobial effect of films loaded with natamycin against Penicillium spp 1, 2 and 3 isolated from cheese. Briefly, 0.5 μL of inoculum containing 1 Â 10 9 CFU/mL of each Penicillium spp, was spread on the surface of Petri plates containing Mueller-Hinton agar. Film disks (10 mm diameter) without natamycin, namely starch/PVA/PU 70:30:0, starch/PVA/PU 70:25:5 and starch/ PVA/PU 70:15:15 (controls), and the same three films with natamycin (0.1 wt. %) were placed on plates previously inoculated. The plates were incubated at 28 C for 96 h. The inhibitory activity was quantified by measuring the total diameter (disk plus inhibition zone). Determinations were made in duplicate.
Statistical analysis of data
Data were analyzed through ANOVA (α ¼ 0.05) and Tukey was the post-hoc test applied using InfoStat software [33]. Results are reported based on their mean and standard deviation.
Results and discussion
3.1. Study of the distribution of natamycin on both sides of the film and coating thickness measurement Fig. 1 A), it can be concluded that the amount of natamycin on the substrate side is greater than the amount in the air face. For films 70:25:5 and 70:20:10, the tendency is reversed (Figure 1 B and C). These films have a larger hydrophobic component due to the replacement of part of the PVA by PU, which leads to the natamycin being preferably distributed towards the air face. Although natamycin is amphiphilic, its structure has a more hydrophobic character, as indicated by the limited solubility in water [22,34].
During the drying process for films starch/PVA/PU 70:30:0, the water evaporation front advances towards the air face. As a result, natamycin in inclined towards the substrate face. In contrast, when PU is present, the medium has a more hydrophobic characteristic and for that reason natamycin is better distributed, so when water is evaporated, natamycin could be left with a preferential distribution towards the air face. For the starch/PVA/PU 70:15:15 films (Fig. 1 D), there is a contribution to the absorption spectrum from the PU that masks natamycin. The modification of natamycin distribution in the film as a consequence of the incorporation of the PU could be useful to reduce the total content of the additive and dispose it selectively.
Coating thickness was measured and the results showed values within 55 AE 5 μm, which was aligned with the film thickness obtained by casting [17].
Diffusion assay of natamycin into the mass of semi hard cheese
Natamycin was found in the mass of every cheese and could be quantified (Fig. 2). Starch/PVA/PU coatings showed less natamycin release than the mixture without PU. Within the mixtures containing PU, the one with the lowest diffusion of natamycin was 70:25:5. Interactions between the film components and natamycin, and the amphiphilic nature of natamycin itself [22], determine in each case a different behavior in the diffusion of natamycin towards the cheese interior. The hydrophobic character of PU can limit the diffusion of natamycin which, despite being considered amphiphilic, has a more hydrophobic character due to its structure [34].
According to the AFC, natamycin should not be detected at 2 mm depth. Considering that the technique has a detection limit of 0.5 mg/kg, the results obtained with 1% wt. of natamycin (maximum detected value 3.65 mg/kg) and the fact that in this study the natamycin migration is over-estimated (ideal conditions for full migration: vacuum seal, time and temperature), it was decided not to proceed with the same study for 0.1 % natamycin, assuming that in this case both AFC conditions would be fulfilled.
. Strain isolation
An interesting practice in evaluating the effectiveness of a potential food coating is to carry out studies with microorganisms isolated directly from related environments: in this case cheese ripening rooms, processing rooms, or directly from the surface of molded cheeses [30,35,36,37]. In this work the isolation of fungi from the surface of molded cheeses from ripening rooms resulted in the collection of three strains of Penicillium: sp. 1, sp. 2 and sp. 3. Basílico et al. and Hocking [35,38] showed that Penicillium spp. is a majoritarian mold that can be isolated from cheese surface. These microorganisms could be potential producers of mycotoxins dangerous to health [30,35,38].
Sensitivity study of the films against Penicillium spp.
The results of the microorganism growth for films are presented in Table 1. The scale ranged between "-" and "þ", where "-" symbolized no growth and "þ" symbolized growth.
For the films exposed to the environment, only growth of environmental molds was registered, since there was no inoculation of the films. These environmental molds were isolated, analyzed and identified. The films incubated at 10 C, presented Penicillium spp. development and additional contamination of environmental molds (Dematiaceae family, Deuteromycetes class), with the most frequent being the genera Alternaria sp. and Cladosporium sp. Less frequently, Aspergillus Niger sp., Fusarium sp. and Mucor sp.
In general, all films containing starch showed a high fungal development after 45 days on agar, possibly because the agar constituted a wet surface in direct contact.
It should be considered that all samples were exposed to high humidity environments, either by the presence of a wet paper in the base of the boxes or by the agar itself, which combined with the temperature offered favorable conditions for the development of molds.
Ripening conditions are here proven favorable to mold development, and starch-based films are attractive substrates, which is why for this product it is crucial to incorporate an antifungal compound. [39] for inhibition effect with natamycin, and, it is important to outline that 0.1 % natamycin in the films is within the 1 mg/dm 2 on cheese surface standard required by the AFC. The inhibition zones obtained against the different strains of Penicillium spp. of environmental molds studied after 96 h incubation are shown in Fig. 3.
It can be observed that the inhibition becomes greater for all samples as the amount of natamycin in solution increases; in all cases, the 0.3 % natamycin solution showed inhibition halos. For lower concentrations, the inhibition efficiency varied, probably because the differences between strains and some characteristics of the diffusing molecule as size, polarity and shape, and also interactions between the natamycin and the polymer chains, can affect the release of the agent.
Evaluation of antifungal performance of natamycin loaded
films. The inhibition halos obtained for starch/PVA/PU films with or without 0.1% natamycin and natamycin solution and PU against Penicillium spp. are presented in Table 2. Fig. 4 shows an example of the inhibition zones against Penicillium sp. 1.
These results showed that the 0.1 % natamycin solution and the pure PU applied in aqueous dispersion had a significant inhibitory effect on the 3 Penicillium spp. strains studied. The hydrophobic feature of the PU dispersion could limit the interaction with Penicillium spp. strains in aqueous suspensions, leading to an "inhibition halo". Films loaded with 0.1 % natamycin showed a significant inhibitory effect, although the inhibition zones were smaller than those showed by natamycin solution; probably because natamycin is less available to act when incorporated into the films. This effect was the same statistically for all formulations of films versus all the strains. A variety of works for soft, semi hard and hard cheeses with coatings or active films (synthetic or natural sources) containing natamycin can be found in the literature. In most cases, good results are obtained depending on the preservation times of the proposed cheese type [8,9,15,30,40]. Hanu sova et al. [30] studied the release of natamycin from PVC films containing an 7.1 mg/dm 2 of natamycin against isolated from the surface of a soft cheese (Penicillium spp. and Cladosporium sp., mainly), finding inhibition zones for all the strains against the active film. These authors also emphasize the importance of studying the difference between the effectiveness of natamycin in solution and natamycin contained in a polymer film.
There are several methods for applying natamycin in cheese preservation to prevent the growth of undesirable and/or potentially hazardous fungi on the surface. In the Argentinean industry, it is a common practice to spray the surface of the cheese with a natamycin solution, which means that there is no control over the amount of active compound per area unit, no control over the amount of natamycin that can migrate into the cheese and over the effectiveness of the treatment. Reps, Jedrychowski, Tomasik, & Wisniewska [41] studied the use of Delvo ® Cid to protect semi hard cheeses against mold growth by different application methods: immersion of cheese in brine containing natamycin, immersion in aqueous suspension of natamycin (before or after brine), coating cheeses with polyvinyl acetate containing natamycin in layers with and without wax, packaging cheeses with films that were immersed in natamycin solution and packaging cheeses after immersion in natamycin solution with vacuum bags. The results showed that the content of natamycin is depleted over time, and that this loss depends on the application conditions of natamycin: the longer time of natamycin presence was obtained when one layer of wax was applied after 3 layers of polyvinyl acetate containing natamycin. De Oliveira, De F atima Ferreira Soares, Pereira, & De Freitas Fraga [40] demonstrated, in agreement with these results, that the use of a layered coating containing natamycin results in a lower amount of natamycin in the cheese shell than if natamycin is directly applied in solution. Therefore, the efficiency of an antimicrobial film depends on: the amount of natamycin released, the contact between the cheese and the film, the barrier properties of the film and the temperature and humidity conditions in the ripening room.
Conclusions
Natamycin showed inhibitory effect against environmental molds including Penicillium spp. isolated from cheese factories. Films loaded with 0.1 % natamycin showed a significant inhibitory effect against Penicillium spp.
This study demonstrated that a corn-starch coating with starch percentage maximized to 70 % with PVA and PU as plasticizers and enriched with natamycin, could be applied as an effective coating to control environmental molds development on the surface of foods that require ripening like semi-hard cheeses. The polymers proportion was optimized to obtain an active coating with good physicochemical properties, and results showed that when PU is present in the formulation, natamycin adopts a preferential distribution towards the air side of the coating, being more available for acting against mold when applied on cheese surface.
Author contribution statement
Lucia Gonz alez-Forte: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.
Javier Amalvy, Nora Bertola: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper. | v3-fos-license |
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} | pes2o/s2orc | MULTIPHOTON IONIZATION AND FRAGMENTATION OF CSz UNDER INTENSE SHORT PULSE LASER RADIATION
The interaction of CS2 with intense short pulse laser radiation is studied, experimentally
using time-of-flight mass spectroscopy. Laser pulses of 0.5 and 5 psec at 248 and 496 nm
have been used in order to investigate the effect of the wavelength and the pulse duration
on the molecular ionization and fragmentation. As shown, for low enough intensities
the parent molecular ion is present as the most important peak in all cases. Increasing
the intensity results in extensive fragmentation, where the molecular parent ion remains
always the more intense mass peak at 496 nm while at 248 nm S
INTRODUCTION
The study ofionization, dissociation and/or fragmentation ofmolecules in the gas phase under irradiation with short laser pulses has received a lot ofinterest during the last few years, especially since the technology of femto-second (fsec) laser systems became readily available.As a result significant information has been revealed towards the understanding of elementary and fundamental physical and chemical processes [1].*Corresponding author.129 Although the use of short laser pulses and high intensities has stimulated intense research interest in the field of molecular fragmentation and dynamics, a lot of questions still remain open, not only due to the lack of appropriate theory but also due to the fragmented and sometimes controversial experimental results appearing in the literature.
The motivation of the present work is to provide a consistent set of data on the multiphoton ionization and fragmentation of a simple and well studied, from the spectroscopic point of view, small molecule like CS2, using different laser pulse durations and wavelengths.The experimental results presented here are consistent in the sense that they have been obtained under the same experimental conditions, changing only the characteristics of the laser excitation.The main question that we are addressing here is the competition between ionization and fragmentation i.e., if the molecule ionizes first and then dissociates or vice versa and what ionization/fragmentation channels are favored depending on the pulse duration and the wavelength used.These questions are investigated here for intensities ranging between 109 and 4 x 1012 W/cm 2.More work is currently underway in order to extend the range of intensities up to 10 a6 W/cm 2.
EXPERIMENTAL
The laser system employed here is a hybrid excimer-dye laser system based on the concept of distributed feedback dye laser (DFDL).Specifically, the 308 nm (XeC1) output of a double-cavity excimer laser (Lambda Physik EMG 150 MSC) pumps a cascade ofdye laser modules to produce 0.5 psec pulses at 496 nm with a corresponding energy of 100 laJ.This seeding pulse is frequency doubled in a BBO crystal and amplified in a double pass by a KrF second cavity of the excimer unit.The resulting UV laser beam at 248 nm has a pulse duration of 500 fsec, a repetition rate up to 20 Hz and a maximum energy of 20 mJ per pulse.By inserting a bandwidth limiting etalon pulses 5 psec long at both 496 and 248 nm can be produced.Laser pulse diagnostics are obtained on- line by a lm Littrow-type spectrograph and a multiple shot autocorre- lator at 248 nm.Attention was paid to clean operation of the system in providing low amplified spontaneous emission (ASE) content in the laser output, which is monitored both by energy measurements of ASE versus total energy in the near field, and through the autocorrela- tion profile having the correct signal-total background ratio.The nanosecond laser system used was a Lambda Physik FL2001 dye laser pumped by a Lambda Physik 201 MSC excimer laser opera- ting at 308 nm.The tunable laser light has a bandwidth of 0.3 cm-1, duration of 15 nsec and provides energies of several mJ.
A home made time-of-flight mass spectrometer has been used, consisting of a 60cm long field-free drift tube and an extracting electrode arrangement of a repeller plate (+ 1900 V) and a guarding electrode (grounded) separated by 1.5 cm.Unit mass-resolved cation signals were detected by a pair of 2.5cm diameter channel plates.Alternatively, another 100 cm long Wiley-McLaren type time-of-flight was employed, having an electrode arrangement consisting of a repeller plate (at + 1900 V) and an acceleration plate (at + 1500V) followed by a grounded electrode.The distance between the electrodes was 2 cm.From the extracted ions, only those that were passing through a mm pinhole placed on the geometrical axis of the field-free tube were allow- ed to reach the detector.A mixture of 50 mbar CS2 in 200 mbar argon was expanded through a 0.3 mm nozzle into the vacuum chamber.The pressure in the chamber was always kept below 10 -6 mbar during the recording of the mass spectra.Spectroscopic grade CS2 was used after several repeated freeze-thaw-degassing cycles under vacuum.The laser beam was focused by means of piano-convex quartz lens, of 15 cm focal length and in the case of the short pulses at 248 nm only the central part of the beam was utilized passing through an aperture.In addition, the polarization was set parallel to the TOF axis for all the experiments presented here.Special care was taken to insure that no space-charge effects would perturb the mass spectra measurements.A previously calibrated Molectron J4-09 energy meter measured the energy of the laser beam.The beam diameter was measured at the focus using a linear diode array and was found to be 40 m and 25 tm at 496 and 248 nm, respectively.
RESULTS AND DISCUSSION
Mass spectra have been recorded under several experimental conditions, at both wavelengths and pulse durations used.Each mass spectrum presented here is the average of the corresponding mass spectra of 100 to 500 laser shots and is normalized with respect to the CSmass peak for comparison purposes.Absolute signals can be obtained by dividing the presented spectra by the appropriate factors indicated in each figure .In Figure 1, we present mass spectra that have been recorded at the lowest possible laser energies used with our experimental setup for which a signal can be recorded.As is shown in all cases, the more intense peak of the spectra is the one corresponding to CS -(m/q 76), the molecular parent ion.Moreover, the presence of the S + (m/q 32) and CS + (m/q 44) mass peaks, even though smaller in intensity, is evident even at the lowest intensities employed for all cases, their contribution being relatively more important at 248 nm and for 5 psec pulses.All sulfur-containing ions were accompanied by a small peak corresponding to the 34S isotope, which has a natural abundance of 4%.
The fragmentation pattern changes dramatically at higher inten- sities, as depicted in Figure 2, where extensive fragmentation is observed in all cases.However, comparing the mass spectra obtained with the 0,2
FIGURE
Typical mass spectra of CS; at low incident intensities corresponding to: (a) 248 nm, 0.5 psec, (a) 248 nm, 0.5 psec, same pulse duration and with similar incident laser intensities, the parent molecular ion remained the most prominent mass peak at 496 nm, while at 248 nm, the sulfur ion dominated the spectra.The CS + and C + (m/q 12) ions were also present in all cases, being in general relatively more important at 248 nm.Appreciable quantities of S 2+ (m! q 16) were also observed for both wavelengths and pulse durations used.Small signals of S 3+ (m/q 10.67) and S-(m/q 64) were present, the former being more intense at 496 and the latter at 248 nm respectively.Furthermore, in the mass spectra at both wavelengths, small intensity signals corresponding to the doubly ionized parent molecular ion CS2+(m/q 38) and C2+(m/q 6) have also been observed.In addition, at even higher intensities, S 4+ (m/q 8) was observed at 496 nm, 0.5 psec.It has to be noted that at the high inten- sities used, the S + and all the multiple charged fragments (S2+, S3+, S4+, C2+) exhibit significant splitting most probably due to Coulomb explosion.Finally, at 248 nm, the presence of H2 O+ (m/q 18) was also clearly distinguishable, being more important for the shorter duration pulses.The appearance of H20 + is most probably due to humidity present in our TOF system, the gas handling apparatus, water traces present in the CS2, or the carrier gas used.The water signature was found to always be present at shorter wavelengths and appears at longer wavelengths only when high enough intensities were employed.
In Figures 3a,b and 4a,b, we present the evolution of the ratio of the signal (i.e., the integrated area of the respective mass peaks) for each principle fragment observed (S +, CS + and C+), divided by the corresponding signal of the molecular parent ion, at the range of in- tensities used.Comparing the values of the ratio of the fragment to the parent molecular ion, it is evident that at 496 nm (Figures 3a and b), CSis the most important peak in all the spectra recorded, the ratio exhibiting values always smaller than one, for both pulse durations.The reduction of the ratio occurring at high intensities is related to the fact that the parent ion signal continuously increases within the in- tensity range used, while the fragments tend to saturate earlier.In ad- dition, at such intensities, the presence of multiply charged fragments becomes significant.
The fragmentation pattern observed here is not in agreement with what has been previously reported [2][3][4][5], where the second harmonic (532nm) of a 35 psec Nd:YAG has been used as the laser excitation source.In these experiments, the relative intensities of S+, C + and CShave been measured at laser intensities similar to the ones used in the present work.As shown in Figures 3 and 5 of Refs.
[1] and [2] respectively, their mass spectra differ significantly from ours since, in their case S + is the major mass peak at low intensity and only at high intensity the CSpeak becomes important.A possible reason for this discrepancy may be the longer pulses used in Refs.[1,2].
Under 248 nm excitation (Figure 4a and b), the principal fragments S+, CS / and C+, were found to be more intense or at least of compa- rable intensity to the parent ion peak, for both pulse durations used, the effect being more pronounced at 5 psec.This observation is in partial agreement with the results presented in Figure 2 of Ref. [3], where the third harmonic (355 nm) of a 35 psec Nd:YAG laser was employed.In these results, the parent ion was found to be much less abundant than the fragment ions, a behavior similar to what we have observed at high intensities.However, it must be pointed out that in our results and at low incident intensities, CSwas found to be the most CS2 UNDER SHORT PULSE LASER RADIATION important feature of the mass spectra, while in Ref. [3] no significant difference had been observed between 532 and 355 nm.
In Figure 5, we present the ratio of the signal corresponding to the sum of all fragments (except CS -) over the CSsignal.As shown, at Intensity (GW/cm2) FIGURE 4 Variation of the ratio of the signal of the principal fragments (S+, CS+, C+) over the signal of the parent molecular ion (CS -) as a function of the incident laser intensity at 248 nm for: (a) 0.5 psec and (b) 5 psec laser pulses.
248 nm the ratio smoothly increases, largely exceeding the value of one, while at 496 nm it is constantly smaller than one and decreases with intensity.It is noteworthy that at low incident intensities for both pulse durations at 248 nm, the ratio approaches unity, its value becoming Intensity (GW/cm2) FIGURE 5 Variation of the ratio of the total ion signal (all fragments except CS -) over the signal of the parent molecular ion (CS -) as a function of the incident laser intensity for: (a) 248 nm, 0.5psec (e), (b) 248nm, 5psec (i), (c)496nm, 0.5psec ('), (d) 496 nm, 5 psec (A) (e) 248 nm, 15 nsec (0).larger for 5 psec ( 60) than for 0.5 psec ( 30 at higher intensities.The increase of the ratio is even more pronounced for 15 nsec pulses, where the ratio attains values two orders of magnitude larger than those obtained at 5 psec, 248 nm.The physical insight of Figure 5 is that shorter wavelengths tend to fragment the molecule more efficiently, while longer wavelengths result in reduced fragmentation and more parent ion production.Also, at the same wavelength, the fragmentation is more efficient for longer pulses.Preliminary experiments currently underway, using 800 and 400nm laser pulses of 200 fsec and 50 fsec duration, confirm again that longer wavelengths and shorter pulses favor the formation of the molecular parent ion, while shorter wave- lengths result in enhanced fragmentation.This is consistent with what has been previously reported in Ref. [6], where the multiphoton multi- electron dissociative ionization of CO at two wavelengths, 305 and 610 nm have been studied. Considering the above experimental results and the low intensity mass spectra of Figure 1, it is irrefutable that the parent molecular ion is produced in considerable amounts and reduced fragmentation is observed at both wavelengths and pulse durations at relatively low intensities.Taking into account the ionization potential (I.P.) of CS2 [7] (Tab.I), the molecule must absorb at least 3 and 5 photons at 248 and 496 nm respectively, in order to be ionized.In the former case, the absorption of three photons corresponding to an energy of 15 eV, brings the molecule slightly above the dissociation limit of the B"Zu / state of the molecular ion, leading to S+(4S) and CS(1E+) (the thresh- old limit being at 14.81 eV [8]).This implies that S + will be inevitably produced when the molecule is ionized.Moreover, sulfur atoms, having an ionization potential of 10.35 eV [9], can be also easily ionized at these intensities, resulting in enhanced S + production.This is nicely depicted in Figures 4a and b, where the ratio S+/CSincreases with incident intensity, approaching the value of 20 and 40 for 0.5 and 5 psec pulses respectively.Meanwhile, the produced neutral CS (1E+) can also absorb three 248-photons [10] (Tab.I) becoming ionized within the laser pulse.Similarly, at the 4-photon level, corresponding to 20eV, CS + and Scan be efficiently produced [7].The intensity dependence of the principal fragments and the parent ion and their saturation ap- pearing in the order CS -, S+, CS + and C+, also support the previous picture.Increasing the intensity of the incident laser radiation, more channels can be reached through the absorption of more photons and consequent fragmentation of the produced molecular ions and/or during the fragmentation of the multiply charged CS+(q _> 1) ions.
In this respect, CS -(after absorption of 3 or more 248 nm photons 24.37 [9] S-9.35 [13] CS2 UNDER SHORT PULSE LASER RADIATION 139 by the neutral molecule) and CS 2+ can be seriously considered as precursors towards the production of the S q+ and C q+ (q _> 1) [5].
In the case of 496 nm photons, the molecule has to absorb at least 5 photons to be ionized, corresponding to an energy of 12.47 eV.The intensity dependence of the ion signals reveals that the saturation regime of the principal fragments is reached almost simultaneously by all of them, the CSsaturating at higher intensities and remaining the most important peak in all cases.Moreover, the production of the doubly charged parent ion CS 22+ [11] and multiply charged fragments, i.e., S2+, S3+, S 4+ and C 2+ [9] (see also Tab.I) occur with relatively large yields compared to the parent ion and at least larger than the value at shorter wavelengths.
In Figure 6, the ratio of the S 2+ and S 3+ signals over the S + signal at 496 and 248 nm is shown for 0.5 psec duration pulses.As depicted, the S 2+ ions appear at lower intensity at 248 nm than at 496 nm, a behavior consistent with the concept of a multiphoton process, where the number of photons required for the ionization at 248 nm is half of that needed at 496 nm.On the other hand, the S 3+ ions seem to be produced at the same intensity of 1012 W/cm 2 at both wavelengths.This should most probably reflect the different mechanism responsible for their produc- tion.The ratio of $2+/S + at 248 nm increases with intensity at the beginning, reaching rapidly a saturation regime, where the S 3+ ion signal appears which in turn exhibits saturation at an intensity of 4 1012 W/cm 2. At the longer wavelength of 496 nm, the ratio $2+/S + increases continuously and even more rapidly than at 248 nm, never reaching saturation (for the intensities used) and exhibits values even larger than those at 248 nm.Similar behavior is seen for the $3+/S + ratio at 496 nm, that attains almost the same saturation value of the $2+/S + ratio at 248nm.These observations strongly indicate that at the shorter wavelengths used the production of the principle fragments is favored in contrast to the longer wavelength, where multiply charged fragments are more efficiently produced.
The dynamics of the laser light-molecule interaction is often characterized by taking into account the adiabatic Keldysh [11] param- eter % which provides an indication whether the ionization process takes place in the multiphoton or in the field ionization regime.Moreover its value, with a dependence on the wavelength A (in lam), the intensity of the incident light I (in W/cm) and the ionization potential Ei (in eV), is given by the following relation: ,v/Ei/(1.8710-131A2).When -y >> 1, multiphoton processes are the dominant mechanism for the ionization of the molecule.When << 1, then field ionization explains better the ionization.For -y values near 1, an intermediate mechanism is generally assumed.By substituting in the formula the parameters that correspond to our experimental conditions, we found that the lowest , values, corresponding to the highest intensities used, were 10 and 16 for 496 and 248 nm respectively.These values being much larger than one indicate that we are operating at the multiphoton regime.
Another interesting point is the presence of the Smass peak [13] (Tab.I) and its dependence on the wavelength and the pulse duration.As shown in Figure 7, shorter wavelength and longer pulses enhanced + the formation of S -.In particular, while at 496 nm the ratio S -/CS 2 was found continuously decreasing with intensity, at 248 nm it was slightly increasing and reached a plateau for both 0.5 and 5 psec.This behavior appears to be even more pronounced when the same wave- length but 15 nsec duration laser pulses were employed, the ratio exhi- biting an almost tenfold increase.The observation of the formation of CS2 Intensity (GW/cm2) FIGURE 7 Ratio of S -/CSas a function of the incident laser intensity for: (a) 248 nm, 0.5 psec (o), (b) 248 nm, 5 psec (m), (c) 496 nm, 0.5 psec ('), (d) 496 nm, 5 psec (,) (e) 248 nm, 15 nsec (0).Sin our experiments contradicts what has been previously reported in Refs.[2,4,5], where Swas observed only in collisions of charged particles (i.e., electrons, positive or negative ions) with CS2 [4].In Ref.
[14], an explanation of the Sformation was given, assuming the fragmentation of the CS-and CS + ions through bent geometry states.Although this mechanism may be probable we believe that it is not very satisfactory, since we have observed Sformation also using 400 and 800 nm, 200 fsec laser pulses.The fact that the dimer ion is formed at four different wavelengths does not favor the argument of potential resonances with bent states.This was also confirmed in studies of the Rydberg states of CS2, where Swas observed at several wavelengths using nsec lasers [15][16][17].
CONCLUSIONS
Experimental results were presented related to the interaction of CS 2 with intense short pulse laser radiation.It has been shown that when short pulses are employed, the parent ion is the most important mass peak at low incident intensities, independently of the wavelength used.This implies that CS2 first ionizes and then dissociates at the wavelengths, pulse durations and intensities used in this study.At higher intensities, at 248 nm, extended fragmentation was observed, S + being the major feature of the mass spectra, while at 496 nm, the ionization of the parent molecule was found to be more efficient.The production of multiply charged fragments was found enhanced at 496 nm.Finally, the formation of Swas enhanced at 248 nm.
6. 7 FIGURE 2
FIGURE 2 Typical mass spectra of CS2 at high incident intensities corresponding to:
10 FIGURE 3
FIGURE 3 Variation of the ratio of the signal of the principal fragments (S+, CS+, C+) over the signal of the parent molecular ion (CS -) as a function of the incident laser intensity at 496 nm for: (a) 0.5 psec and (b) 5 psec laser pulses.
TABLE Species Appearance
UNDER SHORT PULSE LASER RADIATION | v3-fos-license |
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} | pes2o/s2orc | Synthesis, Structural Characterization, and Antitumor Activity of a Ca(II) Coordination Polymer Based on 1,6-Naphthalenedisulfonate and 4,4′-Bipyridyl
A novel Ca(II) coordination polymer, [CaL(4,4′-bipyridyl)(H2O)4]n (L = 1,6-naphthalenedisulfonate), was synthesized by reaction of calcium perchlorate with 1,6-naphthalenedisulfonic acid disodium salt and 4,4′-bipyridyl in CH3CH2OH/H2O. It was characterized by elemental analysis, IR, molar conductivity and thermogravimetric analysis. X-ray crystallography reveals that the Ca(II) coordination polymer belongs to the orthorhombic system, with space group P212121. The geometry of the Ca(II) ion is a distorted CaNO6 pengonal bipyramid, arising from its coordination by four water molecules, one nitrogen atom of 4,4′-bipyridyl molecule, and two oxygen atoms from two L ligands. The complex molecules form a helical chain by self-assembly. The antitumor activity of 1,6-naphthalenedisulfonic acid disodium salt and the Ca(II) coordination polymer against human hepatoma smmc-7721 cell line and human lung adenocarcinoma A549 cell line reveals that the Ca(II) coordination polymer inhibits cell growth of human lung adenocarcinoma A549 cell line with IC50 value of 27 μg/mL, and is more resistive to human lung adenocarcinoma A549 cell line as compared to 1,6-naphthalenedisulfonic acid disodium salt.
Introduction
The last few decades, many studies have focused on the metal-organic hybrid materials constructed by d-block or f-block cations and organic ligands, because they have potential applications in catalysis [1], photoluminescence [2], gas storage [3], molecule-based magnetic materials [4] and biomedical materials [5]. In comparison to d-block cations, the coordination behavior and potential applications of alkaline earth metal complexes has remained largely an unexpored area [6]. As part of our group to explore the synthesis and properties of alkaline earth metal complexes, we have been exploring the preparation of metal-organic hybrid materials by combining alkaline earth metal ions and organic ligands containing multi-oxygen and nitrogen atoms [7][8][9][10][11]. In this paper, a new hybrid material, [CaL(4,4′-bipyridyl)(H 2 O) 4 ] (L = 1,6-naphthalenedisulfonate) was synthesized and characterized by elemental analysis, IR, thermogravimetric analysis and X-ray structure analysis. On the basis that metal-organic frameworks can be used as delivery vehicles for drug molecules [12], the antitumor activity of 1,6-naphthalenedisulfonic acid disodium salt and its Ca(II) coordination polymer against human hepatoma smmc-7721 cell line and human lung adenocarcinoma A549 cell line also have been investigated.
IR Spectra
The IR spectrum of the Ca(II) coordination polymer exhibits ligand bands with the appropriate shifts due to complex formation. The ν(SO 3 − ) vibrations at 1336 cm −1 and 1201 cm −1 in the free 1,6-naphthalenedisulfonate ligand shift to lower frequencies and are observed at 1301 cm −1 and 1186 cm −1 for the complex, indicating that the oxygen atoms of SO 3 − coordinate to Ca(II) ions [14].
The IR spectrum of the Ca(II) coordination polymer displays peaks at 1219 cm −1 , 800 cm −1 and 607 cm −1 which may be attributed to the ν(C=N) stretching of 4,4′-bipyridyl, showing that the nitrogen atoms of 4,4′-bipyridyl also take part in the coordination with calcium atom. In addition, at lower frequency the complex exhibits bands around 519 cm −1 and 417 cm −1 which may be assigned to ν(Ca-N) and ν(Ca-O) vibration.
Structure Description
A single-crystal X-ray crystallographic study reveals that the Ca(II) coordination polymer crystallizes in the space group P2 1 2 1 2 1 with Z value of 4. The basic unit consists of one Ca(II) center with distorted pengonal bipyramidal geometry ( Figure 1). The coordination environment around Ca(II) is afforded by two oxygen atoms (O1 and O4) from two 1,6-naphthalenedisulfonate ligands, four oxygen atoms (O1W, O2W, O3W and O4W) from four coordinated water molecules, and one nitrogen atom (N1) from 4,4′-bipyridyl ligand. The Ca(II) coordination polymer molecules from one-dimensional chained helical structures by the π-π interaction of the bridging ligand, 1,6-naphthalenedisulfonate ( Figure 2). The helical chains are further connected by hydrogen bonds to form a three dimensional network structure ( Figure 3). It is interesting that the 4,4′-bipyridyl only acts as monodentate ligand in the complex molecule. The distances of the Ca-O bonds are in the range of 2.2982(13) ~ 2.4896(14) Å, and that of Ca-N bond is 2.5934(15) Å, which are similar to the Ca-O bond lengths reported previously [15,16]. In addition, the formation of network benefits from the intermolecular and intramolecular hydrogen bonds (Table 1). Table 1. Hydrogen-bond geometry (Å, °). Symmetry code:
Thermogravimetric Analysis
The thermogravimetric analysis of Ca(II) coordination polymer was performed under air atmosphere. The TG measurement confirms that Ca(II) coordination polymer is thermally stable up to 180 °C. The TG curve indicates that Ca(II) coordination polymer starts to loose water molecules at ca. 180 °C and completes dehydration at ca. 200 °C. The mass loss is 12.36% in the range 180-200 °C, which corresponds to the loss of four water molecules. On further heating, the TG curve shows a continuous mass loss up to 650 °C due to decomposition of Ca(II) coordination polymer.
Antitumor Activity
The data of antitumor activities of Ca(II) coordination polymer and 1,6-naphthalenedisulfonic acid disodium salt are given in Table 2. Up to now, there have been no reports that calcium salts have antitumor activity. From Table 2, It can be seen that both Ca(II) coordination polymer and 1,6-naphthalenedisulfonic acid disodium salt exerted cytotoxic effect against human hepatoma SMMC-7721 cells, and the antitumor effect of 1,6-naphthalenedisulfonic acid disodium salt is better than that of Ca(II) coordination polymer. However, Ca(II) coordination polymer has stronger cytotoxicity against human lung adenocarcinoma A549 cells with lower IC 50 (27 ± 1.2 μg/mL) than that of 1,6-naphthalenedisulfonic acid sodium. The result of molar conductivity of the Ca(II) coordination polymer shows that the Ca(II) coordination polymer is a nonelectrolyte, so we think that the antitumor activity of Ca(II) coordination polymer is due to the joint action of the ligand and the Ca(II).
Elemental analysis (C, H, N) was carried out on a Elementar Vario III EL elemental analyzer (Hanau, Germany). Infrared spectra were recorded as KBr discs using a Nicolet AVATAR 360 FTIR spectrophotometer in the range 4000 cm −1 ~ 400 cm −1 . Thermogravimetric analysis was performed on a Shimadzu PT-40 with heating rate programmed at 5 °C min −1 . X-ray diffraction data of the Ca(II) complex was collected on a Bruker smart CCD diffractometer.
Synthesis of Ca(II) Coordination Polymer
A 5 mL methanol solution of 0.5 mmol (0.1555 g) Ca(ClO 4 ) 2 · 4H 2 O was added to a solution containing 0.5 mmol (0.1661 g) of 1,6-naphthalenedisulfonic acid disodium salt in 10 mL CH 3 CH 2 OH. The mixture was stirred for 2 h at refluxing temperature. Then 0.5 mmol (0.07809 g) 4,4′-bipyridyl was added to the above solution. The mixture was continuously stirred for 3 h at refluxing temperature. The white precipitates were collected by filtration. Then the white precipitates redissolved in MeOH, and the single crystal suitable for X-ray determination was obtained from methanol solution after 20 days by evaporation in air at room temperature.
X-ray Crystallography
Single crystal X-ray diffraction data were collected at 273(2) K on a Bruker smart CCD diffractometer using graphite-monochromatic Mo Kα radiation (λ = 0.71073 Å). The structure was solved by direct method and refined using a full-matrix least-squares technique against F 2 with anisotropic displacement parameters for non-hydrogen atoms with the program SHELXL-97 [17]. All hydrogen atoms were placed at calculated positions using suitable riding models with isotropic displacement parameters derived from their carrier atoms. Molecular graphics were drawn with the program package SHELXTL-97 crystallographic software package [18].
Antitumor Activity
Human hepatoma SMMC-7721 cells and human lung adenocarcinoma A549 cells were propagated continuously in culture and grown in RPMI 1640 medium with 10% inactivated fetal calf serum and antibiotics. Cell harvested from exponential phase were seeded equivalently into 96 well plates and incubated for 24 h, then the solid compounds studied were added in a concentration gradient. The final concentrations were maintained at c/(μg mL −1 ) 5, 10, 20, 30, 40, 60 respectively. The plates were maintained at 37 °C in a humidified 5%CO 2 -90%N 2 -5%O 2 atmosphere and incubated for 48 h, MTT solution was added, and the procedure described in [19] was then followed. The measurements of absorption of the solution concerned with the number of live cells were performed on spectrophotometer at 570 nm.
Conclusions
In summary, we have synthesized a new one-dimensional helical chained Ca(II) coordination polymer, [CaL(4,4′-bipyridyl)(H 2 O) 4 ] n . The spectral properties, crystal structure and antitumor activity also have been investigated. Further investigation of the property and application of Ca(II) coordination polymer are currently in progress in our laboratory. | v3-fos-license |
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} | pes2o/s2orc | Membrane binding mechanism of an RNA virus-capping enzyme.
The RNA replication complex of Semliki Forest virus is bound to cytoplasmic membranes via the mRNA-capping enzyme Nsp1. Here we have studied the structure and liposome interactions of a synthetic peptide (245)GSTLYTESRKLLRSWHLPSV(264) corresponding to the membrane binding domain of Nsp1. The peptide interacted with liposomes only if negatively charged lipids were present that induced a structural change in the peptide from a random coil to a partially alpha-helical conformation. NMR structure shows that the alpha-helix is amphipathic, the hydrophobic surface consisting of several leucines, a valine, and a tryptophan moiety (Trp-259). Fluorescence studies revealed that this tryptophan intercalates in the bilayer to the depth of the ninth and tenth carbons of lipid acyl chains. Mutation W259A altered the mode of bilayer association of the peptide and abolished its ability to compete for membrane association of intact Nsp1, demonstrating its crucial role in the membrane association and function of Nsp1.
The RNA replication complex of Semliki Forest virus is bound to cytoplasmic membranes via the mRNA-capping enzyme Nsp1. Here we have studied the structure and liposome interactions of a synthetic peptide 245 GSTLYTESRKLLRSWHLPSV 264 corresponding to the membrane binding domain of Nsp1. The peptide interacted with liposomes only if negatively charged lipids were present that induced a structural change in the peptide from a random coil to a partially ␣-helical conformation. NMR structure shows that the ␣-helix is amphipathic, the hydrophobic surface consisting of several leucines, a valine, and a tryptophan moiety (Trp-259). Fluorescence studies revealed that this tryptophan intercalates in the bilayer to the depth of the ninth and tenth carbons of lipid acyl chains. Mutation W259A altered the mode of bilayer association of the peptide and abolished its ability to compete for membrane association of intact Nsp1, demonstrating its crucial role in the membrane association and function of Nsp1.
Electron microscopic studies of Alphavirus-infected cells have revealed specific cytoplasmic structures designated as cytoplasmic vacuoles (1,2). Electron microscopic autoradiography suggested that cytoplasmic vacuoles might be the sites of virus-specific RNA synthesis (2). Later it was shown that cytoplasmic vacuoles are modified endosomes and lysosomes (3,4). Similar structures have been detected in rubella virus-infected cells (5,6). The following two obvious questions arose. How was the replication complex bound to the membrane, and how was it targeted specifically to the endo/lysosomal membranes? To answer these questions, we have expressed the virus-specific RNA replicase components, i.e. the nonstructural proteins Nsp1-4 of Semliki Forest virus (SFV) 1 in different cells (7,8). Of these proteins only Nsp1 (537 amino acids) was found to attach to the plasma membrane and to some extent also to endosomal/lysosomal membranes (8 -10). The strength of membrane association of Nsp1 was as strong as that of integral membrane proteins due to post-translational palmitoylation of the cysteine residues 418 -420. Mutation of these cysteines to alanines resulted in peripheral-type membrane association (10). Surprisingly, infection of cells with SFV coding for nonacylated Nsp1 resulted in a normal virus production appearance of typical cytoplasmic vacuole structures (11).
Membrane binding of Nsp1 in the absence of acylation was studied in more detail by producing the protein either in Escherichia coli or by translation in vitro (12). Flotation tests in discontinuous sucrose gradients indicated that the synthesized protein was associated with the plasma membrane of E. coli. When the membrane was solubilized with detergents, Nsp1 lost its methyltransferase and guanylyltransferase activities (13)(14)(15). Both activities were restored upon the addition of either liposomes or detergent micelles containing phosphatidylserine or other anionic phospholipids. To map the membrane binding domain within Nsp1, we used deletion and point mutagenesis and in vitro translation in the presence of phosphatidylserine-containing liposomes. By these means, a specific lipid binding region in the middle of Nsp1 was identified. A synthetic peptide corresponding to residues 245-264 was able to compete for the binding of in vitro translated Nsp1 to liposomes (12).
Here we have studied the mechanism of membrane association of the putative membrane binding peptide of Nsp1 and its mutant derivatives using fluorescence and circular dichroism spectrometry. The three-dimensional structure of the peptide was determined by NMR spectroscopy. The results suggest that the membrane association of the peptide and, thus, that of Nsp1 is mediated by polar interactions between positively charged amino acid residues and the negatively charged head groups of the anionic phospholipids. Also, hydrophobic interactions, particularly those between the single tryptophan residue and the lipid acyl chains, were critical. We expect that other positive-strand RNA viruses have similar mechanisms for membrane binding of their replication complexes (see 12).
Peptide Synthesis-The peptides were synthesized at the Division of Biochemistry, Department of Biosciences and at the Haartman Insti-* This work was supported by Academy of Finland Grant 8397 and by the Technology Development Center (TEKES). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The tute Peptide and Protein Laboratory of the University of Helsinki using the Applied Biosystems model 433A peptide synthesizer and 9-fluorenylmethoxycarbonyl chemistry. The peptides were purified on a octadecylsilica reverse-phase column, and their purity and mass were checked by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.
Preparation of Liposomes-The lipids dissolved in chloroform/methanol (9:1) stocks were mixed in small glass tubes and dried under nitrogen flow. Any residual solvent was removed by keeping the residue in a vacuum desiccator for 1 h. The lipids were hydrated by adding 5 mM Tris, pH 7.5, and sonicated on ice with a Branson sonicator (50 W output) equipped with a microtip until the solution clarified (ϳ10 min). The resulting small vesicles were stored at 4°C and used the following day. Multilamellar liposomes for the flotation assays were prepared by vortexing the dried lipids into 50 mM Tris buffer, pH 7.5, containing 50 mM NaCl.
Protein Expression and Flotation Analysis-Combined in vitro transcription-translation was performed with the TnT T7 kit (Promega) using an appropriate circular plasmid containing the Nsp1 gene under the control of the T7 promoter. Nsp1 was translated in vitro in the presence of multilamellar liposomes (1.67 mM lipids), and the translation mixtures were subjected to flotation in discontinuous sucrose gradients (12). The sucrose solutions were prepared in a buffer containing 100 mM NaCl and 50 mM Tris, pH 7.5. When a synthetic peptide (0.5 mM) was present in the translation mixtures, it was also included in the 67, 60, and 50% sucrose layers at 50 M concentration. The gradients were centrifuged overnight at 35,000 rpm in a SW50.1 rotor (Beckman) at 4°C. The vesicles and the associated proteins floated to the top of the 50% sucrose layer, whereas the soluble proteins remained in the 60% layer. Fractions were collected from the top and analyzed by SDSpolyacrylamide gel electrophoresis followed by autoradiography.
Fluorescence and CD Spectroscopy-The fluorescence spectra were recorded on a PTI QuantaMaster fluorescence spectrophotometer with the emission and excitation slits set to 2 nm. The samples were kept in a 1-cm quartz cuvettes thermostatted to 25°C, and they contained 5.0 M peptide and 100 M lipid in 5 mM Tris buffer, pH 7.5, in a total volume of 2 ml. CD spectra were obtained at 25°C with a Jasco 720 spectropolarimeter equipped with a 1-mm quartz cell. The scan rate was 50 nm/min, and 10 spectra were averaged. The samples were in a total volume of 200 l in 5 mM Tris buffer, and the peptide and lipid concentrations were 50 M and 1 mM, respectively, unless otherwise indicated.
NMR Spectroscopy-Samples of freeze-dried peptide were dissolved in D 3 -trifluoroethanol/H 2 O (7:3, v/v) to obtain 1 mM solution. The samples were loaded to 220 l Shigemi micro cells and pH (not corrected for the deuteron or solvent effects) was adjusted to 6.0 by adding dilute HCl. The NMR spectra were acquired with 600 and 800 MHz Varian Unity NMR spectrometers. Homonuclear two-dimensional spectra, correlation spectroscopy (COSY), relayed COSY, total correlation spectroscopy (TOCSY, with mixing times of 30, 55, and 180 ms), and nuclear Overhauser enhancement (NOESY, with mixing times of 50, 100, 200, and 400 ms) data were collected at 10°C and processed using the Felix 98 software.
Structure Generation-Spin systems were assigned from the through-bond correlation spectra, and the sequence-specific assignments were deduced from the NOEs between the adjacent spin systems. Distance restraints were extracted from the homonuclear NOE data by fitting a second-order polynomial to integrated cross-peak volumes of the NOE series. The intra-methylene and helical NH i -NH i ϩ1 NOEs were used for the calibration. Distances were given with ϩ30 and Ϫ40% uncertainties. When a distance could not be extracted from the build-up curve, either owing to an overlap, a poor signal-to-noise ratio, or other disturbances, the distance was restrained to be not more than 5.0 Å. The upper bounds were extended by 1.0 Å for each pseudo-atom in methyl groups and 1.5 Å for pseudo-atoms in aromatic rings. Backbone -dihedral angles characterized by small JH N H␣, measured from the correlation (COSY) spectra, were restrained to the helical conformation (Ϯ30 degrees) on the basis of the Karplus relation. Dihedral angles characterized by intermediate JH N H ␣ or hydrogen bonds were not constrained. The spectra were assigned, and the distance restraints were generated using the Felix 98 software. Structures were generated by DYANA 1.5 using the torsion angle dynamics approach (18). A final set of structures was analyzed for root mean square density, energy, and backbone dihedrals. The coordinates and NMR restraint files of Semliki Forest virus Nsp1 membrane binding peptide will be deposited to the Protein Data Bank (code 1FW5).
Inhibition of Membrane Binding of Nsp1 by Synthetic
Peptides-The peptide corresponding to the previously assigned membrane binding segment (G 245 STLYTE-SRKLLR-SWHLPSV 264 ) of SFV Nsp1 (12) and three variant peptides with single amino acid replacements (R253E, K254E, and W259A) were synthesized. In addition, a peptide containing the same amino acids as the wild type, but in a random sequence (Fig. 1B), was prepared. Secondary structure prediction (19,20) suggested an ␣-helical conformation for residues Thr-250 -Ser-258 of the wild type peptide. At pH 7.5, the net charge of the wild type, the W259A variant, and the random peptide should be ϩ2, and that of R253E and K254E variants should be close to zero.
The wild type peptide, variants W259A and R253E, and the random peptide were tested for their ability to compete with Nsp1 for binding to negatively charged multilamellar liposomes. Nsp1 was translated in vitro in the presence of PS:PC (1:1) liposomes and one of the peptides. The fraction of Nsp1 associating with the vesicles was then determined by subjecting the samples to centrifugation in discontinuous sucrose gradients (12). The wild type peptide inhibited the association of Nsp1 with liposomes by 64% (Fig. 2C), whereas the random peptide (Fig. 2B), W259A (Fig. 2D), or R253E (not shown) variant peptides did not inhibit the binding of Nsp1 to liposomes.
As can be seen in Fig. 2, a relatively large amount of wild Rost and Sander (19,20). The bold region of SFV wild type peptide represents ␣-helix in the presence of trifluoroethanol, as indicated in Fig. 8. SAG, Sagiyma; EEE, eastern equine encephalitis; WEE, western equine encephalitis; Igbo, Igbo Ora; SIN, Sindbis; Aura, Barmah, Barmah Forest; RRV, Ross River; ONN, O'nyon-nyong. B, previously identified mutations in the binding peptide region of SFV Nsp1 protein that affect the liposome binding of in vitro synthesized Nsp1 protein and its methyltransferase activity, as determined previously (12). The bold peptide sequences have been synthesized chemically and used in this study. The random peptide consisted of the same amino acids as the SFV wild type (wt) peptide. type peptide was required to successfully compete with the binding of Nsp1 to the liposomes. In these experiments the surface of the liposomes must be covered by the peptide to obtain inhibition, and the liposomes were in excess to the in vitro translation product.
Binding of the Peptides to Lipid Vesicles with Varying Charge-To study association of the peptides with membranes of varying charge, we made use of tryptophan fluorescence properties (21,22). The addition of PC vesicles containing 30 mol % of a negatively charged phospholipid, i.e. phosphatidylglycerol, PS, or phosphatidic acid, caused a major blue shift of tryptophan emission and a marked increase in the fluorescence intensity (Fig. 3). In contrast, a minor shift of tryptophan emission was observed upon the addition of vesicles consisting of zwitterionic phospholipids only. Since a blue shift and an increase in fluorescence intensity are considered to indicate penetration of a tryptophan residue into a lipid bilayer (e.g. Refs. 21 and 23), these data provide strong evidence that the wild type peptide binds avidly to negatively charged membranes but not to uncharged membranes. The variant peptides behaved similarly, except that the effects on the fluorescence parameters were somewhat less pronounced (see below).
To study further the effect of the density of negative charge on membranes for peptide association, the PS content of the vesicles was varied from zero to 50 mol % (Fig. 4A). With the wild-type peptide, the blue shift increased until PS content reached 35-40 mol % and then leveled off. With the variants, a threshold concentration of about 10 mol % of PS was necessary before any significant shift was observed. In addition, the blue shift was markedly smaller than with the wild type at all PS concentrations (Fig. 4A). The effect of PS content on the intensity of tryptophan fluorescence is shown in Fig. 4B. In case of the wild-type peptide, the intensity increased linearly with PS content until 20 -30 mol % and then declined. With the vari-ants, a minimum of ϳ10 mol % of PS was again required to obtain a significant effect, and the maximal effect was much smaller than with the wild type (Fig. 4B). As with the wild type peptide, the intensity decreased above 30 mol % of PS. The reason for this is not clear, but obviously it does not indicate that less peptide would be binding to the vesicles, since the blue shift did not level off until 40 -50 mol % (Fig. 4A). This decrease could result from self-quenching tryptophan-tryptophan interactions that become significant when the surface concentration of the peptide increases with increasing PS content. The addition of increasing amounts of NaCl in the peptide-PC/PS-vesicle solution diminished the blue shift and abolished it completely when the concentration of NaCl reached 100 mM (Fig. 4C). The effect of NaCl concentration was similar with the wild type and the variant peptides. These data support the importance of ionic interactions for the binding of the peptides to negatively charged membranes.
Depth of Tryptophan Penetration to Membrane-The blue shift observed upon interaction of the peptides with the negatively charged vesicles indicates that the tryptophan moiety penetrates into the vesicle bilayer (23). To estimate the depth of penetration, we made use of PC species containing a brominated fatty acid in the sn-2 position. Bromine quenches tryptophan fluorescence by a collision mechanism, and therefore, it is possible to obtain information on the depth of tryptophan penetration by using a set of phospholipids with the bromines attached to different acyl carbons (23)(24)(25)(26)(27)(28). Accordingly, we prepared vesicles consisting of POPS (50 mol %) and a PC species (50 mol %) with bromines in carbons 6 and 7 (6,7dibromo-PC), 9 and 10 (9,10-dibromo-PC), or 11 and 12 (11,12dibromo-PC) of the sn-2 acyl chain, mixed them with the peptide, and then recorded the tryptophan emission spectrum. As shown in Fig. 5A, the tryptophan fluorescence of the wild-type peptide was quenched most effectively with 9,10-dibromo-PC. Quenching by the 6,7-and 11,12-dibromo-PC species was clearly less efficient. These findings indicate that the tryptophan of the wild-type peptide penetrates to a depth of the bromines attached to carbons 9 and 10 of the PC sn-2 acyl chain. The 9,10-dibromo-PC species was also the most efficient quencher of K254E (Fig. 5B) and R253E (Fig. 5C) variants. However, the fluorescence of the variants was quenched less efficiently with each dibromo-PC species than that of the wild type peptide. This finding implies that the tryptophan residue in the variants penetrates to a similar depth as that of the wild type, but the fraction of the peptide bound to the vesicles was smaller than in the case of the wild type peptide.
Effect of Peptides on the Lateral Mobility of PS and PC-To
study the effect of the peptides on the mobility/lateral organization of the vesicle lipids, we included 5 mol % of either pyrene-labeled PS (PyrPS) or PC (PyrPC) in PS/PC (3:7) vesicles and then determined the effect of the peptides on the pyrene excimer to monomer fluorescence intensity ratio (E/M). The E/M ratio is an indicator of the lateral mobility of the pyrene phospholipid (16,17). Mixing of the wild type peptide with vesicles containing PyrPS caused a significant (10%) decrease in the E/M ratio (Fig. 6, A and B). This phenomenon was not seen with the variant peptides (Fig. 6B). In contrast, a slight increase in the E/M ratio was observed when the wild type peptide or the variant peptides were mixed with vesicles containing PyrPC (Fig. 6C). These results suggest that the wild type peptide interacts preferentially with PS molecules, thus selectively retarding their lateral mobility, whereas interaction of the variant peptides with PS-containing vesicles was weaker. It is intriguing that the W259A variant, even if it contains the same positively charged amino acid residues as the wild type, had no effect on the E/M ratio of PyrPS-containing vesicles.
Conformational Changes-CD spectroscopy was used to determine whether the peptides undergo a conformational change upon interaction with the lipid vesicles. In buffer or in the presence of PC vesicles, all peptides gave spectra with a minimum close to 200 nm and an overall shape suggesting a random coil conformation, as shown for the wild type peptide in Fig. 7A. When PS was included in the vesicles, the spectrum changed remarkably, with new minima appearing at 208 and 220 nm (Fig. 7A). These changes imply that the peptide adopts predominantly an ␣-helical conformation in the presence of PS-containing vesicles. A maximal effect was obtained when the concentration of PS reached 20 -30 mol % (Fig. 7A). Identical results were obtained when PS was replaced by another acidic phospholipid such as phosphatidic acid or phosphatidylglycerol but not when it was replaced by the zwitterionic phospholipid PE. Similar results were obtained for the variant peptides R253E, K254E, and W259A (data not shown), whereas the CD spectrum of the random peptide was not significantly affected in the presence of 50 mol % of either PS or PC in the vesicles (Fig. 7C).
The CD spectroscopy of the peptides was done also in trifluoroethanol, which had to be used for the NMR studies (29). A gradual change from a random coil to an ␣-helical conformation was observed when increasing amounts of trifluoroethanol was added to the wild type peptide in buffer (Fig. 7B). The maximal effect was obtained already with 30% trifluoroethanol. With the variant peptides, similar results were obtained (data not shown). These results indicate that the conformation of the peptides in 30% trifluoroethanol is very similar to that of the liposome-bound peptides.
Structure of the Wild Type Peptide-NMR spectroscopy was used to assess the structure of the wild type peptide. In water, the wild type peptide was predominantly in a random-coil conformation as indicated by the typical crowding of the NH resonances in fast exchange with water (30). This finding agrees with the CD data (see above). We attempted to determine the structure of the wild type peptide in the presence of lipid vesicles, but this was not feasible due to excessive spectral broadening. This broadening, observed also in the presence of SDS micelles, probably resulted from aggregation of the peptide in the presence of the vesicles or with vesicles. Such vesicle-induced peptide aggregation has been observed previously (31).
Because of these difficulties, we decided to perform the NMR measurements in trifluoroethanol/water (3:7 v/v) solution. According to CD measurements (see above) the peptide adopts a similar, i.e. a largely ␣-helical conformation in trifluoroethanol/ water solutions, as in the presence of negatively charged liposomes. The three-dimensional structure of the wild type pep-tide in 3/7 (v/v) trifluoroethanol/water is shown in Fig. 8. The overall structure is compatible with the CD data, indicating that in the presence of trifluoroethanol the peptide forms a slightly bent ␣-helical structure. This helix has an obvious amphiphilic character. One face consist of the hydrophobic residues Leu-248, Leu-255, Leu-256, Leu-261, Val-264 and residues Ser-252 and Trp-259 (Fig. 8B). The other face consists of polar residues mainly, i.e. the positively charged residues Arg-253, Lys-254, Arg-257 and the negatively residue Glu-251. The positively charged residue Arg-253, shown to be important for the interaction with negatively charged membranes (see above), lies close above the hydrophobic cluster consisting of residues Leu-55, Leu-256, and Trp-259. The hydrophobic surface of the peptide is well defined, whereas the hydrophilic surface is less so due to considerable mobility of the amino acid side chains (Fig. 8C). DISCUSSION Recently, we identified a short sequence of the Semliki Forest virus RNA-capping protein Nsp1 that appeared to be responsible for the binding of the protein to the E. coli plasma membrane and to anionic liposomes (Fig. 1B) (12). Here, we have studied the mechanism of membrane binding of a synthetic peptide corresponding to the binding sequence of Nsp1. Parallel studies were carried out with variant peptides with single amino acid replacements corresponding to Nsp1 mutant proteins that lacked enzymatic activity and had a reduced membrane affinity (Fig. 1). All the peptides occurred as a random coil in buffer solutions but attained a partly ␣-helical conformation in the presence of anionic liposomes as well as in 30% trifluoroethanol. This conformational change may reflect the behavior of the wild type Nsp1 protein, which is enzymatically active in the presence of membranes or mixed micelles of detergent and anionic phospholipids but loses activity in the presence of detergents (12). The structure of the wild type binding peptide, determined by NMR spectroscopy in 30% trifluoroethanol, revealed an amphiphilic ␣-helix in which hydrophobic residues concentrate on one side and the polar residues residues concentrate on the other (Fig. 8). As similar CD spectra were obtained both in trifluoroethanol and in the presence of liposomes, we suppose that the NMR structure of the binding peptide is similar in both environments, with the possible exception of polar residues as discussed below.
The tryptophan fluorescence data provide strong evidence that the wild type peptide binds avidly to vesicles containing negatively charged phospholipids but not to vesicles consisting of zwitterionic phospholipids only. The positively charged amino acid residues of the peptide obviously play an important role in this interaction, since the R253E and K254E variants appeared to interact differently with negatively charged vesicles. Other findings supported the importance of ionic interactions in the association of the peptide with membranes. First, the peptide is released from the vesicles upon the addition of salt (Fig. 4C). Second, it adopts an ␣-helical conformation only in the presence of negatively charged but not uncharged vesicles (Fig. 7). Third, the wild type peptide reduces lateral diffusion (excimer formation) of PyrPS but not that of PyrPC (Fig. 6). NMR analysis of the wild type peptide indicated that the side chains of Arg-253 and Lys-254 are not well localized (Fig. 8C), suggesting that they are quite mobile and thus capable of interacting with acidic phospholipids in vivo. We assume that Arg-257 would also interact with anionic phospholipids, since mutation R257E inhibits the enzymatic activities of Nsp1 protein and its binding to liposomes (12).
A marked blue shift and enhancement of tryptophan fluorescence of the wild type peptide was observed upon addition of negatively charged vesicles, indicating that the tryptophan moves to a less polar milieu (22,32). This conclusion is supported by the efficient quenching of the tryptophan fluorescence by bromines attached to a PC acyl chain. The quenching data indicated that tryptophan 259 of the wild type peptide penetrates approximately to the level of 9 -10 carbon atoms of the acyl chains. Placing tryptophan 259 to this level would bring leucines 248, 255, 256, and 261 as well as Val-264 in contact with the acyl chains. Assuming that the helix of wild type peptide lies nearly parallel to the bilayer surface, one can estimate that the maximum distance from the charged side chain groups of Arg-253 to the ␣-carbon of Leu-256 is about 10 Å and about 12-15 Å to the aromatic carbons of Trp-259. These estimates comply with the tryptophan quenching data (Fig. 5) placing the tryptophan moiety close to the acyl carbons 9 and 10, i.e. 12 and 13.5 Å below the phospholipid polar head groups.
This type of membrane association of proteins, i.e. only one leaflet of a lipid bilayer has been designated as monotopic (33). Alignment of Nsp1 s of the Alphavirus family suggests that a similar amphipathic ␣-helix is present in each protein (34). The amino acid residues that form the hydrophobic surface of the Nsp1 wild type peptide are conserved, as is the critical arginine residue 253 (Fig. 1). Thus, all Alphavirus Nsp1 proteins most probably have a similar monotopic membrane binding mechanism. Several other proteins seem to associate with membranes monotopically. One of the best characterized among these is prostaglandin H synthase, which binds to the endoplasmic reticulum membrane via four short amphipathic helices with hydrophobic surfaces interacting with the outer leaflet of the lipid bilayer (35,36). CTP:phosphocholine cytidyltransferase (EC2.7.7.15) associates with membranes via an amphipathic ␣-helical peptide (23,37,38). The binding of human coagulation factors V and VIII to phosphatidylserine-rich platelet membranes is mediated by a C2 domain that is homologous in both proteins. The crystal structures of the C2 domains of factors V and VIII determined recently revealed finger-like loop structures that enable hydrophobic interactions within the outer leaflet of the lipid bilayer as well as polar interactions with the phosphatidylserine head groups (39,40).
The importance of tryptophan 259 for the membrane associ-FIG. 8. Solution structure of wild type peptide. A, energetically most preferred conformation from an ensemble of 15 out of 20 calculated structures. B, surface presentation of the wild type peptide in the amino acid residues are color-coded according to their hydrophobicity; blue represents hydrophobic residues and red represents hydrophilic residues. C, conformational ensemble of 15 out of 20 calculated structures. The backbone root mean square deviation for the structure ensemble is 0.62 Å and the overall heavy atom root mean square deviation is 1.19 Å. 304 NOE distances and 17 angle restraints were used in the calculation.
ation of Nsp1 was confirmed by the finding that its replacement by alanine diminished the ability of the peptide to retard lateral diffusion of PyrPS and abolished binding of intact Nsp1 to membranes (12). Variant peptide W259A also failed to compete with binding of wild type Nsp1 protein to liposomes (Fig. 2D). It is becoming increasingly apparent that tryptophan plays an important role in membrane association of many cellular proteins, such as phospholipases (41)(42)(43)(44). Notably, replacement of valine by tryptophan (V3W) in human phospholipase PLA 2 resulted in 250-fold enhancement of the activity due to enhanced membrane association (41). Two of the amphipathic helices of prostaglandin H synthases have tryptophans in the hydrophobic surface (36). The hydrophobic loops of factors V and VIII described above also have tryptophans. Tryptophan appears to possess several characteristics (including aromaticity) that make its disposition at the membrane interphase particularly favorable (for a detailed discussion, see Ref. 45).
In conclusion, we have shown that the binding peptide of Nsp1 attains an ␣-helical conformation in the presence of anionic liposomes. Its structure in the presence of trifluoroethanol reveals an amphipathic ␣-helix with a protruding tryptophan residue and a hydrophobic surface, which interact with acyl chains in the liposomes. Tryptophan 259 is necessary for proper binding of the peptide to lipids. Residues Arg-253, Lys-254, and probably also Arg-257 are needed for the interaction with head groups of anionic phospholipids, indicating that lipid binding of the peptide and most probably also of the entire Nsp1 protein is a finely tuned process perhaps initiated by polar interactions that lead to conformational change, allowing hydrophobic interactions to take place (cf. Ref. 45). Our intention is to introduce the present mutations of the binding peptide to the infectious clone of SFV to analyze the localization of the replication complex, putative infectivity, and pathogenicity of the transcribed RNAs.
In virus-infected cells, Nsp1 protein is found attached to the cytoplasmic side of the plasma membrane (8, 10) containing high concentrations of phosphatidylserine (46). High concentrations of phosphatidylserine are also needed to restore the methyltransferase activity of detergent-inactivated isolated Nsp1 (12) and for the optimal binding of the synthetic peptide to phospholipid bilayers (this study). Thus, we propose that membrane binding, enzymatic activation and intracellular targeting of Nsp1, and consequently, the entire RNA polymerase complex is regulated by this monotopic binding peptide. This proposition is supported by the RNA synthesis of Sindbis virus, another Alphavirus, is severely inhibited in the Chinese hamster ovary cell mutants with a lowered PS content (47). | v3-fos-license |
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} | pes2o/s2orc | Neonatal Fc receptor is involved in the protection of fibrinogen after its intake in peripheral blood mononuclear cells
Background Fibrinogen is a central player in the blood coagulation cascade and one of the most abundant plasma proteins. This glycoprotein also triggers important events (e.g., cell spreading, the respiratory burst and degranulation) in neutrophil cells via a αMβ2 integrin-mediated binding to the cell surface. Yet, little is known about the interaction of fibrinogen with leukocytes other than neutrophils or stimulated monocytes, although high amounts of fibrinogen protein can also be found in lymphocytes, particularly in T-cells. The aim of the present work is to unveil the dynamics and the function of fibrinogen intake in T-cells. Methods Using the Jurkat cell line as a T-cells model we performed fibrinogen intake/competition experiments. Moreover, by means of a targeted gene knock-down by RNA-interference, we investigated the dynamics of the intake mechanism. Results Here we show that (i) fibrinogen, although not expressed in human peripheral blood mononuclear cells, can be internalized by these cells; (ii) fibrinogen internalization curves show a hyperbolic behavior, which is affected by the presence of serum in the medium, (iii) FITC-conjugated fibrinogen is released and re-internalized by adjacent cells, (iv) the presence of human serum albumin (HSA) or immunoglobulin G (IgG), which are both protected from intracellular degradation by the interaction with the neonatal Fc receptor (FcRn), results in a decreased amount of internalized fibrinogen, and (v) FcRn-knockdown affects the dynamics of fibrinogen internalization. Conclusions We demonstrated here for the first time that fibrinogen can be internalized and released by T-lymphocyte cells. Moreover, we showed that the presence of serum, HSA or IgG in the culture medium results in a reduction of the amount of internalized fibrinogen in these cells. Thus, we obtained experimental evidence for the expression of FcRn in T-lymphocyte cells and we propose this receptor as involved in the protection of fibrinogen from intracellular lysosomal degradation. Electronic supplementary material The online version of this article (10.1186/s12967-018-1446-2) contains supplementary material, which is available to authorized users.
Background
Fibrinogen is one of the most abundant plasma proteins, with a concentration of about 10 μM (340 mg/dl) in healthy subjects [1]. This plasma glycoprotein is synthesized mainly in hepatocytes through the secretory pathway and it is comprised of two sets of three polypeptide chains (namely Aα, Bβ and γ) of 610, 461 and 411 residues, respectively, joined together by disulfide bridges [2]. Fibrinogen plays a central role in the blood coagulation cascade, which is triggered by the conversion of fibrinogen to fibrin by limited proteolysis. Additionally, fibrinogen expression is induced by IL-6 as part of the acute phase reaction. Eventually, fibrinogen participates, together with fibrin, in several biological processes including fibrinolysis, cellular and matrix interactions, inflammation and wound healing [1][2][3].
In severe inflammatory conditions the plasma concentration of fibrinogen can increase up to 10 mg/ml [4] and this is necessary for processes other than coagulation. Indeed, fibrinogen binds to integrin α M β 2 (Mac-1) on both neutrophil and stimulated monocyte cells surface, thus mediating enhanced binding of leukocytes at sites of damaged endothelium and promoting leukocyte extravasation [4][5][6]. The binding of fibrinogen to neutrophil β 2 integrin receptors induces the tyrosine phosphorylation of neutrophil proteins which, in turn, provides the signal for the initiation of various important cellular events, such as cell spreading, the respiratory burst and degranulation [7]. Eventually, it has been suggested that the interaction of fibrinogen with neutrophils may also lead to its degradation after internalization by non-specific pinocytosis [7].
Interestingly, fibrinogen protein β and γ chains have been also found in isolated lymphocytes [8], particularly in T-cells [9], in which the observed amounts were relevant when compared to the most abundant cytoskeletal protein β-actin. Nevertheless, to date nothing is known about the interaction of fibrinogen with leukocytes other than neutrophils and monocytes.
The neonatal Fc receptor (FcRn) has been initially identified as the receptor involved in the IgG transmission from mother to offspring [10,11]. Subsequently, it has been shown that FcRn is expressed in many tissues and cell types beyond neonatal life [12], including polarized epithelia (intestines, lung, breast, kidney) as well as parenchymal cells (hepatocytes, endothelial cells and hematopoietic cells) [13,14]. FcRn receptor has been extensively characterized, thus unveiling important roles in several biological functions. Indeed, FcRn transports IgG across epithelia [15][16][17], provides passive immunity to the newborn and also participates in the development of the adaptive immune system [18,19] and is deeply involved in the intracellular trafficking of IgG through the endolysosomal pathway [20,21]. In particular, FcRn binds IgG with high affinity at low pH, a way to prolong IgG half-life by preventing in part their lysosomal degradation [22][23][24].
It is interesting that FcRn was recognized as the receptor of albumin, another plasma protein with long half-life [23,25]. In this case albumin is internalized by pinocytosis and subsequently bound by FcRn in the acidic pH environment of the early endosome, thus rescuing it from degradation when the complex migrates to the lysosome [26]. Albumin is then released by exocytosis in the extracellular space, where the neutral pH counteracts its binding to the FcRn receptor [27].
Here, we report on the dynamics of internalization of fibrinogen within lymphocyte cells and demonstrate that FcRn is involved in the rescue of fibrinogen from lysosomal degradation.
Non-T and T-cells fractions preparation from peripheral blood
Five volunteers have been enrolled by the Department of Neuroscience, University of Torino and signed an informed consent before being recruited, following approval by the Institutional Review Board of the University Hospital and according to the Declaration of Helsinki. The subjects (3 females and 2 males; average age: 54 ± 9 years) underwent a venous blood sampling (20 ml) from the antecubital vein, between 9 and 10 a.m., after an overnight fast. The five subjects declared no inflammatory diseases and/or drug treatments within 2 weeks before sampling. Whole blood was collected into vacuum tubes containing EDTA, diluted with 50 ml of phosphatebuffered saline (PBS) and stratified in two 50 ml tubes on top of 15 ml of Lympholyte ® -H (Cedarlane, Burlington, Canada) each. After centrifugation (800×g, 20 min, 20 °C), peripheral blood mononuclear cells (PBMCs) were collected, centrifuged at 400×g, 15 min, 20 °C and washed twice with 10 ml of magnetic-activated cell sorting (MACS) buffer (Miltenyi Biotec, Cologne, Germany). The isolation of T-cells was achieved by MACS with the Pan T cell isolation kit (Miltenyi Biotec) using the manufacturer's protocol. The non-T fractions (all PBMC but T-cells) have been also collected.
Cells and treatments
The Jurkat T-cell leukemia cells (kindly provided by Prof. Jean-Pierre Mach, University of Lausanne, Switzerland) were maintained at 37 °C in a 5% CO 2 humidified atmosphere in RPMI 1640, supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine. Human neuroblastoma SH-SY5Y (ECACC 94030304) cell line was maintained at 37 °C in a 5% CO 2 humidified atmosphere in DMEM, supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ ml streptomycin, and 2 mM l-glutamine. All cell culture media and reagents were from Euroclone (Pero, Milano, Italy). Stock flasks were transferred for culture twice weekly or as required to maintain optimal cell growth.
For the concomitant incubation with fibrinogen and human serum albumin (HSA), IgG, hemoglobin or catalase, Jurkat cells have been incubated for 4 h with 0.4 mg/ ml fibrinogen and 0.4 mg/ml HSA, 50 µg/10 6 cells of IgG, hemoglobin or catalase, respectively.
Enzymatic protein deglycosylation was performed using the PNGase F enzyme, following the manufacturers' instructions (EDEGLY kit, Sigma-Aldrich). Briefly, total cell lysates from SH-SY5Y cells were clarified by protein precipitation (acetone/methanol) and proteins were resuspended in 200 mM HEPES/NaOH. After protein quantification, 100 µg of total proteins were used for the deglycosylation reaction (24 h, 37 °C).
Co-culture and flow cytofluorimetric analysis (FACS)
Jurkat or SH-SY5Y cells were loaded with Fibrinogen-FITC (Axxora, NZY-F006) for 4 h at 37 °C (0.4 mg/ml in RPMI medium w/o FBS). Loaded cells were washed three times with RPMI w/o FBS and the fibrinogen uptake was analyzed by flow cytometry (EPICS XL flow cytometer) and EXPO32 analysis software (Beckman Coulter, Pasadena, California) at different time points as previously described [28].
For co-culture experiments, 20% of untreated Jurkat cells were mixed with 80% of Fibrinogen-FITC-loaded Jurkat cells in RPMI medium w/o FBS. Cells were analyzed by flow cytometry as indicated above.
RNA-interference assay
SH-SY5Y cells were seeded in 12-well plate and transfected with Lipofectamine ® MessengerMAX ™ reagent (Life Technologies, Carlsbad, California) using 100 nM siRNAs (final concentration) according to the manufacturer's instructions.
When incubation with either HSA or fibrinogen was performed, 44 h after transfection the cells were starved in fresh serum-free medium for 1 h and then incubated with fibrinogen or HSA (0.4 mg/ml in DMEM w/o FBS) for 3 h. After incubation (48 h after transfection), the cells were washed in ice-cold PBS and harvested for western blotting and qRT-PCR assays. Three independent knock-down experiments were performed.
RNA extraction, reverse transcription and PCR
Total RNA was extracted from SH-SY5Y, T-cells and PBMCs of the same control subject and from Jurkat and HepG2 cells using the ReliaPrep ™ RNA cell miniprep system (Z6011, Promega, Milano, Italy) following the manufacturer's instructions. Two micrograms of DNA-free total RNA were reverse transcribed into first-strand cDNA with random primers in a 20 μl final volume using the GoScript ™ reverse transcription system (Promega, A5000).
Primer design was performed by using Primer3 software (http://primer3.sourceforge.net/) and manually adjusted, if needed, to avoid the amplification of undesired sequences and to have comparable melting temperature and reaction efficiency. Primer specificity was tested by BLAST (http://blast.ncbi.nlm.nih. gov/Blast.cgi) and experimentally by the positive control amplification. Primer pairs sequences: ACTB Fw: The semiquantitative RT-PCR was performed using 20 ng of cDNA as template and the amplification products were separated on a 2% agarose gel stained with ethidium bromide and acquired.
The relative gene-expression qPCR analysis was performed in triplicate using 20 ng of cDNA in 25 μl final volume/well in 96-well plates, using the GoTaq ® qPCR master mix (Promega, A6001) following the manufacturer's instructions. The ABI PRISM 7000 sequence detection system (Thermo Fisher) was used as real-time PCR instrument. Beta-actin (ACTB) was quantified as reference housekeeping gene. The amplification steps were set as follows: a first step at 95 °C for 10 min, 40 cycles (95 °C for 15 s, 60 °C for 1 min) and a final dissociation step (95 °C for 15 s, 60 °C for 20 s, 95 °C for 15 s). The relative expression levels of the FcRn transcript were calculated using the ΔΔCt method. Statistical significance was verified by Student's t-test.
Data analysis
Time-dependent cellular intake of fibrinogen was analyzed using simple exponential association: where Y max is the maximum fibrinogen signal observed in the experiment, k in is the first-order kinetics constant for fibrinogen intake. The cell-bound fibrinogen fraction has been described by a simple equilibrium isotherm: where K d is the fibrinogen concentration needed to reach half-saturation and [Fib] is the fibrinogen concentration.
Fibrinogen is present in PBMCs but is not synthesized by these cells
In order to assess the presence of fibrinogen in peripheral blood mononuclear cells (PBMCs), these cells have been isolated from peripheral blood of donors and then fractionated into two major components (defined here as T and non-T cells).
A western blot analysis (Fig. 1a) has been performed on total cell lysates and fibrinogen resulted to be present in both the T-and the non-T cell fractions, the latter being mainly composed by B-cells, natural killer (NK) cells and monocytes. On the contrary, fibrinogen protein was not present in the human T-cell leukemia Jurkat cell line.
Thus, we verified whether the Fibrinogen β-chain (FGB) transcript was expressed in PBMCs by performing a semiquantitative RT-PCR, using the human hepatocellular carcinoma HepG2 cell line as a positive control. As shown in Fig. 1b, fibrinogen β chain was not expressed in PBMCs, thus suggesting the exogenous derivation of the protein.
Fibrinogen intake in Jurkat cells shows a hyperbolic behavior and is affected by the presence of serum in the culture medium
Since fibrinogen protein was abundantly present in PBMCs, but not expressed by these cells, we decided to assess whether the presence of fibrinogen was due to its uptake from the extracellular milieu (i.e., plasma). To this purpose, we used the Jurkat cell line, in which fibrinogen is not expressed, and we cultured these cells in medium supplemented with fibrinogen.
Firstly, we investigated the thermodynamics and kinetics aspects of the possible intake. Jurkat cells were incubated with increasing doses of fibrinogen for 4 h, to determine the intake equilibrium. The experiments were performed either in the presence or in the absence of serum in the culture medium and, as shown in Fig. 2a, fibrinogen was incorporated into Jurkat cells and the intake showed a hyperbolic behavior, consistent with a simple equilibrium. Intake curves were generated (Fig. 2b) and the calculated apparent K d in the presence of serum was 1.2 ± 0.1 mg/ml, whereas in the absence of serum an apparent K d of 0.60 ± 0.15 mg/ml was observed.
To assess the intake kinetics, Jurkat cells were then incubated at the same concentration of fibrinogen (0.4 mg/ml) for different time points, either in the presence or in the absence of serum. The amount of internalized protein was quantified by immunoblotting (Fig. 3a). As a result, fibrinogen intake in the absence of serum followed a fast kinetics (k in = 12 ± 6/h) while, in the presence of serum in the culture medium, the intake showed a slower kinetics, with k in = 0.16 ± 0.02/h (Fig. 3b).
Thus, fibrinogen can be internalized by Jurkat cells and the internalization curves show a hyperbolic behavior, which is influenced by the presence of serum in the culture medium.
Fibrinogen is released and re-internalized by Jurkat cells
To assess the fibrinogen fate after internalization, Jurkat cells were incubated with 0.4 mg/ml fibrinogen for 4 h, washed and analyzed after a 24 h recovery period. We observed that fibrinogen was almost completely undetectable after 24 h, thus suggesting that the internalized fibrinogen was either degraded within the cells or protected from degradation and eventually released in the extracellular space.
To verify these hypotheses, we assessed the ability of Jurkat cells to secrete the internalized fibrinogen, and, if this was the case, whether it could be re-uptaken by At time zero (Fig. 4, upper panel), the fluorescence profile of the co-culture was represented by two different cell populations, one FITC-positive (corresponding to the stimulator "S" cells, about 80% of the cells) and one FITCnegative (corresponding to the responder "R" cells, about 20% of the cells). The mean fluorescence (m.f.) values of the two populations in the co-culture corresponded to that measured in the untreated cells and in the fibrinogen-FITC loaded cells cultured alone.
After 2 h (Fig. 4, middle panel) we could appreciate only a slight decrease in the amount of fibrinogen-FITC in the population of stimulator cells, as indicated by the m.f. values at T0 and T2h (64 and 52 arbitrary unit (a.u.), respectively). This reduction was accompanied by a slight shift of the fluorescence profile of the co-culture, with an increase of the m.f. value of the responder cells (from 4 to 6.4 a.u.) and a parallel decrease of the m.f. value of stimulator cells (from 63 to 50 a.u.), thus suggesting a possible exchange of fibrinogen between the two cell populations.
After 19 h (Fig. 4, lower panel), the amount of fibrinogen found in the stimulator cells was significantly lower when compared to that found at T2h, as indicated by the m.f. values (31 a.u. at T19h; 52 a.u. at T2h). Interestingly, in the co-culture we found a single cell population, in which the mean fluorescence was nearly half of that measured at T2h in the FITC-positive cells (29 a.u. with respect to 50 a.u.) and comparable to that found in the stimulator cells at 19 h (m.f. = 31), thus demonstrating that fibrinogen can be secreted and re-uptaken by Jurkat cells.
Human serum albumin (HSA) and immunoglobulin G (IgG) modify fibrinogen internalization in Jurkat cells
Since (i) the internalization of fibrinogen was affected by the presence of serum in the culture medium, and (ii) the internalized fibrinogen was protected from intracellular degradation, released and re-uptaken from adjacent cells, we decided to perform some targeted experiments aimed at clarifying the dynamics of fibrinogen intake, transport and secretion in Jurkat cells.
To this end, we first evaluated the effects of the concomitant incubation of Jurkat cells with fibrinogen and either HSA or IgG. These proteins were selected because they are abundantly present in the serum and also because a well-characterized mechanism of rescue from degradation and recycling, mediated by the neonatal Fc receptor (FcRn), has been recently described for both of them. As shown in Fig. 5, the amount of internalized fibrinogen in Jurkat cells after a 4 h incubation was strongly reduced both in the presence of HSA (Fig. 5a) and IgG (Fig. 5b). Strikingly, this effect was restricted to HSA and IgG, since other non-plasmatic proteins (i.e., catalase and hemoglobin) did not affect the amount of internalized fibrinogen in Jurkat cells (Fig. 5c).
The neonatal Fc receptor (FcRn) is involved in fibrinogen protection in SH-SY5Y cells
Since it has been demonstrated that FcRn is the receptor of both albumin and IgG, shown above to decrease the amount of internalized fibrinogen in Jurkat cells, we hypothesized that FcRn could be responsible for the protection of fibrinogen from lysosomal degradation as well. FcRn resulted to be expressed in all the analyzed cell lines, as well as in T cells isolated from peripheral blood of donors (Fig. 6a, b). In particular, two distinct protein forms were detected in promyelocytic cell lines and in SH-SY5Y cells, with respect to lymphocyte cells (Fig. 6b). Since human FcRn protein can be post-translationally modified by N-glycosylation [29], we assessed whether the difference in molecular mass between the two protein forms was due to the presence of N-glycan moieties. To this end, we performed PNGase F treatment in SH-SY5Y total protein lysates and we observed that a single lower weight protein form was detectable after treatment, thus demonstrating that the higher molecule weight form corresponds to the N-glycosylated FcRn protein (Fig. 6c).
SH-SY5Y cells showed FcRn protein levels higher than Jurkat cells (Fig. 6b) and fibrinogen was neither expressed at the transcript level nor detectable at the protein level in this cell line. Moreover, SH-SY5Y cells, such as Jurkat cells, were shown to be able to uptake and release fibrinogen-FITC (Fig. 6d). Therefore, this cell line was selected for subsequent experiments.
If the FcRn is a receptor protecting fibrinogen from degradation, the absence of FcRn is expected to cause a decrease in the amount of intracellular fibrinogen after internalization. To address this point, we performed FcRn knock-down in SH-SY5Y cells by RNA-interference and verified by qRT-PCR that the transcript level was reduced by 70% (Fig. 7a). Upon transcript depletion, we also observed a significant reduction of FcRn protein (up to 40%), as assessed by western blotting (Fig. 7b, c). Strikingly, in cells incubated with either HSA (positive control) or fibrinogen, FcRn depletion caused a significant reduction in the intracellular amount of both proteins (Fig. 7b, c), thus supporting the notion of FcRn as a receptor for fibrinogen protection as well.
Discussion
Our previous studies clearly indicated the presence of very high levels of fibrinogen protein inside PBMCs [8,9]. This observation was somehow surprising since fibrinogen is a highly abundant protein in plasma, with almost no roles described in peripheral cells. Therefore, we set to investigate the biochemical basis and the possible biological implications of this experimental observation.
Here, we demonstrate that fibrinogen is not synthesized by PBMCs, rather it is internalized from plasma.
Fig. 5 HSA and IgG influence fibrinogen internalization in Jurkat cells. A western blot analysis was performed in Jurkat cells after 4 h incubation
with fibrinogen (0.4 mg/ml), in the presence or in the absence of HSA (a) and IgG (b). c Fibrinogen intake was also assessed in the presence of two non-plasmatic proteins, i.e., catalase (CATA) and hemoglobin (HB) Alberio et al. J Transl Med (2018) 16:64 In order to better characterize fibrinogen intake levels and kinetics, we took advantage of the human T cell line Jurkat. Indeed, in this working model of the major component of human PBMCs, standardized conditions can be maintained. On the contrary, PBMCs isolated from human subjects suffer from intrinsic biological variability. Jurkat cells, as T-cells in general, do not express fibrinogen, however we demonstrated that they are able to internalize it from the extracellular milieu. The fibrinogen intake in Jurkat cells showed a hyperbolic behavior, consistent with a simple equilibrium, where the presence of FBS in the medium influenced the thermodynamics of the intake. Indeed, if cells were maintained in standard medium containing FBS, the calculated apparent K d was twofold compared to that obtained in the absence of serum.
Of note, fibrinogen-exposed Jurkat cells completely eliminated the protein within 24 h. Co-culture of Jurkat cells loaded or not with FITC-conjugated fibrinogen allowed us to follow the two phases of internalization and secretion of fibrinogen in an ordered sequence. These experiments not only demonstrated that fibrinogen is secreted, but also that it can be re-uptaken by adjacent cells.
As far as the internalization phase, we showed that while a very fast kinetics was observed in the absence of serum, a slower kinetics characterized the intake of fibrinogen in the presence of FBS in the culture medium. The fact that either HSA or IgG, which are both serum proteins, could mimic the presence of FBS in conditions of serum-free medium suggested that a common mechanism could orchestrate the dynamics of internalization, transport and secretion of these proteins. This was further corroborated by the fact that other non-plasmatic proteins, such as catalase or hemoglobin, did not alter fibrinogen intake in Jurkat cells.
Importantly, the evidence that fibrinogen could be internalized and re-externalized implied a mechanism of protection from intracellular degradation. Within this frame, it has been well documented by literature [22][23][24][25] that both HSA and IgG half-lives in serum result to be extended by the pH-dependent interaction with the neonatal Fc receptor (FcRn), which protects these proteins from intracellular lysosomal degradation and recycles them to the extracellular space. We thus hypothesized that FcRn could subserve the same function also for fibrinogen.
FcRn was originally identified as the receptor in charge to regulate IgG transport from a mother to fetus, thereby the definition as "neonatal" receptor [10,11]. Hereafter, it has been related to IgG homeostasis and transport across polarized epithelial tissues also in adults. Therefore, it 16:64 has been described as the receptor able to prevent or at least minimize IgG degradation in the lysosomes, being responsible for their long half-life in the serum [22][23][24]. IgG are internalized aspecifically by pinocytosis; however, IgG bind to FcRn upon acidification of the endosome, thus allowing IgG to escape lysosomal degradation. After vesicle docking with the plasma membrane, the pH returns to neutral with consequent release of bound IgG to the serum. The same mechanism was described to protect albumin from degradation and consequently to increase the half-life of the protein [30].
The results presented in this investigation indeed strongly support the idea that also fibrinogen, another abundant serum protein, could be internalized and then protected by degradation through a mechanism involving FcRn. A series of relevant observations in this direction were made. First, fibrinogen can be re-externalized and uptaken by neighboring cells, as demonstrated by co-culture experiments. Second, silencing of FcRn by siRNAs clearly showed a decreased accumulation of fibrinogen in SH-SY5Y cells.
FcRn interacts with IgG and albumin through residues located in opposite surfaces, such that FcRn can simultaneously bind IgG and albumin with neither competition nor cooperation occurring [31]. As for the interaction between FcRn and IgG, upon binding at pH 6, the protonation of three histidine residues (H310, H435, H436) in the C H2 -C H3 hinge region of IgG allows for the formation of salt bridges at the FcRn-Fc interface [32]. By contrast, FcRn binding to albumin is mostly hydrophobic in nature and is stabilized by a pH-dependent hydrogenbonding network internal to each protein. This interaction involves two tryptophan (W53, W59) residues of FcRn and three histidine (H464, H510, H535) residues of albumin [33]. In this frame, it is quite surprising that we observed a decreased amount of internalized fibrinogen in Jurkat cells in the presence of either HSA or IgG. The biochemical and structural characterization of the binding between fibrinogen and FcRn is beyond the scope of the present work. However, in order to explain mechanistically our results, it can be hypothesized that a steric hindrance is involved, due to the large size of the protein molecules. In Additional file 1: Figure S1, we show the interaction interfaces of HSA and Fc with FcRn. As it may be noticed, the two interactors bind at opposite regions of the α1-α2 subunits. As mentioned above, hindrance is not expected to occur since the size of both ligands is quite small. On the other hand, we may postulate that large sized ligands such as exameric fibrinogen may interfere with the binding of both HSA and Fc, either competitively or non-competitively.
Another important aspect is the pH dependency of the binding. Indeed, FcRn binds IgG and albumin at acidic pH, which can be found in early and late endosomes, in the proximal tract of intestine during neonatal life and, eventually, in the extracellular space of inflamed tissues [34]. It is well documented by literature that FcRn is expressed in dendritic cells, where it directs immune complexes to lysosomes for facilitation of antigen presentation, in monocytes/macrophages, in polymorphonuclear leukocytes and also in B lymphocytes [35,36]. Here we presented clear evidence for the expression of FcRn in T cells as well, where the protein is present in its glycosylated form. A single N-glycosylation site (N102 residue within NTS motif ) is present in human FcRn, where the addition of a glycan moiety increases the molecular mass by 1.5-3 kDa [29], which is in keeping with the two discrete protein forms that we detected in promyelocytic and SH-SY5Y cell lines. The biological significance of the addition of glycan moieties to human FcRn remains unclear. N-Glycans have been suggested to be apical targeting signals in other proteins [37] and it has been recently proposed that this modification may play a role in mediating apical membrane distribution of FcRn and enhancing either stabilization of FcRn on the cell surface or movement of FcRn to the cell surface [29].
In this context, within sites of inflammation fibrinogen could bind FcRn on the surface of T cells, be internalized, protected from degradation and eventually recycled extracellularly.
Conclusions
Collectively, the biological and functional implications of our findings are important. Indeed, three of the most abundant proteins (i.e., HSA, IgG and fibrinogen) regulating key processes in the homeostasis of the cell and of the biological fluids, as well as of the immunity of the individual, can be protected by drastic degradation using the FcRn-mediated mechanism. This allows these proteins to increase their half-life and thus prevent an excessive biosynthesis of already highly expressed molecules. It is important to underline that a major cellular component in the blood involved in the internalization and protection from degradation of fibrinogen is the T cell compartment. T cells are involved in crucial functions of the immune response. In particular the CD4+ T cells, which represent up to 40% of the total circulating mononuclear cells, are involved in the initial phases of the adaptive immune response against foreign aggressors by recognizing antigens and subsequently triggering the effector phases of the immune response [38][39][40]. As such, T cells are among the first cells to infiltrate inflamed tissues to patrol the presence of foreign aggressors. As it has been postulated for granulocytes, during inflammation the requirement of high concentration of fibrinogen may be key in the process of recruitment and extravasation of the inflammatory cells [3,41]. The fact that lymphocytes, and particularly T-cells, will eventually colonize these tissues to contribute to the elimination of the insult makes them ideal candidates to maintain a high concentration of fibrinogen in situ without the need of continuous support of new protein synthesis by the liver. Future investigation on this aspect is certainly warranted. Authors' contributions TA and MF conceived the project. TA performed the kinetics experiments. GF, GT and RSA conceived and performed the co-culture experiments. ML performed the knock-down experiments. All authors analyzed the data, wrote and revised the manuscript. All authors read and approved the final manuscript. | v3-fos-license |
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} | pes2o/s2orc | The 18 kDa Translocator Protein (TSPO) Overexpression in Hippocampal Dentate Gyrus Elicits Anxiolytic-Like Effects in a Mouse Model of Post-traumatic Stress Disorder
The translocator protein (18 kDa) (TSPO) recently attracted increasing attention in the pathogenesis of post-traumatic stress disorder (PTSD). This study is testing the hypothesis that the overexpression of TSPO in hippocampus dentate gyrus (DG) could alleviate the anxiogenic-like response in the mice model of PTSD induced by foot-shock. In this study, hippocampal DG overexpression of TSPO significantly reversed the increase of the contextual freezing response, the decrease of the percentage of both entries into and time spent in the open arms in elevated plus maze test and the decrease of the account of crossings from the dark to light compartments in light–dark transition test induced by electric foot-shocks procedure. It was further showed that the behavioral effects of TSPO overexpression were blocked by PK11195, a selective TSPO antagonist. In addition, the expression of TSPO and level of allopregnanolone (Allo) decreased in the mouse model of PTSD, which was blocked by overexpression of TSPO in hippocampal dentate gyrus. The difference of neurogenesis among groups was consistent with the changes of TSPO and Allo, as evidenced by bromodeoxyuridine (BrdU)- positive cells in the hippocampal dentate gyrus. These results firstly suggested that TSPO in hippocampal dentate gyrus could exert a great effect on the occurrence and recovery of PTSD in this animal model, and the anti-PTSD-like effect of hippocampal TSPO over-expression could be at least partially mediated by up-regulation of Allo and subsequent stimulation of the adult hippocampal neurogenesis.
The translocator protein (18 kDa) (TSPO) recently attracted increasing attention in the pathogenesis of post-traumatic stress disorder (PTSD). This study is testing the hypothesis that the overexpression of TSPO in hippocampus dentate gyrus (DG) could alleviate the anxiogenic-like response in the mice model of PTSD induced by footshock. In this study, hippocampal DG overexpression of TSPO significantly reversed the increase of the contextual freezing response, the decrease of the percentage of both entries into and time spent in the open arms in elevated plus maze test and the decrease of the account of crossings from the dark to light compartments in light-dark transition test induced by electric foot-shocks procedure. It was further showed that the behavioral effects of TSPO overexpression were blocked by PK11195, a selective TSPO antagonist. In addition, the expression of TSPO and level of allopregnanolone (Allo) decreased in the mouse model of PTSD, which was blocked by overexpression of TSPO in hippocampal dentate gyrus. The difference of neurogenesis among groups was consistent with the changes of TSPO and Allo, as evidenced by bromodeoxyuridine (BrdU)-positive cells in the hippocampal dentate gyrus. These results firstly suggested that TSPO in hippocampal dentate gyrus could exert a great effect on the occurrence and recovery of PTSD in this animal model, and the anti-PTSD-like effect of hippocampal TSPO over-expression could be at least partially mediated by up-regulation of Allo and subsequent stimulation of the adult hippocampal neurogenesis.
INTRODUCTION
Post-traumatic stress disorder (PTSD) is a chronic and debilitating mental disorder that develops in survivors of traumatic events and PTSD can cause disturbing thoughts, helpless, and the attempt to avoid trauma-related cues (Bisson et al., 2015). However, the precise mechanisms of the intricate biological and psychological symptoms of PTSD remain unclear. Over the last two decades,the down-regulation of neurosteroid biosynthesis has been shown in several mental disorders including PTSD (Ceremuga et al., 2013;Locci and Pinna, 2017). Additionally, quite many clinical trials have shown that the decreased level of neuroactive steroids allopregnanolone (Allo) may play an important role in the pathology of PTSD (Rasmusson et al., 2006;Pinna, 2010;Pinna and Rasmusson, 2012). Pre-clinical studies have also found that corticolimbic Allo content remarkably reduced in patients of anxiety and aggression and the Allo decrease was positively related with the impaired behavioral performance (Pibiri et al., 2008;Pinna and Rasmusson, 2012). Previous results showed that the infusion of Allo into the dorsal hippocampus induced vigorous anxiolyticlike behavior in the elevated plus-maze test (Modol et al., 2011). These interesting studies gave reason to the hypothesis that downregulation of neurosteroids could contribute to the etiology of PTSD.
In the central nervous system, the translocator protein (18 kDa) (TSPO) are mainly expressed in glial cells. It mediates the translocation of cholesterol from the outer to the inner mitochondrial membrane, and thus regulates the synthesis of neurosteriods (Nothdurfter et al., 2012;Hatty and Banati, 2015). Studies demonstrated that long-term stress induced a decrease in the TSPO expression in the central nervous system in rodents (Milenkovic et al., 2015;Wang et al., 2015;Zhang et al., 2016). Interestingly, our group was the first to show that oral administration of certain TSPO ligands, including AC-5216 and YL-IPA08, enhanced synthesis of neurosteroids (such as Allo) in the brain and exerted anti-PTSD-like effect in some PTSD animal models with a favorable side effect Zhang et al., 2014aZhang et al., , 2016. Hence, TSPO protein might provide a promising target for novel anti-PTSD drug, but the specific mechanism remains to be determined.
The prefrontal cortex, the amygdala and the hippocampus are three brain regions in the limbic system which have been identified as the most clearly involved regions in PTSD (Wingenfeld and Wolf, 2014). Among them, the hippocampus plays an important role in remembrance of traumatic events and correlation of learned responses to contextual cues. Indeed, hippocampal reduction was reported to happen in patients with PTSD in many structural neuroimaging studies (Rodrigues et al., 2011). We therefore proposed that an up-regulation of TSPO expression in hippocampus, which could then enhance neurosteroidogenesis (such as Allo), may contribute to the behavioral adaptation to PTSD. To address our hypothesis, we used foot-shock procedure, an established mice model of PTSD to specifically examine the role of TSPO in PTSD (Bali and Jaggi, 2015). Furthermore, we examined a possible interference in PTSD-like behavior after application of a lentivirus-mediated overexpression of TSPO into the dentate gyrus (DG) of hippocampus. To explore the possible mechanisms in mediating the anti-PTSD effect of hippocampal TSPO overexpression, we then tested the changes of Allo and the hippocampal neurogenesis after behavioral tests.
Animals
Adult male ICR mice (18-22 g) were obtained from the Beijing SPF Animal Technology Company (Animal License No. SCXK 2016-0002;Beijing, China). All animals were housed in groups of 3 to 5 per plastic cage (320 mm × 220 mm × 160 mm) in an air-conditioned room of controlled temperature (23 ± 1 • C) and a 12-h light/dark circle (lights on at 6:00 AM). Mice had access to food and water ad libitum. All procedures were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition
Drugs and Treatments
Lentiviral vectors containing the non-targeting negative control (Lv-NC) or TSPO (Lv-TSPO) sequence were generated by Genechem Company (Genechem, Co., Ltd., Shanghai, China). Recombinant and packaging lentiviruses encoding the TSPO gene was constructed as our previous studies by the Genechem Company (Genechem, Co., Ltd., Shanghai, China) (Wang et al., 2016;Li et al., 2017). EGFP was added to all viral vectors to track lentivirus-mediated target gene as a marker expression. TSPO is expressed in different organs, including the adrenal cortex, luteal cells, the testis, ovarian granulosa cells, the placenta and glial cells in the brain. Specifically, we overexpressed TSPO in the bilateral hippocampus using a lentivirus to study the important role of TSPO in the hippocampal dentate gyrus and confirm the overexpression site by tracking the lentivirus-mediated target gene under an inverted fluorescence microscope.
Experiment Design
Sixty mice were randomly assigned to five groups: Lv-negative control (NC), Lv-NC + foot-shock (FS), Lv-NC + Ser + FS, Lv-TSPO + FS and Lv-TSPO + PK11195 + FS (n = 12 for each). A schematic overview of the experiment is depicted in Figure 1A. First, BrdU (100 mg/kg, i.p.) was administered for 3 Frontiers in Pharmacology | www.frontiersin.org And the effects were reversed by either Ser administration or Lv-TSPO injection. All the effects of Lv-TSPO in (D,E,H,I) was blocked by PK11195. Data were presented as the means ± SEM (n = 8-11). * P < 0.05 compared with the Lv-NC+foot-shock (-) group; # P < 0.05, ## P < 0.01 compared with the Lv-NC+FS group; $ P < 0.05 compared with the Lv-TSPO+FS group. times at a 3 h interval 24 h before lentiviral vector administration. Then animals were subjected to microinjection of lentiviral vectors containing the non-targeting negative control (Lv-NC) or TSPO (Lv-TSPO) into the DG of hippocampus. Following a recovery period of 2 weeks, we conducted the electric foot-shock procedures and assessed the behavioral effects of over-expression of TSPO on anxiety-like behaviors induced by the inescapable electric foot shock, an established mouse model of PTSD.
To observe and confirm the microinjection sites, three vectortreated mice in each group were randomly chosen and perfused transcardially following the behavioral experiments. The brains were removed, post-fixed and dehydrated. Serial coronal brain sections (30 µm thick) were cut. The microinjection sites and infected zones were defined by direct visualization with a fluorescence microscope (Olympus AX70 Provis, Center Valley, PA, United States) for the benefit of the green fluorescent protein (GFP) tag as described previously (Li et al., 2009). To detect the TSPO protein expression and allopregnanolone (Allo) level after hippocampus injection of Lv-NC or Lv-TSPO, hippocampal tissues (3 mm in diameter around the injection site on both sides) were removed and Western blot analysis (n = 3) and enzyme-linked immunosorbent assay (ELISA) (n = 3) were performed respectively as described previously. The neurogenesis in hippocampus DG was evaluated by the immunohistochemistry of BrdU/NeuN-positive cells in DG (n = 3).
Mouse Surgery and Lentiviral Microinjections
After 2-week acclimatization period and the following BrdU administration, mice received lentiviral microinjection under anesthesia with chloral hydrate (400 mg/kg, i.p.). The craniotomy was created aimed for bilateral DG according to the coordinates of the mouse brain atlas (Franklin and Paxinos, 2008): (AP, −1.7 mm; ML, ±1.8 mm; DV, −2.0 mm). The bilateral DG were both injected by a 10 µl microsyringe on the stereotaxic apparatus. The 30-gauge-needle was lowered into the dorsal DG. Lentiviral vectors containing NC or TSPO (2 × 10 8 TU/µl, 1 µl/side) were infused at a rate of 0.2 µl/min by a microsyringe injector and Micro4 controller (World Precision, Ins., Sarasota, FL, United States). The amount of lentivirus and the duration of infusion were determined by the repeated preliminary experiments and previous studies. The needle stayed in place after injection for 5 min to guarantee proper diffusion of the vectors. From the day (Day 1) of lentiviral microinjection, Ser (15 mg/kg, i.g.) or PK-11195 (3 mg/kg, i.p.) was administered once per day. The dosages were selected according to our previous studies (Miao et al., 2014;Zhang et al., 2014b;Qiu et al., 2015).
Electric Foot-Shock Procedures
Two weeks after lentiviral vectors injection, the electric footshock procedure was conducted as per Zhang et al. (2012) and Qiu et al. (2013). Electric foot-shocks were delivered through a stainless-steel grid floor (9 mm interval) by an isolated shock generator (Med Associates, Inc., United States) in a Plexiglas chamber (20 cm × 10 cm × 10 cm). Each mouse received 15 intermittent inescapable foot-shocks (intensity: 0.8 mA, interval: 10 s, and duration: 10 s) for 5 min following a 5-min adaptation period. Mice in the control group were placed in the same chambers without electric foot-shocks for total 10 min to adapt to and remember the same circumstance without trauma. Ethyl alcohol was used to wipe the chamber to avoid the effect of feces and smell between mice.
Contextual Freezing Measurement
The rodents of electric foot-shock model will freeze intermittently when re-exposed to the shock context and the freezing behavior is associated with the fear memory induced by the trauma-related cues, as the symptoms of PTSD patients. Thus, contextual freezing measurement was reported as one of the effective methods to evaluate PTSD (Maier, 1990;Siegmund and Wotjak, 2007;Zhang et al., 2012). All mice were re-exposed to the same chamber, which is the reminder situation of footshocks for 5 min 8 days after electric foot-shock procedures (Day 23; Figure 1A). The total cumulative freezing time was recorded and analyzed by computer (Med Associates, Inc., Video Freeze SOF-843, United States).
Open Field Test
To evaluate whether the reversion of PTSD-like behavior by over-expression of TSPO depends on locomotor activity change in mice, the number of line crossings and rears were assessed 10 days after electric foot-shock procedures (Day 25; Figure 1A) as in our previous study Zhang et al., 2016). Mice were placed in the center of a transparent box (36 cm × 29 cm × 23 cm) of which the base were divided into nice equal section. And then an observer who was blind to the study design counted and recorded the number of crossings (all four paws placed into a new square) and rears (both front paws raised from the floor) for 5 min. Ethyl alcohol was used to wipe the interior wall and floor to avoid the effect of feces and smell between mice.
Light-Dark Transition Test
Twelve days after electric foot-shock procedures (Day 27; Figure 1A), each mouse started in the dark side of the lightdark chamber and was free to cross through an opening between the light compartment and the dark one (30 cm × 23 cm for each side). The illumination of the light compartment was 720 lux. This test pits mice' innate aversion to bright areas against their natural drive to explore in response to mild stressors such as a novel environment. The transitions were only defined by the number of crossings from the dark to the light compartments. An observer who was blind to the study design counted and recorded the transitions for 5 min.
Elevated Plus Maze Test
Fourteen days after electric foot-shock procedures (Day 29; Figure 1A), the elevated plus maze test was performed. It is a widely-applied method to assess the anxiogenic-like behavior of PTSD model rodents (Li et al., 2009;Zhang et al., 2012). The four branching arms (30 cm × 5 cm) composed the cross-shaped maze. The maze was set at 40 cm above the ground. The opposite two open arms without wall and two closed arms with dark walls (10 cm high) were connected by a central platform (5 cm × 5 cm). Each mouse was placed on the central platform and recorded by video for the time spent on the open/closed arms and number of entries into any arm. The entrance was defined by all four paws on the arm base. We calculated the ratio of time spent in the open arms to the total time spent in all arms and the ratio of the number of entries into open arms to that into any arm. The maze was cleaned with 5% ethanol between tests to avoid the effect of feces and smell between mice.
Western Blot Analysis
The western blot analysis was conducted as described previously (Wang et al., 2016). Briefly, hippocampal tissues (3 mm in diameter around both injection sites) were removed and then extracted by RIPA lysis buffer (Applygen, China). Fifty microgram of protein were separated by SDS-PAGE, measured and analyzed by western blot with primary antibody rabbit anti-TSPO (1:1000; Abcam, Cambridge, MA, United States) and β-actin (1:3000; Santa Cruz, CA, United States). The expression of protein was measured by Gel-Pro Analyzer software, Version 3.1 (Media Cybernetics, Rockville, MD, United States) and the TSPO expression was normalized to β-actin. Every experiment was independently repeated no less than four times.
Enzyme-Linked Immunosorbent Assay (ELISA)
The removed hippocampal tissues (3 mm in diameter around both injection sites) were extracted on ice with the lysis buffer containing 137 mM NaCl, 0.5 mM sodium vanadate, 10 µg/mL aprotinin, 1 µg/mL leupeptin1% NP40, 10% glycerol, 1 mM PMSF, 20 mM Tris-HCl (pH 8.0). Allo concentrations were quantified using ELISA kits according to the manufacturer's protocol (Arbor Assays, United States), and the density values were detected by the spectrophotometer at a wavelength of 450 nm and a reference wavelength of 650 nm.
Immunohistochemistry
Immunohistochemistry was conducted as Li et al. (2009). Mice were anesthetized deeply with chloral hydrate (500 mg/kg, i.p.), transcardially perfused with ice-cold 0.9% NaCl and then 4% buffered formalin. Brains of mice that received intraperitoneal injections of BrdU were carefully and quickly removed and fixed in 4% paraformaldehyde at 22 • C for 48 h for histochemistry of BrdU. Coronal 12-µm sections were cut and incubated freefloating for 24 h at 4 • C in PBS containing both rat anti-BrdU antibody (1:200; Abcam, Cambridge, MA, United States) and mouse anti-NeuN antibody (1:1000; Chemicon, Temecula, CA, United States). After rinsing with PBS for three times, the sections were then incubated with Red-X-conjugated goat antirat IgG and FITC-conjugated goat anti-mouse IgG (1:200 for both; Jackson, MS, United States) to react to the corresponding primary antibody in PBS for 2 h at 22 • C before mounting. The sections were photographed and analyzed by confocal microscope (Zeiss LSM510, Thornwood, NY, United States). The BrdU-positive cells were counted as described previously (Li et al., 2009). Briefly, the BrdU immunohistochemistry was performed for every sixth section throughout the entire hippocampus. All BrdU-positive cells in the hippocampus DG were counted by a blind observer and multiplied by 6, recorded as the total number of labeled cells in the DG.
Statistical Analysis
Data were analyzed using GraphPad Prism 6 software (Graphpad Prism Institute, Inc., La Jolla, CA, United States). Results are expressed as means ± SEM. Outliers were removed according to the interquartile range (IQR) test (Zijlmans et al., 2018). Outliers here were defined as observations that fall below data set median -1.5 IQR or above data set median + 1.5 IQR. Data were analyzed by Mann-Whitney U-test for multiple comparisons, followed by the Holm-Sidak test as post hoc analyses to adjust. Values of P < 0.05 were considered statistically significant.
TSPO Overexpression in the DG Elicited Anxiolytic-Like Effect in the Mice Exposed to Electric Foot-Shocks
There was no significant difference in the line crossings and rears between groups in the open field test. These results indicated that none of Lenti, Ser (15 mg/kg) or PK11195 (3 mg/kg) significantly did harm to locomotor activity in this animal model (Figures 1B,C).
A significant increase in the contextual freezing time was observed in Lv-NC + Foot Shock group compared to the non-shocked Lv-NC group, indicating that the anxiogenic-like mouse model of PTSD was successfully established. The freezing behavior was alleviated in the Lv-NC + Ser + FS group as the positive control compared with Lv-NC + FS group. After Holm-Sidak correction was used to calibrate the error from multiple tests, the significant difference remained, demonstrating that the validity of this model (P = 0.0272 for Lv-NC+FS vs. Lv-NC; P = 0.0019 for Lv-NC+Ser+FS vs. Lv-NC+FS; Figure 1D). The contextual freezing response was also decreased in mice that received an intra-hippocampal injection Lv-TSPO compared with foot-shock vehicle group (P = 0.0038 for Lv-TSPO+FS vs. Lv-NC+FS; Figure 1D). These results demonstrated that TSPO overexpression in DG of hippocampus attenuated the contextual freezing behavior in post-shocked mice.
In the light-dark transition test, the number of crossings from dark to light compartment decreased in the foot-shock-exposed Lv-NC + FS mice compared to the non-shocked Lv-NC mice (P = 0.0134 for Lv-NC+FS vs. Lv-NC; Figure 1E). It was also shown that either repeated administrations of Ser or Lv-TSPO injection increased the number of transitions (P = 0.0018 for Lv-NC+Ser+FS vs. Lv-NC+FS; P = 0.0461 for Lv-TSPO+FS vs. Lv-NC+FS; Figure 1E), suggesting that TSPO overexpression in DG of hippocampus significantly ameliorated PTSD-associated anxiogenic-like behaviors. Figures 1F,G, no significant differences were observed between groups for total arm entries or total time spent in all arms in the EPM test. It was further showed that these above anti-PTSD behavioral effects of TSPO overexpression was reversed by PK11195 administration (P = 0.0412 for freezing time in contextual freezing test, Figure 1D; P = 0.0486 for dark-to-light transition in light-dark transition test, Figure 1E; P = 0.0355 for the percentage of entries into open arms, P = 0.0432 for percentage of time spent in open arms in EPM test, Figures 1H,I). These results indicated that TSPO overexpression in DG of hippocampus attenuated the anxiogenic-like behavior induced by electric footshock procedures in mouse model of PTSD, but these effects could be blocked by the TSPO ligand PK11195, suggesting that these effects might be at least partially attributed to TSPO activation.
Targeted Overexpression of TSPO in the Hippocampus
To confirm the overexpression of TSPO in vivo, the lentivirusmediated overexpression was traced by the expression of GFP using fluorescence microscopy (Figures 2A,B). The results showed that fluorescence obtained with this lentiviral vector was localized to the subgranular layer and hilus of the DG, as indicated by GFP-positive cells (green), while no expression was detected outside the hippocampus. Combined with the immunoblots of TSPO in punched hippocampus tissue, the results showed that foot-shock procedure significantly reduced the expression of TSPO (P = 0.0143), and chronic administration of Ser or Lv-TSPO injection clearly increased the TSPO expression (P = 0.0286 for Lv-NC+Ser+FS vs. Lv-NC+FS; P = 0.0143 for Lv-TSPO+FS vs. Lv-NC+FS) at the same time of exerting the anti-PTSD-like effect. The overexpression of TSPO by administration of LV-TSPO was blocked by TSPO antagonist PK11195 (P = 0.0143; Figure 2C).
Effects of Hippocampal TSPO Overexpression on the Level Allo After Electric Foot-Shock
The endogenous Allo level in the hippocampus tissues (3 mm in diameter around both injection sites) of post-shock mice were measured at the end of the behavioral tests to further confirm the role of Allo in the anti-PTSD-like behavior effect of Lv-TSPO. As shown in Figure 2D, the foot-shock procedure significantly reduced the Allo level in the hippocampus compared to shockfree control mice (P = 0.0140), which was clearly reversed by daily administration of Ser or Lv-TSPO injection (P = 0.0475 for Lv-NC+Ser+FS vs. Lv-NC+FS; P = 0.0375 for Lv-TSPO+FS vs. Lv-NC+FS). And the increase of Allo by administration of LV-TSPO was blocked by TSPO antagonist PK11195 (P = 0.0312; Figure 2D).
Effect of Hippocampal TSPO Overexpression on the Number of BrdU-Positive Cells in the Hippocampus in Mice After Electric Foot-Shocks
Given the view that neurogenesis could be reduced by PTSD, we then labeled BrdU-positive cells in the hippocampus DG to determine the effect of TSPO on neurogenesis. Mice were sacrificed 30 day after the beginning of BrdU labeling. Cells labeled with BrdU were counted per bilateral, entire hippocampal dentate gyri. BrdU-positive cells were predominantly localized in the subgranular layer ( Figure 3A) and co-localized with NeuNcells ( Figure 3B). Statistical analysis revealed that the foot-shock procedure significantly decreased the number of BrdU (+) cells present in the DG when compared with foot-shocked (−) mice (P = 0.0269 for Lv-NC+FS vs. Lv-NC). And this effect could be reversed by the chronic administration of Ser or Lv-TSPO injection which exhibited significantly more BrdU (+) cells than Lv-NC treated animals (P = 0.0085 for Lv-NC+Ser+FS vs. Lv-NC+FS; P = 0.0334 for Lv-TSPO+FS vs. Lv-NC+FS; Figure 3C). These data indicated that the overexpression of TSPO alleviated the impaired hippocampal neurogenesis induced by foot-shocked procedure.
DISCUSSION
In this study, we showed that Lv-TSPO mediated overexpression of TSPO in the DG attenuated PTSD-like behaviors without significantly affecting locomotor activity. These results suggested that overexpression of TSPO in the DG may reverse the PTSDlike behaviors, which can be reversed by a TSPO antagonist PK11195. Furthermore, hippocampal TSPO overexpression increased the level of Allo and improved hippocampal neurogenesis.
In the present study, the lentivirus was selected for its stable and long-term gene overexpression as the vector (Schratt et al., 2006;Krassnig et al., 2015;Chen et al., 2016). It was reported that lentiviral vectors can receive a big fragment of exogenous target gene, express constantly in cells, and possess satisfactory safety (Escors and Breckpot, 2010;Sauer et al., 2014). Till now, no studies about the application of lentiviral vectors to PTSD therapy were reported worldwide. Our previous study suggested that the lentiviral vectors carrying TSPO gene were well-expressed in the DG of hippocampus (Wang et al., 2016), which fits the evidence of our present study. As the lentiviral vectors efficiently inspired hippocampal TSPO signaling, it was examined whether TSPO expression change can modify the PTSD-like responses of mice after foot-shock paradigms, a reliable animal model for PTSD. Interestingly, our studies showed that intra-hippocampal injection of the Lv-TSPO reversed the behavioral impairment including the anxiogenic-like effect in mice after foot-shock exposure, which is consistent with the effect of chronic administration of sertraline. Sertraline, as a selective serotonin reuptake inhibitor (SSRI), is a FDA-approved medication for PTSD. It has shown its certain anti-PTSD effect independent on the shock exposure in many rodent models (Miao et al., 2014;Zhang et al., 2015;Qiu et al., 2017;Zhang Z.S. et al., 2017). Our results suggested that the dose of intra-hippocampal injection of the Lv-TSPO was efficient to exert the anti-PTSD effect as sertraline. We also found that neither the foot-shock procedure nor the intrahippocampal injection of the Lv-TSPO significantly impacted on the locomotor activity in mice, suggesting that the observed behavioral differences were independent on the basal locomotor activity changes.
In order to investigate the anti-PTSD-like effect of intrahippocampal injection of the Lv-TSPO, it was tested whether blocking the TSPO pathway by PK11195 affected the behavioral effects in the foot-shock procedure. According to our previous studies, chronic administration of PK11195 alone would not change the foot-shock induced PTSD-like behavior or the level of Allo in serum or brain (Zhang et al., 2014b). However, in this study when PK11195 intervened the effect of intrahippocampal injection of Lv-TSPO in foot-shock model, it was shown that the TSPO antagonist PK11195 administration reversed all the attenuated behavioral effects induced by DG Lv-TSPO. These results were in line with our previous studies which demonstrated that PK11195 completely blocked the anti-PTSD-like effects of TSPO ligand YL-IPA08 . Overall, these findings indicated that the anti-PTSD effects of intra-hippocampal injection of the Lv-TSPO could be mediated by TSPO activation.
The expression level of TSPO protein were measured and further verified the overexpression of TSPO in DG in the Lv-TSPO+FS group. The decreased TSPO expression induced by foot shock in PTSD mice model and increased TSPO expression by sertraline agree with our previous studies Zhang et al., 2014b;Li et al., 2017;Zhang L.M. et al., 2017). Interestingly, we found that PK11195 blocked the effects of TPSO over-expression on TPSO protein levels in the hippocampus. We think it might be some compensatory mechanism, for that TSPO is technically not a classic receptor. On the other hand, PK11195 was reported to be able to induce changes in expression of immediate early genes and transcription factors in U118MG glioblastoma cells which were studied for TSPO functions for years. These changes also included gene products that are part of the canonical pathway serving to modulate general gene expression (Yasin et al., 2017). It might be other reason for the TSPO expression change and needs further investigation.
Allo is the most abundant neurosteroid in the central nervous system potently and selectively acting with GABA A the receptors and modulating of GABA A signaling action (Puia et al., 1990;Lambert et al., 1995Lambert et al., , 2003. The decreased level of Allo in the central nervous system was reported to associate with the symptoms of PTSD (Vaiva et al., 2004;Rasmusson et al., 2006). . The improvement of Lv-TSPO was reversed by PK11195 (Lv-TSPO + PK11195 +FS). Data were presented as the means ± SEM (n = 3). * P < 0.05 compared with the Lv-N+foot-shock (-) group; # P < 0.05 compared with the Lv-NC+FS group; $ P < 0.05 compared with the Lv-TSPO+FS group. ## P < 0.01 compared with the Lv-NC+FS group.
Numerous studies have demonstrated that Allo plays a pivotal role in the mediation of contextual fear memory cued by the trauma-related events and the incapacity of Allo biosynthesis may be one of the molecular mechanisms underlying the etiology of PTSD (Uzunova et al., 1998;Rasmusson et al., 2006;Pibiri et al., 2008;Pinna and Rasmusson, 2012). In this current study, we detected the level of Allo in mice to substantiate this hypothesis that the normalization of brain Allo levels may underlie the anti-PTSD-like effects of intra-hippocampal injection of the Lv-TSPO. Our results showed a remarkable decreased Allo level in the hippocampus in post-foot-shock mice, which was reversed by sertraline in hippocampus, and the result is in line with the previous knowledge that anti-PTSD-like activities of sertraline were closely associated with elevated biosynthesis of Allo (Pinna and Rasmusson, 2012;Xu et al., 2018). Likewise, the intra-hippocampal injection of the Lv-TSPO reversed the decreased Allo level in the hippocampus in post-foot-shock mice, further suggesting that the anti-PTSDlike effects of Lv-TSPO could be mediated by the subsequent synthesis of Allo in hippocampus DG. It is consistent with the results of previous studies on TSPO ligands, such as YL-IPA08 and AC-5216, which have been shown to display efficacy toward the treatment of psychiatric disorders by increasing neurosteroid biosynthesis in the brain, inducing anxiolytic and antidepressant activities in some rodent models and improving behavioral deficits in a mouse model of PTSD Zhang et al., 2014a;Wang et al., 2016;Li et al., 2017;Zhang L.M. et al., 2017). It is important to note that other neurosteroids were not examined in our present study, but their roles cannot be excluded and their potential contribution need further studies to identify. Our present study also found that sertraline could reverse the lowered Allo levels in post-foot-shock mice and further suggested that the anti-PTSD-like effects of sertraline were partly mediated by the subsequent synthesis of Allo.
A vast literature demonstrated the important role of adult-born neurons in buffering stress responses and in mediating anti-PTSD-like effects. Functional studies that have described the impaired adult hippocampal neurogenesis in PTSD patients and different animal models and the hippocampal neurogenesis became one of the treatment targets of anti-PTSD interventions (Kheirbek et al., 2012;Peng et al., 2013;Besnard and Sahay, 2016). This study showed that similar as sertraline, intra-hippocampal injection of the Lv-TSPO in post-foot-shock mice induced a significant proliferation of progenitor cells as shown by BrdU immunohistochemistry. Interestingly, evidences reported that exogenous administration of Allo could prevent the occurrence of depression/anxietylike behavior, as well as alleviate the damage of hippocampal neurogenesis (Evans et al., 2012) and promote cell survival (Brinton, 1994;Djebaili et al., 2005). Furthermore, recent studies have demonstrated the neuroprotective role of Allo against the hippocampal neurogenesis impairment in a transgenic mouse model of Alzheimer's disease (Wang et al., 2010;Singh et al., 2012). In addition, it was reported that besides GABAergic mechanisms, Allo could also enhance neurogenesis, which contribute to the regulation of depression and anxiety (Wang et al., 2005). These observations suggested that impaired hippocampal neurogenesis may participate the pathology of PTSD, and thus the hippocampal neurogenesis also provides a promising target for anti-PTSD treatment (Kempermann and Kronenberg, 2003).
In summary, the over-expression of TSPO in hippocampal DG exerted anti-PTSD effect in mice submitted to the foot-shock, which may be related to the up-regulation of Allo synthesis and subsequent stimulation of the adult hippocampal neurogenesis. It should also be stated that in this present study, we did not measure other neurosteroids or the new-born neurons in hippocampus DG, so we could not attribute the anti-PTSD effect of TSPO overexpression completely to increased Allo level. Nevertheless, our study advances our knowledge of the PTSD theory and provide a promising implication for the treatment of this mental disorder.
AUTHOR CONTRIBUTIONS
X-YZ helped to conceive the study, carried out the study execution and data analysis, and contributed to the manuscript draft. WW and QF contributed to the analysis of immunohistochemistry. L-MZ participated in the research design, the construction of recombinant lentiviruses, and the draft of manuscript. Y-ZZ, W-DM, and Y-FL contributed to the research design, data analysis, and manuscript revision. | v3-fos-license |
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} | pes2o/s2orc | Antioxidant Capacity and HPLC-DAD-MS Profiling of Chilean Peumo (Cryptocarya alba) Fruits and Comparison with German Peumo (Crataegus monogyna) from Southern Chile
Liquid chromatography (LC) coupled with UV detection and electrospray ionization (ESI) tandem mass spectrometry (MS/MS) was used for the generation of chemical fingerprints and the identification of phenolic compounds in peumo fruits and aerial parts from southern Chile. Thirty three compounds (19 of these detected in C. alba and 23 in C. monogyna) were identified, mainly flavonoid glycosides, phenolic acids, anthocyanins and flavonoid aglycons. Total phenolic content and total flavonoid content was measured for both species, and were higher in the extracts from C. monogyna fruits and aerial parts than extracts from C. alba. The fruits of Cryptocarya alba (Chilean peumo) presented high antioxidant capacity (9.12 ± 0.01 μg/mL in the DPPH assay), but was three times lower to that of Crataegus monogyna (German peumo) (3.61 ± 0.01 μg/mL in the DPPH assay).
Introduction
Cryptocarya alba (Chilean peumo), is a shade-tolerant evergreen tree endemic of Chile, distributed from Coquimbo province (IV Region) to Valdivia province (XIV Region) mainly inhabiting streams and humid shady valleys in the forest. It produces edible red-colored berries, called peumos, collected wild and consumed by the Mapuche Amerindians since pre-Colombian times. It is considered a OPEN ACCESS threatened species in some areas of Chile, mainly due to overexploitation and habitat destruction [1]. The essential oil of this species was reported to be composed mainly of p-cimol and 1-terpinen-4-ol [2] while the -pyrone cryptofolione and a cryptofolione derivative were the only two compounds isolated from the edible fruits [3].
The genus Crataegus is the largest genus among the subfamily Maloideae in the family Rosaceae which comprises 265 species, which are generally known as the hawthorns [4]. The Chilean hawthorn (Crataegus monogyna Jacq. (Lindt.) local name German peumo, peumo Alemán or Majuelo) is a thorny European shrub introduced to Chile and widely used as sedative, diuretic, anti-inflammatory and cardiotonic [5,6] which is prescribed by the Pharmacopoeia Europaea and recommended by the World Health Organization [7]. There are several reports the antioxidant capacity of and phenolic compounds present in several hawthorn species, including C. monogyna, which were analyzed by HPLC-MS [5,8]. However, the fruits from both species called peumo in Chile and are similar in appearance (Figure 1), yet the species are not related, even though the fruits look similar and are used for edible purposes in Chile, thus a chemical comparison and HPLC fingerprint of phenolics from both species collected in the same location (Southern hemisphere) could be a valuable tool for the differentiation of the different species and prove the health benefits of the fruits. In the present study we assessed the qualitative and quantitative phenolic profile of both edible fruits (C. alba and C. monogyna) called peumo in Chile by spectroscopic and spectrometric methods, evaluated their antioxidant power and compared the phenolic content with the leaves of both species. The phenolic compounds of aerial parts and fruits of the peumos were investigated by high-performance liquid chromatography paired with UV photodiode array and electrospray ionization ion trap tandem mass spectrometry detectors (HPLC-DAD-ESI/MS-MS).
Total Phenolic, Total Flavonoid Content and Antioxidant Power of Peumo Fruits and Aerial Parts
Dietary antioxidants have been shown to be effective scavengers of harmful free radicals, preventing the oxidation of biomolecules, such as DNA and low-density lipoprotein [9,10]. Fruits and vegetables are a good source of dietary antioxidants, such as vitamin E, vitamin C and β-carotene. The best-known phytochemical antioxidants are traditional nutrients; However, the contribution of some of these nutrients and/or vitamins in different edible fruits analyzed was estimated as being lower than 15 percent [11]. The antioxidant properties of fruits and vegetables are maily due to the polyphenolic content, and several cross-cultural epidemiological studies have supported the chemoprotective properties of polyphenolics [12][13][14]. In this work methanolic extracts of fruits and leaves from Chilean peumo (Cryptocarya alba) and German peumo (Crataegus monogyna) collected in Re-Re, Chile were evaluated for antioxidant power by the DPPH scavenging activity and the ferric reducing antioxidant power assay (FRAP) and the results were compared. Both fruits showed high antioxidant power but the leaves presented the highest activity ( Table 1). The fruits of C. alba showed total phenolic content of 17.70 ± 0.02 mg GAE (gallic acid equivalents) per g dry material. This value is 1.6 times lower than the content in C. monogyna fruits (28.30 ± 0.02 mg GAE/g dry material), collected in the same location. The aerial parts showed similar trend but for C. alba the value was 5.65 times higher (100.12 ± 0.83 mg GAE/g dry material), than its fruits, while for C. monogyna was 4 times higher (114.38 ± 1.62 mg GAE/g dry material), than its fruits. German peumo fruits (C. monogyna) also showed a higher value in total flavonoids (8.77 ± 0.00 mg QE (quercetin equivalents)/g dry material) than Chilean peumo (C. alba) fruits (8.22 ± 0.04 mg QE/g dry material), while the highest content of flavonoids was found in C. monogyna aerial parts (64.9 ± 0.00 mg QE/g dry material). C. monogyna fruits and aerial parts showed higher DPPH scavenging capacity (3.61 ± 0.01 and 3.34 ± 0.38 µg/mL, respectively, Table 1) and higher ferric reducing antioxidant power (85.65 ± 0.09 and 95.05 ± 0.15 µmol TE(trolox equivalents)/g, respectively, Table 1) than C. alba fruits and aerial parts. The antioxidant activities of polyphenolic compounds are mainly due to their ability to act as hydrogen donors, reducing agents, singlet oxygen quenchers and radical scavengers [9,10]. As reported here, the antioxidant activity of fruits and aerial parts significantly increases with high total polyphenol and flavonoid contents, however no association could be found between both antioxidant assays for these species (FRAP and DPPH, R 2 = 0.283) and between TPC and DPPH reduction was observed positive correlation (R 2 = 0.420), but it was not significant, as well as between FRAP and TFC (R 2 = 0.364) and between TPC and TFC (R 2 = 0.570) at p < 0.05. The low linear relationship or low correlation between the antioxidant assays and phenolic or flavonoid content as published for other plants [15][16][17][18] can be due to the different antioxidant capacity (The FRAP assay is based on the ability of the substance to reduce Fe 3+ to Fe 2+ while the DPPH assay the hydrogen donating capacity to scavenge DPPH radicals) or different redox properties of the mixtures of antioxidant compounds found in the organic extracts. The fruits of C. monogyna from Chile showed better DPPH scavenging activity than that reported for a sample from Portugal (15 ± 1% scavenging activity at 100 g/mL) [19], but the content of phenolics and flavonoids were lower than that reported (83 ± 2 and 51 ±14 mg GAE) for that fruit sample [19].
HPLC DAD and MS Analysis of Phenolic Compounds from Edible Peumo Fruits and Aerial Parts
In the last years, several biological samples such as plant and fruit extracts containing mixtures of phenolic compounds have been analyzed with the use of hyphenated techniques such as liquid chromatography (HPLC, UPLC) coupled to DAD or PDA, (photodiode array detectors), and time of flight (ToF) or electrospray ionization-ion trap (ESI) mass spectrometers [20,21]. In this context we have analyzed using these precise tools several South American fruits including the white strawberry (Fragaria chiloensis) [22] the mountain papaya (Vasconcellea pubescens) [23], as well as several Mapuche Amerindian's herbal medicines [19,24].
In the present work and following our chemical studies on South American fruits [22,23] phenolic compounds that might be responsible [22] for the antioxidant capacity of the extracts from both peumo plants (C. monogyna and C. alba) with edible fruits growing in the VIII region of Chile were identified by HPLC using UV/visible (DAD) and tandem mass spectrometry detectors (ESI-MS-MS). For this purposes the methanolic extracts (see experimental) were injected into the HPLC system to obtain the HPLC-DAD chromatograms ( Figure 2). For mass spectrometry analysis all compounds were detected in both ESI positive and negative modes. Since both fruits have a red-brown color and taking into account that the orange or red pigmentation of fruits were due generally to anthocyanins (as in blueberries, strawberries, cherries, etc.) or carotenoids (as in tomato, carrots, chiles, physalis, etc.) we searched for these compounds in the fruits under study. We found several anthocyanin derivatives (Figures 3 and 4) that can be responsible for the red pigmentation in Crataegus monogyna (German peumo) fruits. However, we were not able to find any of those pigments (anthocyanins or carotenoids) in detectable amounts in Cryptocarya alba (Chilean peumo) fruits. The color of the peel of this species can thus be produced by tannins or a combination of other compounds detected in this species, since we found several groups of flavanols, C-and O-glycoside flavonoids and phenolic acids ( Table 2). The mobile phase used was acidic in order to avoid the broadening of peaks due to the presence of the deprotonated form of the acid groups (carboxylic and phenolic) and to improve the retention of those compounds in the HPLC column. In addition, anthocyanins are stable in the flavilium form at a pH 1-4, so these compounds were detected in ESI positive mode, while the other phenolic compounds were detected in negative mode. In particular using the ESI ion trap detector, we could analyze cross-ring cleavages of sugar residues of three C-glycosyl flavones which produced main MS ions [25] that allowed differentiation with several O-glycosyl flavones detected ( Table 2). The HPLC DAD fingerprints from the methanolic extracts of the fruits and leaves of both species are shown in Figure 2, the structures of the tentatively identified compounds are presented in Figure 5 and MS spectra are shown in Figures 5-11. In this study we identified or tentatively identified 4 anthocyanins (peaks 24-
Phenolic Acids and Related Phenolic Compounds
Peak 2 with a molecular anion at m/z 191 was identified as quinic acid (MS 2 at m/z 110), while peak 6 was assigned as chlorogenic acid (5-O-caffeoyl quinic acid, Figure 6) [26] by co-elution with authentic compound. Peak 7 present in the same fruits, with a MW of 368 a.m.u. could be assigned as feruloyl quinic acid [27], however the presence of an entire caffeic acid ion at m/z 179 (with MS 3 at m/z 135) instead of a quinic acid ion at m/z 191 in MS experiments led to the assignment of the compound as methyl (5-caffeoyl)quinate ( Figure 6). Peaks 12 and 18 with the same UV and MS characteristics as peak 7 could be assigned as the other isomers of this compound, methyl (3-caffeoyl)quinate ( Figure 6) and methyl (4-caffeoyl)quinate, respectively [28]. Peak 10 was assigned as the hydroxycinnamic acid derivative sinapoyl glucose [29].
LC-DAD analyses were carried out using a Merck-Hitachi equipment with a quaternary L-7100 pump, a L-7455 UV diode array detector, and a D-7000 chromato-integrator (LaChrom, Tokyo, Japan). A 250 × 4.6 mm i.d., 5 m, Purospher star-C18 column (Merck, Germany) set at 25 °C was used for the separation of all phenolics. Detection was carried out at 280, 354 and 520 nm, with peak scanning between 200 and 600 nm. Gradient elution was performed with water/1% formic acid
Plant Material
The study was carried out with ripe fruits and aerial parts (leaves and stems) of Cryptocarya alba (Molina) Looser (local name: peumo chileno), and Crataegus monogyna (Molina) A. Gray (local name: peumo Alemán), which were collected by Luis Bermedo Guzmán and Mario J. Simirgiotis in Re-Re, Región del Bio-Bio, Chile in May 2011. Examples were deposited at the Laboratorio de Productos Naturales, Universidad de Antofagasta, Antofagasta, Chile, with the numbers Ca-111505-1 and Cm-111505-1, respectively.
Sample Preparation
Fresh peumo fruits and aerial parts (leaves and stems) were separately homogenized in a blender and freeze-dried (Labconco Freezone 4.5 L, Kansas, MO, USA). One gram of lyophilized material was finally pulverized in a mortar and extracted thrice with 25 mL of 0.1 % HCl in MeOH in the dark for one hour each time. The extracts were combined, filtered and evaporated in vacuo (40 °C). The extracts were suspended in 10 mL ultrapure water and loaded onto a reverse phase solid phase extraction cartridge (SPE, Varian Bond Elut C-18, 500 mg/6 mL). The cartridge was rinsed with water (10 mL) and phenolic compounds were eluted with 10 mL MeOH acidified with 0.1 % HCl. The solutions were evaporated to dryness under reduced pressure to give 184.6 mg of C. alba fruits, 127.7 mg of C. alba aerial parts, 146.8 mg of C. monogyna fruits and 118.3 mg of C. monogyna aerial parts, respectively (for extraction yields see Table 1). The extracts were then dissolved in MeOH:water 7:3 (approximately 2 mg/mL) filtered through a 0.45 m micropore membrane (PTFE, Waters) before use and 20 l were injected into the HPLC instrument for analysis.
Polyphenolic Content
A precisely weighed amount of each extract (approximately 2 mg/mL) as explained in Section 3.3 was used for total phenolic (TPC) and total flavonoid (TFC) content. Extracts were dissolved in a MeOH:water 7:3 v/v solution. Appropriate dilutions were prepared and absorbance was measured using a spectrophotometer (see section 3.1). The TPCs were determined by the Folin and Ciocalteu's reagent method [42]. Briefly, the appropriate extract dilution was oxidized with the Folin-Ciocalteu reagent (2 mL, 10 % v/v), and the reaction was neutralized with sodium carbonate. The calibration curve was performed with gallic acid (concentrations ranging from 16.0 to 500.0 g/mL, R 2 = 0.999). The absorbance of the resulting blue color of the complex formed was measured at 740 nm after 30 min, and the results were expressed as mg of gallic acid equivalents per g dry material. The TFCs in the samples were determined as previously reported [43]. The absorbance of the reaction mixture (2.5 mL) was measured at 430 nm and quercetin was used as a reference for the calibration curve (concentrations ranging from 16.0 to 800.0 µg/mL, R 2 = 0.994). Results were expressed as mg quercetin equivalents per g dry weight. Data are reported as mean ± SD for at least three replications.
Bleaching of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) Radical Assay
Free radical scavenging capacity was evaluated according to the method described previously [27] Briefly, aliquots of samples (100 L) were assessed by their reactivity with a methanol solution of 100 M DPPH. The reaction mixtures (2 mL) were kept for 30 min at room temperature in the dark. The decrease in the absorbance (n = 3) was measured at 517 nm, in a Unico 2800 UV-vis spectrophotometer (Shanghai, Unico instruments, Co, Ltd). The percent DPPH scavenging ability was calculated as: DPPH scavenging ability = (A control -A sample /A control ) × 100. Afterwards, a curve of % DPPH scavenging capacity versus concentration was plotted and IC 50 values were calculated. IC 50 denotes the concentration of sample required to scavenge 50 % of DPPH free radicals. The lower the IC 50 value the more powerful the antioxidant capacity. If IC 50 ≤ 50 g/mL the sample has high antioxidant capacity, if 50 g/mL < IC 50 ≤ 100 g/mL the sample has moderate antioxidant capacity and if IC 50 > 200 g/mL the sample has no relevant antioxidant capacity. In this assay, the standard antioxidant compound gallic acid showed an IC 50 value of 1.16 g/mL (6.81 M).
Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP assay was done according to [44] with some modifications. The stock solutions included 300 mM acetate buffer pH 3.6, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl, and 20 mM FeCl 3 ·6H 2 O solution. The working solution was prepared by mixing 50 mL acetate buffer, 10 mL TPTZ solution, and 15 mL FeCl 3 ·6H 2 O solution and then warmed at 37 °C before using. Tumbo fruit extracts (100 L) were allowed to react with 2 mL of the fresh FRAP solution for 30 min in the dark. Readings of the coloured product ferrous tripyridyltriazine complex were then taken at 593 nm (n = 3). The standard curve was performed with the standard antioxidant Trolox (R 2 = 0.9995). Results are expressed in mM TE (Trolox equivalents)/ g dry mass.
Statistical Analysis
The statistical analysis was carried out using the originPro 9.0 software packages (Originlab Corporation, Northampton, MA, USA). The determination was repeated at least three times for each sample solution. Analysis of variance was performed using one way ANOVA and Tukey test (p values < 0.05 were regarded as significant).
Conclusions
The HPLC fingerprints showed in this work can be used to authenticate and differentiate the edible fruits of the two species called peumo from the VIII region of Chile, which are similar in appearance and are grown in the same location and used for similar food purposes. Furthermore, based on our LC/DAD and LC/MS experiments, the distribution of different phenolics in the two species has been analyzed and a total of 33 phenolic compounds were detected and characterized, or tentatively identified for the first time for both species from Chile (19 of those detected in C. alba and 23 in C. monogyna) many of which have not been described hitherto in these plant materials, especially for C. alba. The extracts obtained from C. alba fruits (Chilean peumo) and aerial parts showed high antioxidant capacity which is three times lower to that found for C. monogyna fruits, but was higher for aerial parts, which might be related with the number of phenolic compounds and total phenolic content found in these extracts. The compounds identified can be also used as biomarkers especially for C. alba since little research has been published for this species. The phenolic profiles of the different plant parts revealed high predominance of flavonoids, which are antioxidant compounds that modulate a variety of beneficial biological events. Therefore, C. alba edible fruits and aerial parts may be considered a source of important phytochemicals (mainly flavonoids and phenolic acids) with bioactive properties to be explored for pharmaceutical applications. | v3-fos-license |
2018-09-15T14:04:28.566Z | 2018-09-12T00:00:00.000 | 52195390 | {
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} | pes2o/s2orc | A microfluidic device for isolating intact chromosomes from single mammalian cells and probing their folding stability by controlling solution conditions
Chromatin folding shows spatio-temporal fluctuations in living undifferentiated cells, but fixed spatial heterogeneity in differentiated cells. However, little is known about variation in folding stability along the chromatin fibres during differentiation. In addition, effective methods to investigate folding stability at the single cell level are lacking. In the present study, we developed a microfluidic device that enables non-destructive isolation of chromosomes from single mammalian cells as well as real-time microscopic monitoring of the partial unfolding and stretching of individual chromosomes with increasing salt concentrations under a gentle flow. Using this device, we compared the folding stability of chromosomes between non-differentiated and differentiated cells and found that the salt concentration which induces the chromosome unfolding was lower (≤500 mM NaCl) for chromosomes derived from undifferentiated cells, suggesting that the chromatin folding stability of these cells is lower than that of differentiated cells. In addition, individual unfolded chromosomes, i.e., chromatin fibres, were stretched to 150–800 µm non-destructively under 750 mM NaCl and showed distributions of highly/less folded regions along the fibres. Thus, our technique can provide insights into the aspects of chromatin folding that influence the epigenetic control of cell differentiation.
Results
A microfluidic device with an optically-driven microtool for the extraction of chromosomes and investigation of their stability. For the isolation and investigation of chromosomes from single mammalian cells, a microfluidic device was assembled by bonding a microfabricated polydimethylsiloxane (PDMS, commonly known as silicone rubber) chip onto a coverslip (Fig. 1a). The PDMS chip was fabricated using standard soft lithography 24 , as described in the Supplementary Material. The device had two main channels with micropockets along the side walls and micropillars on the floor of the channels (Fig. 1b). The micropockets served as reaction chambers in which solution conditions were altered by introducing different solutions into the main channels, with solutes diffusing between the main channels and the micropockets [25][26][27][28] . The basic structure of the device was similar to the one reported in our previous study, which focused on yeast spheroplasts 22 . However, since mammalian cells are much larger (diameter: ca. 20-30 µm), the height of the microchannel was increased to 60 µm and larger micropockets were created (entrance width: 50 µm, depth: 250 µm, length of the trapezoid base: 290 µm). Micropillars were used to tether isolated chromosomes by suspending chromosome-attached antibody-conjugated microspheres within the slit between the micropillars against the solution flow in the main channels 22 , which enabled direct observation of changes in the higher-order structures of the isolated chromosomes. It should be noted that the micropillars appear fork-shaped when viewed from the side, but concave or convex (fan-shaped) from viewed from underneath, through the coverslip of the device (Fig. 1b right, 1c (i)). Here, the gap between the micropillars was slightly larger from the coverslip (floor) side to PDMS (ceiling) side ( Fig. 1c(i)), which was due to our photolithographic exposure system. Consequently, microspheres were usually captured at the base of micropillars under the flow (Fig. 1c(i), c(ii)). This ensures that the tethered chromosomes are in a suspended position away from the PDMS ceiling to prevent non-specific binding while permitting maximum exposure to the flow (assuming a plane Poiseuille flow with maximum flow velocity at the half height of the microchannel). Here, emphasis was on achieving chromosome suspension away from the channel ceiling/ floor; the shape of the micropillars was of little importance. To realise this, in the present study, we employed fork-shaped micropillars to achieve both tethering and suspension of the chromosomes within the microchannel, allowing for the direct observation of changes in their higher-order structures. In our previous work, we used convex micropillars with slits for trapping antibody-conjugated microspheres attached to individual chromosomes 22 . Since the fabrication of the convex micropillars was already established in our previous work, we used them as references for assessing the successful fabrication of the concave micropillars used in the present study. For this reason, in Fig. 1b device. It should be noted that, in comparison to the convex micropillars, the newly-designed concave micropillars used in this study improved the visibility of the chromosome, especially around the tethering point. The detailed size and layout of the microstructures are described in the Supplementary Material ( Figure S1).
MethylRO mouse cells expressing a methyl-CpG-binding domain (MBD)-red fluorescent protein (RFP) fusion protein that binds to methylated DNA were used in these experiments (Fig. 2a) 29 . Cell suspension was placed into one of the three inlet wells (Fig. 1a, left end) and sucked into the main channels. A hypotonic solution or a solution of interest was then introduced through the two remaining ports. Using two ports helped to prevent backflow during solution exchange, and the selective use of the three ports reduced the influx of cell debris into the two main channels (details are described in the Methods section). To manipulate cells and chromosomes in the enclosed space of the microchannels and micropockets, optical tweezers were used. Optical tweezers use laser light to trap a micrometre-sized object at the laser focus point formed by an objective lens when the refractive index of the object is greater than that of the surrounding medium 30 . Using this method, cells and compacted chromosomes suspended in aqueous solution can be trapped and translocated individually in a non-destructive manner. However, unfolded chromosomes cannot be trapped by optical tweezers owing to their lower chromatin packing density. Hence, we developed an optically-driven microtool consisting of antibody-conjugated microspheres which can attach to a target protein associated with the chromatin, and which can be manipulated individually by optical tweezers 22 . By utilising the property of antigen-antibody binding, the micromanipulation of individual chromosomes through the microspheres was achieved. In this study, anti-RFP antibody-conjugated microspheres were prepared and used as the optically-driven microtool (Fig. 2b). It should be noted that the peri-centromeric region of mouse chromosomes contains hypermethylated DNA region 31,32 , and the short arms of mouse chromosomes are very short, i.e., chromosomes are telocentric, giving them a narrow V-shaped morphology. Thus, the peri-centromeric region where MBD-RFP proteins accumulate, i.e., the vertex of the V-shaped morphology, is easily captured using the antibody-conjugated microspheres.
The basic procedure for the manipulation of single cells and single chromosomes by the optically-driven microtool within the specially designed microfluidic device is as follows: (a) Schematic illustration of the microfluidic device. The microfabricated PDMS chip (~2 mm thick) with micrometre-scale grooves on one side surface was bonded to a coverslip to form enclosed microchannels. At one end of the microchannels, through-holes (inner diameter: 2.5 mm) were made by punching the PDMS chip to obtain inlet wells. The other end of the microchannels was connected to a silicone tube to form an outlet. Solutions were dropped into the inlet wells and drawn into the main channels by creating suction through the silicone tube. The size of the main channel was as follows: width = 600 μm, height = 60 μm, length = ca. 6 mm. The schematic is not drawn to scale. (b) Scanning electron micrograph of the main channel region of the PDMS chip. Enlarged pictures: a pair of concave micropillars was used in this study. A pair of convex micropillars, first employed in our previous report 22 , was fabricated as a supplementary additional microstructure in the vicinity of thinner concave micropillars and was used as a reference in examining exposure and baking conditions for the fabrication of the mould. Circular micropillars (indicated by red arrowheads) were set at the upper stream side of the slit between another pair of micropillars (used for chromosome tethering) to prevent clogging of the slit by the introduced cells and antibody-conjugated microspheres. (c (i)) Schematic side view of the main channel around the micropillars in a fork-shaped structure for capturing the antibody-conjugated microsphere. The flow direction is from the front of the page to the back, and an antibody-conjugated microsphere is caught in the slit between the micropillars. The gap between micropillars was set to 3 or 5 μm, which was smaller than the diameter of the antibody-conjugated microspheres. (I) Cells synchronised in M phase were introduced into the main channels of the microfluidic device and some cells were individually placed in separate micropockets using optical tweezers (Fig. 2a). Chromosomes-the highly compacted form of individual genomic DNA molecules-in M phase can be individually distinguished and manipulated. (II) The main channels were flushed with isotonic solution to remove excess cells, after which a hypotonic solution was then introduced, causing cells to burst from osmotic shock; as a result, chromosomes were released from the cells. After chromosome extraction in each micropocket, antibody-conjugated microspheres were introduced into the main channels, and individual chromosomes were captured at the peri-centromeric region using optical tweezers. Captured chromosomes were transported from the micropockets to the main channel ( Fig. 2b). (III) Translocated chromosomes were then tethered to the slit between micropillars via the antibody-conjugated microspheres and exposed to solutions of interest, e.g., high-salt solutions, which were introduced into the main channels. Tethered chromosomes were oriented along the direction of the flow; therefore, when the chromosomes unfolded, the spatial resolution along the chromosomes was improved (Fig. 2c).
It should be noted that this tethering method utilising micropillars is suitable for observation of individual non-fragmented chromosomes/chromatins that span multiple frames of a video microscope.
Extraction, translocation, and tethering of chromosomes from single MEFs and ES cells.
Chromosomes were extracted from mouse embryonic fibroblasts (MEFs) (Fig. 3a) and embryonic stem (ES) cells ( Fig. 3b) in each micropocket by introducing a hypotonic solution into the main channels. This resulted in the swelling of cells, which eventually burst within 3-5 min, rapidly releasing intact chromosomes (Supplementary Movie 1). Soon after bursting, Triton X solution was introduced into the device for 10-15 min to lyse the cell membrane. As a result, some chromosomes became dispersed, while others remained attached to one other. In subsequent experiments, we focused on the separated chromosomes, which typically accounted for ≤10% of the total number of chromosomes. It should be noted that there was no major difference between MEFs and ES cells in terms of the success rate for the extraction of individual chromosomes. After the establishment of one-to-one binding between separated chromosomes and anti-RFP antibody-conjugated microspheres, Triton X solution containing anti-RFP antibody (2 µg/ml) was introduced into the main channels for 10-15 min to flush out excess microspheres, and to prevent multiple microspheres from binding to the captured chromosomes as well as the dissociation of MBD-RFP from methylated DNA as the salt concentration increased. Following this, the main channels were once again flushed with Triton X solution and chromosome tethering was performed. Figure 3c shows an example of the translocation and tethering of a chromosome from a MEF (hereafter, MEF chromosomes). The corresponding movie file is available in the Supplementary Material (Movie 2). This tethering process took 5-10 min and 1-3 chromosomes were usually tethered individually at the micropillars. During translocation, the morphology of the captured MEF chromosomes remained unaltered, despite the shear force, and they maintained the typical telocentric chromosome shape (e.g., Fig. 3c left, 6 s).
Chromosomes extracted from ES cells (hereafter, ES cell chromosomes) swelled slightly during lysis of cell membrane with Triton X solution, becoming less compact. In fact, the captured chromosomes became unfolded and stretched (by 5-6 times, ~40 µm in contour length) during translocation using antibody-conjugated microspheres due to hydrodynamic shear force (Supplementary Movie 3), making it almost impossible to tether were extracted from single cells by subjecting the cells to osmotic shock, and some of the released chromosomes were captured and translocated individually to the micropillar region for tethering using optically-driven antibody-conjugated microspheres. (c) Translocated chromosomes were tethered to the slit between micropillars via antibody-conjugated microspheres whose diameter is slightly larger than the gap of the slit. Solutions of interest were sequentially introduced into the main channels (e.g., solutions with stepwise increases in the salt concentration), and the responses, including changes in chromosome unfolding, were evaluated. The schematics are not drawn to scale. them to the slit between the micropillars from their trailing end. To stabilise ES cell chromosome morphology according to a chromosome sorting method 33 , we exposed extracted ES cell chromosomes to a Triton X solution containing 100 mM KCl. The addition of salt prevented the unfolding of chromosomes obtained from ES cells Orange arrows indicate the direction of translocation using optical tweezers. One of the isolated chromosomes was captured by the anti-RFP antibody-conjugated microsphere in the micropocket (0 s). Subsequently, the chromosome (a pair of chromatids) was dragged out of the micropocket using the antibody-conjugated microsphere driven by the optical tweezers (6 s) and transported to the micropillar region in the main channel (13 s). Then, the transported chromosome was moved to approach a slit between the micropillars upstream of the main channel (17 s). With the flow in the main channel, the free ends of the long arms of the trapped chromosome were forced through the slit (21 s). When passing through the slit, the optical trapping ceased, the microsphere moved between the pair of micropillars under flow, and tethering was achieved (27 s Folding stability of MEF chromosomes. After tethering MEF chromosomes to the micropillars, we investigated their folding stability using stepwise increases in the salt concentration (Fig. 4). During the experiments, the flow rate in the main channels was maintained at around 100 µm/s by a water head difference between the solution in the inlet well and the waste solution connected to the outlet of the device via a silicone tube. In our microfluidic device, the channel distance between the solution inlet wells and the positions of tethered chromosomes was approximately 7-8 mm. Thus, it is estimated that tethered chromosomes were exposed to the new conditions within 2 min after solution exchange at the inlet well, considering the effect of the diffusion of solutes.
When the salt concentration of the solution introduced into the device was increased from 0 to 0.5 M, there was no significant change in chromosome morphology. Slight shrinkage of the chromosome was observed upon exposure to 0.5 M NaCl for 4 min (Fig. 4a), which was presumably an artefact of fluorescence visualisation resulting from a decrease in the binding constant of the cationic fluorescent DNA dye YO-PRO-1 at higher salt concentrations 34 . Nonetheless, when the concentration was further increased from 0.5 to 0.75 M, the chromosome gradually swelled, and the length of the long arms increased over time (Fig. 4a, from 4 to 7 min). The swollen chromosome began to unfold and stretch under the influence of the solution flow, and 11 min after the introduction of 0.75 M NaCl, both long arms were stretched to ca. 150 µm, almost 30 times their initial length (in 0 M NaCl). This drastic conformational change occurred at 0.75 M NaCl in all successfully tethered MEF chromosomes (N = 14). It should be noted that MEF chromosomes whose results are outlined above were prepared in a slightly different condition from that of ES cell chromosomes as described in Method. When isolation of MEF chromosomes was carried out under the same conditions as ES cell chromosomes (cells were incubated for 1 h in the presence of demecolcine without Latrunculin A treatment and subsequently washed with 0.5% Triton X after bursting, as described in the Methods section), we could not obtain dispersed MEF chromosomes (as they clumped together). In an alternative experiment, we tried to investigate salt-dependent morphological stability using non-dispersed MEF chromosomes, i.e., chromosomes clumped together in micropockets. However, we did not observe changes in morphology or apparent size even when these chromosomes were exposed to 0.5 M NaCl in the micropocket. On the other hand, when the salt concentration was increased to 0.75 M NaCl, the chromosomes gradually began to swell, and the morphology of individual chromosomes became unclear (Supplementary Figure S2). Thus, considering the range of conditions tested in this study, we established that neither treatment with demecolcine (for 1-4 h), the presence or absence of Latrunculin A, nor treatment with Triton X (0.5-1 wt%) had effect on the critical salt concentration at which the unfolding of the MEF chromosomes began. Additionally, at 0.75 M NaCl, non-uniformity of fluorescence intensity along the stretched chromosome was observed, suggesting differences in folding stability along the length of the chromatin fibres. In a separate experiment, we observed further unfolding and stretching of the MEF chromosomes in the presence of 1.5 M NaCl; the chromosome length reached ca. 630 µm, almost 90 times the initial value (Supplementary Figure S3). Moreover, the stretched chromosome exhibited variations in the degree of folding along its length. Considering that the contour length of genomic DNA for the shortest chromosome in mouse (chromosome 19) is ca. 21 mm 35 , the observed length of 630 µm indicates that the chromatin is still highly compacted (more folded, on average, than the 10 nm fibre chromatin structure).
According to earlier studies, most MBDs dissociate from methylated DNA in a 0.5-0.8 M salt solution 36,37 . In this study, using 0.75 and 1.0 M NaCl solutions, most chromosomes detached from the anchored antibody-conjugated microspheres and flowed away, although some remained tethered, even in solutions containing up to 1.5 M NaCl. This was likely due to the formation of multiple bonds between MBD-RFP proteins in the peri-centromeric region and anti-RFP antibodies on the microsphere, which created steric hindrance that prevented chromosome dissociation.
Folding stability of ES cell chromosomes. We examined changes in the higher-order structure of tethered ES cell chromosomes with stepwise increases in salt concentration (Fig. 5). Conformational changes, i.e., unfolding and stretching, occurred immediately upon exposure to the 0.5 M NaCl solution (This drastic conformational change occurred at 0.5 M NaCl in all successfully tethered ES cell chromosomes [N = 5]). This salt concentration was lower than that at which changes in MEF chromosome conformation were induced. In one representative case, contour length of the stretched chromosome was >700 µm (Fig. 5a, 0.5 M NaCl for 8 min), which was ca. 100 times the initial length. When the salt concentration was increased to 0.75 M NaCl, the chromosome stretched even further, to over 800 µm. Two strands of chromatin fibre from the long arms of daughter chromosomes were visible (white arrows), and bright fluorescent spots, i.e., highly folded regions, were distributed along the stretched chromatin fibres. However, since we were unable to distinguish chromosome number, we could not directly compare the differences in the distribution of fluorescent spots between stretched ES cell and MEF chromosomes of the same chromosome number. Nonetheless, ES cell chromosomes also exhibited variation in chromatin folding stability, although the sensitivity to the salt concentration differed between MEF and ES cell chromosomes.
Discussion
The folding of ES cell chromosomes was sufficiently stable for manipulation under flow (≤100 µm/s) with the optically-driven microtool in the presence of 100 mM KCl. It has been reported that arrays of reconstructed nucleosomes show a compact form under quasi-physiological salt conditions (150-200 mM NaCl) relative to the apparent size of complexes seen under lower salt concentrations (≤50 mM NaCl) 38,39 . This folding of reconstituted chromatin comes from a reduction in the electrostatic repulsion between negatively charged chromatin fibres 38 . This explanation can be applied to our experimental results, i.e., the observed stabilization of ES cell chromosome folding. Thus, by direct micromanipulation and imaging of individual native chromosomes in a microchannel, we confirmed the folding stabilisation of chromosomes by tuning the salt concentration. Whereas chromosomes from both cell types exhibited the same highly-compacted morphology in M phase (Fig. 3a, 3b), ES cell chromosomes were relatively less stable under high-salt conditions (Fig. 5) compared to MEF chromosomes (Fig. 4), even though ES cell chromosomes were prepared with the Triton X solution containing 100 mM KCl for morphological stabilization. It has been reported that the chromatin-binding protein heterochromatin protein 1 (HP1) and histone H2B exhibit more hypermobility in mouse ES cells than in primary MEFs, as revealed by fluorescence recovery after photobleaching (FRAP) experiments 40 . Moreover, there was no difference in FRAP recovery of histone H1 0 -green fluorescent protein at any point in the cell cycle of mouse ES cells 41 . FRAP is powerful tool for investigating the turnover of chromatin-associated proteins in living cells 42,43 , although individual chromatin fibres cannot be distinguished owing to crowding within the nucleus. In addition, H1 and HP1 proteins in murine R1 ES, but not neural progenitor cells, were released at low salt concentrations 41 . These reports suggest that folding stability differs significantly between chromosomes from differentiated and undifferentiated cells and support our observations from single cell/single non-fragmented native chromosome-based experiments.
By comparing the degree of folding under 0.75 M NaCl conditions between MEF chromosomes (e.g., Fig. 4a, 0.75 M NaCl for 11 min) and ES cell chromosomes (e.g., Fig. 5a, 0.75 M NaCl for 8 min), it is likely that partially-unfolded MEF chromosomes maintained much higher-order folding structures than did ES cell chromosomes (for the comparison, an additional example is presented in the Supplementary information, Figure S4). This is consistent with previous findings which indicated that mouse ES chromatin shows greater fluctuations in spatio-temporal condensation within the nucleus than does MEF chromatin, which shows a more consistent distribution of chromatin condensation 44 . By single cell/single non-fragmented native chromosome-based experiments, we demonstrate that folding stability varies along the length of individual chromosomes, and that ES cell chromosomes exhibit greater stretching upon the replacement of a 100 mM KCl solution with one containing 0.5 M NaCl as compared to that of MEF chromosomes exposed to solutions with NaCl concentrations increasing from 0 to 0.5 M (Figs 4 and 5). Thus, ES cell chromosomes have more regions that may be easily unfolded, which may be related to the maintenance of a pluripotent state and their capacity to differentiate into any cell type.
In conclusion, we developed a method for investigating the distribution of folding stability along individual chromosomes at single-cell resolution without fragmentation. This system can serve as a powerful tool for evaluating the relationship between the progression of cell differentiation and changes in the distribution of folding stability along chromatin. Future studies will employ appropriate DNA sequence-specific fluorescence probes, such as dCas9-based probes 45,46 , to more clearly observe sites along tethered chromatin fibres and to discriminate between chromosomes, which will provide valuable information for epigenetic studies.
Methods
Cell culture and cell cycle arrest. Mouse embryonic fibroblasts (MEFs) and embryonic stem (ES) cells were obtained from a MethylRO mouse expressing MBD-RFP 29 . Details pertaining to cell culture can be found in the Supplementary Material. MEFs were incubated for 4 h in the presence of 0.2 µg/ml demecolcine (Wako Pure Chemical Industries, Osaka, Japan), an inhibitor of tubulin filament polymerisation. Latrunculin A (Cayman Chemical, Ann Arbor, MI, USA; final concentration: 2 µM), an inhibitor of actin filament polymerisation, and Hoechst 33342 (Dojindo Laboratories, Kumamoto, Japan; final concentration: 5 µM) were added to the cells, followed by incubation for 30 min. ES cells were incubated for 1 h in the presence of 0.2 µg/ml demecolcine before adding Hoechst 33342 (final concentration: 5 µM) for 30 min. Following cell cycle arrest, cells were collected, and the solution was replaced with 300 mM sorbitol containing 5 μM Hoechst 33342 (referred to as isotonic solution in this manuscript). The cells were then immediately introduced into the microfluidic device for experiments. It should be noted that the cell cycle of MEFs is about 24 h, whereas that of ES cells is about 12 h. Therefore, in order to increase the yield of M phase cells, the incubation time of MEFs with demecolcine was made longer than that of ES cells (in fact, even in the case of treatment with MEF for 1 hour, cells in M phase was obtained, but acquisition efficiency was very low). We empirically determined the use of Latrunculin A and 1 wt% Triton X solution (composition is described in the next subsection) was optimal for obtaining dispersed MEF chromosomes that could be micro-manipulated individually using optical tweezers. In fact, under the determined conditions we managed to obtain a few dispersed chromosomes per cell (others remained clumped to each other).
Antibody-conjugated microspheres, solutions for chromosome isolation, and investigation of structural stability. Antibody-conjugated microspheres were prepared as previously described 22 . Briefly, biotin-conjugated anti-RFP antibody (ab34771, rabbit polyclonal anti-RFP, Abcam, Cambridge, UK) and streptavidin-coated microspheres (diameter: 6 μm; PolyScience, Niles, IL, USA) were reacted to obtain anti-RFP antibody-conjugated microspheres that were dispersed in Triton X solution. The obtained microspheres were evaluated by fluorescence-labelled anti-rabbit IgG antibody (described in the Supplementary Material, Table S1). The hypotonic solution used to induce cell bursting was composed of 5 μM Hoechst 33342 and 30 mM dithiothreitol (DTT). The detergent solution for lysing the burst cell membrane (referred to as Triton X solution in this manuscript) was composed of 1 wt% (for MEFs) or 0.5 wt% (for ES cells) Triton X-100, 20 mM HEPES-KOH (pH 7.6), 1 mM EDTA, 30 mM DTT, and 0.5 µM YO-PRO-1. To investigate the stability of the higher-order structure of tethered chromosomes, Triton X solutions containing 0, 0.5, 0.75, 1, or 1.5 M NaCl were employed.
Chromosome extraction in the microfluidic device. Selective use of the three ports of the microfluidic device reduced the influx of cell debris into the two main channels. First, the interior of the microfluidic device was filled with isotonic solution, after which the cell suspension was introduced into inlet well #1 or #2 (indicated in Fig. 1a), and sucked into the main channels. After placing cells in M phase into the micropockets by optical tweezers, the inlet well from which the cells were introduced was sealed using a silicone rubber sheet SCIEntIFIC RepoRts | (2018) 8:13684 | DOI:10.1038/s41598-018-31975-5 with a thickness of 1 mm. This was followed by introducing an isotonic solution from the other two inlet wells to flush out excess cells from the main channels. Hypotonic solution was then introduced from inlet well #3. By closing the inlet well used to introduce cells, influx of cell debris was reduced. Usually, when the inlet #1 was used for cell introduction, the main channel #2′ contained less cell debris, and when inlet #2 was used, the main channel #1′ contained less cell debris. Using two ports helped to prevent backflow in the main channels during solution exchange. The problem with backflow was that it usually dislodged the chromosome-attached antibody-conjugated microspheres from the tethering slits or caused entanglement of the tethered chromosomes/ stretched chromatin fibres. The loading solutions, e.g., high-salt solutions, were replaced in each inlet well one by one. This procedure of solution exchange restricted backflow within the microfluidic channels to branch channels between the inlet ports and main channels. | v3-fos-license |
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} | pes2o/s2orc | Nrf2 Is a Key Regulator on Puerarin Preventing Cardiac Fibrosis and Upregulating Metabolic Enzymes UGT1A1 in Rats
Puerarin is an isoflavone isolated from Radix puerariae. Emerging evidence shown that puerarin possesses therapeutic benefits that aid in the prevention of cardiovascular diseases. In this study, we evaluated the effects of puerarin on oxidative stress and cardiac fibrosis induced by abdominal aortic banding (AB) and angiotensin II (AngII). We also investigated the mechanisms underlying this phenomenon. The results of histopathological analysis, as well as measurements of collagen expression and cardiac fibroblast proliferation indicated that puerarin administration significantly inhibited cardiac fibrosis induced by AB and AngII. These effects of puerarin may reflect activation of Nrf2/ROS pathway. This hypothesis is supported by observed decreases of reactive oxygen species (ROS), decreases Keap 1, increases Nrf2 expression and nuclear translocation, and decreases of collagen expressions in cardiac fibroblasts treated with a combination of puerarin and AngII. Inhibition of Nrf2 with specific Nrf2 siRNA or Nrf2 inhibitor brusatol attenuated anti-fibrotic and anti-oxidant effects of puerarin. The metabolic effects of puerarin were mediated by Nrf2 through upregulation of UDP-glucuronosyltransferase (UGT) 1A1. The Nrf2 agonist tBHQ upregulated protein expression of UGT1A1 over time in cardiac fibroblasts. Treatment with Nrf2 siRNA or brusatol dramatically decreased UGT1A1 expression in puerarin-treated fibroblasts. The results of chromatin immunoprecipitation–qPCR further confirmed that puerarin significantly increased binding of Nrf2 to the promoter region of Ugt1a1. Western blot analysis showed that puerarin significantly inhibited AngII-induced phosphorylation of p38-MAPK. A specific inhibitor of p38-MAPK, SB203580, decreased collagen expression, and ROS generation induced by AngII in cardiac fibroblast. Together, these results suggest that puerarin prevents cardiac fibrosis via activation of Nrf2 and inactivation of p38-MAPK. Nrf2 is the key regulator of anti-fibrotic effects and upregulates metabolic enzymes UGT1A1. Autoregulatory circuits between puerarin and Nrf2-regulated UGT1A1 attenuates side effects associated with treatment, but it does not weaken puerarin’s pharmacological effects.
INTRODUCTION
The heart manifests robust plasticity in the context of heart disease. This process is donated as pathological remodeling (Burchfield et al., 2013). Pathological myocardial remodeling is characterized by excessive accumulation of extracellular matrix, through a process called cardiac fibrosis (Kong et al., 2014;Travers et al., 2016). Physiologically, extracellular matrix provides a structural scaffold of cardiomyocytes, distributes mechanical forces throughout cardiac tissue, and mediates conduction of electrical impulses (Camelliti et al., 2005;Porter and Turner, 2009;Souders et al., 2009). Cardiac fibrosis is a final common pathway of many heart diseases. Extensive cardiac fibrosis increases myocardial stiffness, worsens diastolic function, and eventually results in progression to heart failure (Kong et al., 2014;Travers et al., 2016). Some drugs, including angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, aldosterone antagonists, and β-blockers, reduce morbidity and mortality in patients with chronic systolic heart failure (Fonarow et al., 2011). However, the progression of heart failure cannot be completely suspended. New pharmacological therapies need to be discovered. Puerarin (7,4 -dihydroxyisoflavone-8β-glucopyranoside) is a major active ingredient in the Chinese medicine Pueraria radix which is extracted from the kudzu root [Pueraria lobata (wild) Howe]. Puerarin has been widely prescribed to treat cardiovascular diseases, including hypertension (Tan et al., 2017), coronary heart disease (Xie et al., 2003) and heart failure (Duan et al., 2000). Supporting findings published previously, our laboratory has reported that puerarin may prevent cardiac fibrosis induced by pressure overload (Yuan et al., 2014;Liu et al., 2015;Tan et al., 2017). In a mouse model of cardiac fibrosis, the inhibition of myocardial fibrosis by puerarin involved transforming growth factor (TGF)-β1, monocyte chemoattractant protein (MCP)-1, and peroxisome proliferator-activated receptor (PPAR) α/γ (Chen R. et al., 2012;Tao et al., 2016). Jin et al. (2017) have demonstrated that puerarin mitigates cardiac fibrosis induced by transverse aorta constriction. This protective effect may be attributed to the upregulation of PPAR-γ and inhibition of TGF-β1/Smad2mediated endothelial-to-mesenchymal transition. However, the effects of puerarin on cardiac fibrosis and the related mechanism remain unclear.
Puerarin is largely insoluble in water, so its oral bioavailability is low (Luo et al., 2011a,b). Understanding the metabolic pathway of puerarin may be conducive to illuminating its pharmacological effects. The results published previously by our laboratory indicated that UDP-glucuronosyltransferase (UGT) 1A1 is the primary enzyme responsible for catalysis of puerarin's glucuronidation in human liver microsomes to form its major metabolite, puerarin-7-O-glucuronide (Luo et al., 2012). UGT1A1 can be upregulated by transcription factors such as transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) (Buckley and Klaassen, 2009;Gong et al., 2014). Nrf2 is a member of the cap-n-collar family of transcription factors. It is an essential modulator of celluar detoxification responses and redox status, contributing to antioxidant response elementregulated physiologic expression of numerous genes (Wang et al., 2012). Nrf2 plays an essential role in preventing fibrosis. In mice subjected to transverse aortic constriction surgery, Nrf2 deficiency exacerbated left ventricular fibrosis. Conversely, Nrf2 overexpression inhibits proliferation of cardiac fibroblasts (Li et al., 2009). In this study, we explored the roles of Nrf2 in puerarin's preventive effect against cardiac fibrosis, as well as regulation of UGT1A1 and its pathway, in neonatal rat cardiac fibroblasts (NRCF) induced by AngII and a mouse model of cardiac fibrosis induced by abdominal aortic banding (AB).
Animal Model
Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (United States and National Institutes of Health). Specific pathogen-free Sprague-Dawley rats weighting 150-180 g (Guangdong Medical Laboratory Animal Center, Guangzhou, China) were used. The animal use and care protocol was reviewed and approved by the Ethics Committee of Guangzhou Medical University. Rats were randomly divided into three groups, each of which included six rats: the sham-operated group (Sham), abdominal aortic banding group (AB) and puerarin treatment for 6 weeks in AB rats group (Pue). Myocardial fibrosis was induced by AB . Rats assigned to Sham group underwent a similar procedure, except for arterial ligation. Intraperitoneal injection of puerarin (50 mg/kg/day) was started from 1 week after the AB procedure. Rats in the Sham group received an equal volume of normal saline.
Echocardiography
Following anesthetization with isoflurane, transthoracic two-dimensionally guided M-mode echocardiography was performed, 6 weeks after treatment was administered, by (I) Quantitative analysis of collagen I and collagen III in vivo. Sham, sham-operated group; AB, aortic banding group; Pue, puerarin-treated aortic banding group.
an experienced technician blinded to the study groups. Transthoracic echocardiography was performed with a 250 MHz ultrasound transducer (Vevo 2100, VisualSonics).
Organ Weight
Body weight (BW) and tibia length (TL) of each rat were measured after 6 weeks of puerarin administration. After rats were euthanized using cervical dislocation under anaesthetization, hearts were perfused briefly with 10% KCl to arrest the heart in diastole, then removed. Heart weight to body weight ratio (HW/BW) and heart weight to tibia length ratios (HW/TL) were calculated.
Hematoxylin-Eosin (HE) Staining, Masson's Trichrome Staining and Immunohistochemistry
After fixation with 10% formalin in phosphate-buffered saline (PBS) for 24 h, the heart tissues were subjected to alcoholic dehydration and embedded in 4% paraffin. Heart sections (5 µm) were sliced and subjected to HE and Masson's trichrome staining. Collagen volume fraction (CVF) was determined by Image Pro Plus software to evaluate the degree of myocardial fibrosis. Mean CVF values were determined by one investigator blinded to the group assignment. Immunohistochemical staining was performed as previously described . Briefly, heart sections were stained with anti-collagen I antibody (1:200), anti-collagen III antibody (1:200) and anti-Nrf2 antibody (1:200) at 37 • C for 2 h. After three times wash with PBS, secondary antibody was added. Then the samples were incubated at 37 • C for 2 h and washed with PBS before addition of 3,3 Diaminobenzidine (DAB) for 5 min. After hematoxylin counterstaining, dehydration in graded alcohols and stepping in xylene, neutral gum was used for mounting. Brown granules in the cells were observed by microscope (Nikon Eclipse TS 100, Japan), and six fields were chosen randomly.
Cell Culture and Treatment
Neonatal SD rats (1-2 days old) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China). Primary culture of NRCFs were prepared from ventricles of neonatal rats (Yi et al., 2014). Briefly, hearts were removed from thorax and immediately placed in cold Dulbecco's Hanks' balanced salt solution (D-HBSS), and ventricles were minced, pooled, digested with 0.25% trypsin overnight at 4 • C. At the next day, Dulbecco's Modified Eagle Media (DMEM) medium supplemented with 10% fetal bovine serum (FBS) was added to stop the digestion. Then ventricles were digested with 1% collagenase type II and 5% bovine serum albumin (BSA) for 15 min at 37 • C with rotation at the speed of 250-300 rpm. DMEM with 10% FBS were added to arrest digestion. The cells were collected and suspended in DMEM medium supplemented with 10% FBS and incubated with 95% O 2 + 5% CO 2 . After 2 h, weakly attached or unattached cells were rinsed free and discarded, attached cardiac fibroblasts continued to culture in fresh DMEM medium supplemented with 10% FBS. When the confluence of NRCF in culture wells was up to 80-90%, the cells were digested by 0.25% trypsin and then passaged at 1:3 dilutions. And passages 2-4 were used for the subsequent experiments.
Cell Viability Analysis
Cell viability was measured by using CCK-8. Briefly, NRCF were plated in 96-well plates with a density of 5 × 10 5 cell/well. After different treatments, medium (90 µl) was incubated with 10 µl of CCK-8 solution for 2 h at 37 • C in the dark. Absorbance was determined at 450 nm on a microplate reader (VARIOSKAN LUX, Thermo Scientific, United States).
Immunofluorescence Microscopy
Neonatal rat cardiac fibroblasts were cultured on sterile glass and treated by different agents. NRCF were washed with PBS for once and fixed with 4% formaldehyde in PBS for 15 min at room temperature. The cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and blocked with 5% goat serum for 1 h at room temperature and then incubated with anti-Nrf2 antibody (1:200, CST) overnight at 4 • C. NRCF were incubated with the fluorescent secondary antibody in 3% BSA in PBS and counterstained with DAPI for 10 min at room temperature in the dark. The cells were imaged with an inverted fluorescence microscope (Nikon Eclipse Ni-u, Japan). Green fluorescence was considered a marker of Nrf2 positivity.
Chromatin Immunoprecipitation (ChIP) Assay
Chromatin immunoprecipitation analysis was performed using a Chromatin Immunoprecipitation Kit (EZ-ChIP, Catalog # 17-371, Thermo Fisher Scientific, United States) according to manufacturer's instruction. Briefly, cells were treated with 1% formaldehyde for 15 min for crosslinking. Then sonication was performed to shear the chromatin to a 200-1000 bp of DNA. And the size of DNA was verified by agarose gel electrophoresis. Next, chromatin samples were immunoprecipitated using anti-Nrf2 antibody (1:100, CST). Immunoprecipitated DNA was purified and amplified across the Ugt1a1 promoter region by Real-time PCR using primers: forward: CATCCTCAAAGGGCCTGATTTAT and reverse: GGTTTCAAGATGGCAGCTGAG.
Measurement of Intracellular Reactive Oxygen Species in Cardiac Fibroblasts
The level of intracellular reactive oxygen species (ROS) was measured using the ROSs Assay Kit. NRCF were plated in 24-well plates at a density of 5 × 10 5 cell/well. After different treatments, medium was removed, and the cells were washed with PBS. A solution of 10 µM fluorescent probe 2 ,7 -dichlorofluorescin diacetate (DCFH-DA) in protein-, serum-free medium was added for 30 min at 37 • C in the dark. Then intracellular ROS were detected by immunofluorescence microscope. The OD value of intracellular ROS was also checked by fluorometer in opaque-walled 96 well plates after different treatment.
Statistical Analyses
Data were expressed as the means ± standard error (SEM). The differences in means between groups were evaluated using one-way analysis of variance (ANOVA), followed by the Tukey-Kramer HSD post hoc test for multiple comparisons. Differences with p < 0.05 were considered statistically significant.
Puerarin Inhibited AB Induced-Cardiac Fibrosis in Rats
Rats subjected to AB surgery 7 weeks showed cardiac hypertrophy and myocardial remodeling as evidenced by increased cardiac mass (Figure 1A), myocyte cross sectional area (Figure 1B), heart weight/body weight (HW/BW) ratio, and heart weight/tibial length (HW/TL) ratio ( Figure 1C) compared to sham. These measurements were significantly decreased in puerarin-treated rats. Comparison of ultrasonic data (Figures 1D,E,F) between groups revealed no obvious trend in left ventricular ejection fraction (LVEF) or left ventricular fractional shortening (LVFS) (Figure 1E). Compared to Sham, AB animals showed increased left ventricular posterior wall dimension (LVPWd), interventricular end-diastolic septum thickness (IVSd), and interventricular end-systolic septum thickness (IVSs). However, AB animals showed decreased left ventricular internal end-diastolic diameter (LVIDd) and end-systole diameter (LVIDs). Puerarin could reverse these changes in LVPWd, IVSd, and IVSs, but not in LVIDd and LVIDs ( Figure 1F). AB rats also exhibited manifest cardiac fibrosis as evidenced by collagen deposit, increase of collagen volume fraction (Figures 1G,H), and increasing collagen I and collagen III (Figures 1G,I). Puerarin significantly attenuated cardiac fibrosis response induced by AB (Figures 1G-I).
Puerarin Inhibited the Proliferation of Cultured Neonatal Rat Cardiac Fibroblasts
In order to investigate the mechanism of puerarin protecting against cardiac fibrosis, we did some experiments in cardiac fibroblasts. First of all, we explored the effective concentration of AngII and puerarin by CCK-8 assay. NRCF were treated with different concentrations of AngII (0.1-10 µM) for 24 h. The results showed that 1 µM AngII significantly promoted the cell proliferation (Figure 2A) as similar to the previous report (Stacy et al., 2007). So, 1 µM of AngII was selected to establish a cell model of cardiac fibrosis. Then, NRCF were pre-incubated with various concentrations of puerarin (10-1000 µM) for 24 h. 1000 µM of puerarin reduced the cell viability, but not for 1-100 µM of puerarin (Supplementary Figure S2). Treatment with puerarin inhibited AngII-induced cell proliferation of NRCF. This effect was concentration-dependent ( Figure 2B). Based on the results, a 100 µM dose of puerarin was used for subsequent experiments. Similar concentration was selected in other in vitro studies (Yeung et al., 2006;Chen Y.-Y. et al., 2012).
Puerarin Protected Against Cardiac Fibrosis Through Nrf2/ROS Pathway in Cultured Neonatal Rat Cardiac Fibroblasts
It is widely agreed that oxidant stress participated in the activation or differentiation of fibroblasts. We used AngII to induce oxidative stress, and ROS was detected by fluorescent probe DCFH-DA and the relative fluorescence intensity (OD value) was assessed. As shown in Figure 3, the relative fluorescence intensity of ROS increased significantly when NRCF were treated with AngII. Puerarin, pretreating before AngII administration, decreased the relative fluorescence intensity of ROS in NRCF.
Nrf2 mediates the cell's reaction to oxidative stress by binding to an antioxidant responsive element (ARE). Loss of Nrf2 results in increased susceptibility to reactive oxygen in both cardiac fibroblasts and cardiomyocytes (Li et al., 2009). A detailed examination of the time courses of the effect of AngII on Nrf2, collagen I and collagen III were done. It revealed a timedependent downregulation of protein level of Nrf2 after exposure to AngII (Figure 4A). And conversely, a notable raise of collagen I and collagen III expression after AngII treatment (Figure 4A). The immunofluorescence results also showed that AngII reduced the expression of Nrf2 in the nucleus after exposure to AngII for 24 h (Figure 4B).
We then determined the role of puerarin on Nrf2 expression and cardiac fibrosis. We found that compared with AB group, puerarin promoted the expression of Nrf2 in heart tissue (Supplementary Figure S1). As shown in Figure 5A, the time courses of the effect of puerarin on cardiac fibroblasts indicated an increased expression of Nrf2 in time-dependent manner, and a decreased expression of collagen III. After co-incubation of puerarin and AngII 24 h, puerarin markedly increased the protein level of Nrf2 in AngII-treated NRCF, while it decreased the protein level of collagen III ( Figure 5B). Similar to puerarin, an agonist of Nrf2, tBHQ alone or co-treatment with AngII promoted higher protein expression of Nrf2 in a time-dependent manner in NRCF while notably downregulated collagen I and collagen III (Figures 5C,D). These data indicated that puerarin probably activated Nrf2 to attenuate AngII-induced cardiac fibrosis.
To confirm that whether Nrf2 participated in the protective effect of puerarin against cardiac fibrosis, cardiac fibroblasts were exposed to Nrf2 siRNA or AngII, Brusatol (an inhibitor of Nrf2) and AngII alone or combination with puerarin. Results shown a markedly depression of Nrf2 and significant raise of collagen I and collagen III in 24 h after exposure to coincubation of siNrf2, puerarin and AngII (Figure 5B), or co-treatment of brusatol, puerarin and AngII ( Figure 5D) in NRCF.
We further detected whether Nrf2 participated in the antioxidant effects of puerarin. As shown in Figure 5E, AngII treatment increased the relative intensity of ROS, which was blocked by puerarin treatment. tBHQ also could decreased the relative fluorescence intensity of ROS induced by AngII. Brusatol blocked the effects of puerarin.
Under normal conditions, Nrf2 is bound in the cytoplasm to Kelch-Like ECH-Associated Protein 1 (Keap 1). Upon stimulation, Nrf2 escapes from Keap 1-mediated repression and is translocated to the nucleus (Gao et al., 2015). Ang II increased the protein level of Keap 1 in NRCF, which was reversed by puerarin. Brusatol blocked the effect of puerarin (Figure 5F). The western blot of nuclear protein and immunofluorescence results also revealed that both puerarin and tBHQ provoked the expression of Nrf2 in the nucleus, and brusatol reversed the increase of Nrf2 in the nucleus induced by puerarin, compared with AngII group. But there was no obvious change in the expression of Nrf2 in the cytoplasm between different groups (Figure 6 and Supplementary Figure S4). It indicated that puerarin enhanced the expression in NRCF treated with Ang II. Simultaneously, puerarin promoted the activation of Nrf2 via downregulation of Keap 1 and translocation of Nrf2 to nucleus. These results suggest that Nrf2/ROS pathway may be an important route for puerarin to fight against cardiac fibrosis.
Puerarin Upregulated UGT1A1 Levels Through Activation of Nrf2 in Cultured Neonatal Rat Cardiac Fibroblasts
UGT1A1 is one of UDP-glucuronosyltransferases. Our previous study has reported that UGT1A1 significantly catalyzed the formation of puerarin metabolites, and its activity was significantly higher than other catalyzing enzyme (Luo et al., 2012). We detected UGT1A1 expression by Western blotting in NRCF which were subjected different treatments. The protein level of UGT1A1 were dramatically upregulated by puerarin or tBHQ treatment in a time-dependent manner (Figures 5A,C). In contrast, a significant decrease in UGT1A1 was observed in NRCF from 3 to 24 h after exposure to AngII (especially at 12 and 24 h) ( Figure 4A). UGT1A1 upregulation was clearly observed after 24 h co-incubation with puerarin and AngII. After siRNA or inhibitor specific downregulation of Nrf2, the puerarin-induced upregulations of UGT1A1 was partially abolished (Figures 5B,D). These data suggested that puerarin upregulated the expression of UGT1A1 via transcription factor Nrf2.
In order to confirm that puerarin increased expression of UGT1A1 via Nrf2, we studied contribution of Nrf2 to Ugt1a1, gene expression using ChIP. The ChIP results shown that puerarin significantly increased Nrf2-associated Ugt1a1 promoter activity (Figure 7, Supplementary Figure S5, and Supplementary Table S1).
Puerarin Protected Against Cardiac Fibrosis Through p38 MAPK in Cultured Neonatal Cardiac Fibroblasts p38-MAPK exerts an important impact on the proliferation of NRCF. We investigated whether p38-MAPK participated in anti-fibrotic effect of puerarin. The results shown that AngII significantly increased phosphorylation of p38-MAPK in NRCF, and puerarin significantly decreased the protein level of phosphorylated p38-MAPK ( Figure 8A). The protein level of p38-MAPK did not show difference in each group ( Figure 8A). SB203580, a specific inhibitor of p38-MAPK, decreased protein levels of collagen I and collagen III, inhibit the proliferation of NRCF induced by AngII basing on puerarin administration (Figures 8A,B). Similar effect of SB203580 on ROS generation was also observed (Figures 8C,D). These results indicated that puerarin prevented the proliferation and oxidative stress of NRCF induced by AngII at least partly via inactivation of p38-MAPK. Some studies reported that p38-MAPK was involved in regulation of the phosphorylation and activation of Nrf2 (Ho et al., 2011). In our study, in the present of puerarin, levels of phosphorylation of p38-MAPK decreased. The upregulation of Nrf2 and UGT1A1 in puerarin treated-NRCF persisted after 24-h exposure to SB203580 24 h. Thus, in the context of cardiac fibrosis, the activation of Nrf2 induced by puerarin is likely independent of the p38-MAPK pathway.
DISCUSSION
Cardiac fibrosis is an important component of cardiac remodeling that may be related to adverse cardiovascular outcomes (Chin et al., 2016). A previous study from our laboratory reported that puerarin decreased deposition of collagen in hypertrophy rats . In the present study, experiments in rats with AB-induced cardiac fibrosis indicated that puerarin could significantly reduce the deposition of collagen I and collagen III.
Frontiers in Pharmacology | www.frontiersin.org the proliferation of NRCF induced by AngII, and reduced AngII-induced increases in expression of collagen I and collagen III. AngII may induce ROS generation and oxidative stress (Lijnen et al., 2012), and the latter of which is involved in cardiac fibrosis (Wu et al., 2016). We have reported that puerarin may prevent oxidative stress in neonatal rat cardiomyocytes induced by AngII (Hou et al., 2017). In the present study, puerarin also decreased ROS generation in NRCF induced by AngII.
Nrf2 is a member of the cap-n-collar family of transcription factors. Under physiologic conditions, Nrf2 is retained in the cytoplasm. Upon activation, Nrf2 rapidly translocates to the nucleus, where it binds to the antioxidant response element (ARE) in the upstream promoter region. Binding promotes transcription of a battery of antioxidant genes (Kensler et al., 2007;Zhou et al., 2014). Accumulating data indicate that cardiac fibrosis involves signaling pathway mediated by Nrf2 (Li et al., 2009. AngII decreased protein expression of Nrf2 in NRCF. In NRCF treated with AngII, puerarin significantly increased protein expression of Nrf2, accompanied by decreases in levels of collagen I, III, and ROS. Immunofluorescence and western blot analyses showed that puerarin increased nuclear levels of Nrf2. Brusatol, an inhibitor of Nrf2, reversed these effects. These results indicated that puerarin enhanced the expression of Nrf2, as well as tansclocation of Nrf2 into nucleus. These results suggest that puerarin prevented cardiac fibrosis via Nrf2/ROS pathway. Interestingly, protein levels of UGT1A1, a major metabolic enzyme of puerarin, exhibited a pattern similar to that of Nrf2 protein expression in NRCF treated by AngII, AngII + puerarin, AngII + puerarin + brusatol. The results of ChIP assay confirmed that puerarin enhanced Nrf2 binding to the Ugt1a1 promoter in NRCF. In rats, puerarin promotes the expression of UGT1A1 via activation of Nrf2. Nrf2 was the common transcription factor for puerarin to protect against cardiac fibrosis and upregulate the metabolic enzyme UGT1A1. After administration of puerarin, drug levels must remain relatively constant. Severe adverse events may occur after intravenous injection of puerarin (Hou et al., 2011). In the present study, we found that puerarin upregulated its major metabolic enzyme UGT1A1 via activation of Nrf2. This autoregulatory circuit helps to maintain the concentration of puerarin within appropriate limits. Certainly, upregulation of UGT1A1 catalyzes metabolism of puerarin to puerarin-7-O-glucuronide. The anti-hypertrophy effect of puerarin-7-O-glucuronide is similar to that of its precursor, puerarin. Therefore, the autoregulatory circuit between puerarin and Nrf2-regulated UGT1A1 do not weaken its pharmacological effects.
The p38-MAPK pathway strongly affects proliferation of NRCF and may be activated by oxidative stress (Akiyama-Uchida et al., 2002;Yin et al., 2015;Hu et al., 2016). In our study, puerarin downregulated phosphorylation of p38-MAPK induced by AngII, but not p-ERK1/2 or p-JNK (Supplementary Figure S3). In the presence of a p38-MAPK inhibitor, protein levels of phosphorylated p38-MAPK in AngIItreated NRCF could not be further inhibited by puerarin. Conversely, Nrf2 protein expression could not be further increased by puerarin. These findings suggested that puerarin prevented cardiac fibrosis induced by Ang II at least partly via inactivation of p38-MAPK, and the activation of Nrf2 by puerarin is likely independent of the p38-MAPK pathway in NRCF. The decreased p38-MAPK phosphorylation (induced by AngII) inhibited collagen expression and cell proliferation in NRCF.
It has been demonstrated that the activation of Nrf2 typically involves the ERK and p38-MAPK pathways. reported that curcumin induced activation of Nrf2 in a p38-dependent manner. Ho et al. (2012) also observed that diallyl sulfide activated Nrf2-driven ARE activation via the p38 pathway. In contrast, some investigators also found that p38-MAPK plays a negative role on Nrf2 activation. It has been reported that activated Nrf2 may induce expression of antioxidant-genes and inhibit expression of adhesion molecule by decreasing phosphorylation/activation of p38 (Huang et al., 2001). Ma et al. (2014) shown that puerarin induced activation of Nrf2 and inhibited phosphorylation of ERK in carbon tetrachloride-induced cell death in mouse kidney. Our study also shown that puerarin activated Nrf2 and inhibit phosphorylation of p38 in AngII-treated NRCF. Hence, it is important to discriminate crosstalk among various signaling pathways involved in the cardioprotective effects of puerarin.
CONCLUSION
Puerarin prevents cardiac fibrosis via downregulation of Keap 1, promoting expression of Nrf2 and its nuclear translocation. The inactivation of p38-MAPK also contributes to the anti-fibrotic effects of puerarin. Nrf2 is the key regulator of anti-fibrotic effects and upregulates metabolic enzymes UGT1A1 in NRCF (Figure 9).
AUTHOR CONTRIBUTIONS
M-SC and C-FL conceived and designed the experiments. S-AC and G-JZ performed the experiments and analyzed the data. X-WL, Y-YH, H-LL, Y-QH, L-RL, YH, and C-WO contributed reagents, materials, and analysis tools. C-FL and NH wrote the paper. | v3-fos-license |
2018-04-03T00:30:42.084Z | 2017-12-04T00:00:00.000 | 22051505 | {
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} | pes2o/s2orc | 6-(Methylsulfonyl) hexyl isothiocyanate as potential chemopreventive agent: molecular and cellular profile in leukaemia cell lines
Numerous laboratory and epidemiological studies show that the risk of developing several types of cancer can be reduced with the employment of natural substances that act with multiple mechanisms. In this context, an important role is played by the isothiocyanates. Recently, 6-(methylsulfonyl)hexyl isothiocyanate (6-MITC), present in the root of Wasabia Japonica, has stimulated the interest of researchers as a chemopreventive agent. In this particular study we have focused on evaluating 6-MITC’s in vitro cytotoxic, cytostatic and cytodifferentiating activities, as well as its pro-apoptotic potential. These effects were investigated by way of flow cytometric analysis of Jurkat and HL-60 cells as well as of healthy lymphocytes extracted from the blood of AVIS donors, in order to verify a potential selectivity of action. The results demonstrate that 6-MITC exerts a stronger cytotoxic effect on tumour cells than on healthy cells. The apoptosis induction exerted by 6-MITC on transformed cells is triggered by an extrinsic pathway, as demonstrated by the statistically significant increase in the percentage of cells with activated caspase-8. It was also observed that 6-MITC is able to limit tumour growth by slowing down and blocking the cell cycle of Jurkat and HL-60 cells respectively, in a dose- and time-related manner, while exerting no activity of any kind on the replication of healthy cells. Finally, by measuring the expression levels of CD-14 and CD-15, 6-MITC showed the ability to induce cytodifferentiation of HL-60 cells into macrophage and granulocytic phenotypes.
INTRODUCTION
In the late 1970's Dr. Sporn coined the scientific term "chemoprevention" to represent the possible prevention, halting or reversing of the cancerogenic process through the use of synthetic or natural compounds [1][2][3]. The development and progression of cancer is associated with a series of events, including dysregulation of tumour suppressor genes, cellular differentiation, excessive proliferation and dysfunction of apoptotic genes [4,5]. In particular, apoptosis, a physiological process of programmed cell death that plays a key role in homeostasis, is suppressed in many cancer cells. Restoring this ability, therefore, is one of the most important strategies for fighting cancer [6][7][8][9]. Phytochemicals derived from edible and medicinal plants have been extensively studied for cancer chemoprevention thanks to their demonstrated ability to modulate the enzymes involved in xenobiotic activation/detoxification, to inhibit cell proliferation and/or to induce apoptosis and the www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 67), pp: 111697-111714 Research Paper differentiation of neoplastic cells [10][11][12][13]. A large number of pure compounds and extracts have also been tested in various experimental models due to their long history of human exposure, high tolerability and low toxicity. In fact, a promising chemopreventive agent should act selectively on cancer cells and cause low toxicity in non-transformed cells [14,15]. In recent years, growing interest has been focused on isothiocyanates (ITCs), the main pharmacologically active ingredients of cruciferous vegetables that are able to modulate a large number of cancer-related targets including cytochrome P450 enzymes, proteins involved in the antioxidant response, tumorigenesis, apoptosis, the cell cycle and metastasis [16,17].
More recently, another ITC has stimulated the interest of researchers: 6-(methylsulfonyl)hexyl isothiocyanate , present in high concentrations in Wasabia Japonica rhizome. Wasabia Japonica, better known as Wasabi, is becoming increasingly important in the scientific field due to the presence, at high concentrations, of numerous ITCs including 2, 4, 6 and 8-MITCs. Among them, 6-MITC is the one present at the highest concentration and is the most interesting bioactive compound. Numerous scientific and epidemiological studies have confirmed, for example, its significant antiinflammatory [18][19][20] and antioxidant [21][22][23] properties, leading to its hypothesised use in chemoprevention. The aim of this study, therefore, was to evaluate the potential of 6-MITC as a chemopreventive agent in leukaemia cell lines. More specifically, its antiproliferative and proapoptotic effects were analysed in human lymphoblastic leukaemia cells (Jurkat cells) and in human promyelocytic leukaemia cells (HL-60 cells). In addition to evaluating its cytostatic and cytotoxic effects on transformed cells, its selectivity for non-transformed human peripheral blood lymphocytes (PBL) was also tested using the same endpoints.
There are two main apoptotic pathways: the extrinsic (or death receptor) pathway, and the intrinsic (or mitochondrial) pathway that is regulated by the activation or deactivation of BCL-2 family genes [8,9,24]. For this reason, possible molecular mechanisms, such as the intrinsic and extrinsic apoptosis pathways, and apoptotic markers regulating certain tumourigenesis and cell proliferation mechanisms, such as p53, BAX/BCL-2 ratio, cytochrome c, cyclin E2 and cyclin D3 levels, were analysed.
Lastly, most tumour cells exhibit an altered ability to mature into adult non-proliferating cells, thus maintaining a high proliferative state. In contrast, the induction of terminal differentiation generates cells with no or limited replicative capacity, which can then be more easily induced towards apoptosis [25][26][27]. The study, therefore, concluded with an evaluation of 6-MITC's ability to stimulate differentiation in HL-60 cells, considered an ideal model for investigating this effect [28].
Determination of the 6-MITC concentrations used in subsequent experiments/Guava ViaCount assay
The research began with a preliminary study of the cytotoxic and cytostatic potential of 6-MITC by way of a Guava ViaCount assay to determine the concentrations of ITC to be used in subsequent experiments. To this end, the Jurkat and HL-60 cells were treated for 24h, 48h and 72h with 6-MITC at concentrations of 0 to 64μM while the PBL cells were treated for 24h. The results obtained demonstrated that necrosis of the cancer cells was restrained up to 16μM at 24h and up to 8μM at 48h and 72h. Moreover, the viability of the healthy cells at 24h remained greater than 50% at all concentrations tested (data not shown).
Effect of 6-MITC on viability, apoptosis and necrosis of Jurkat cells, HL-60 cells and PBL/ guava nexin assay
On the basis of the results previously obtained, a Guava Nexin assay was conducted to measure the percentage of live, apoptotic and necrotic cells following the 6-MITC 0-16μM treatment for 24h, 48h and 72h of Jurkat cells (Table 1), the 6-MITC 0-32μM treatment for 24h, 48h and 72h of HL-60 cells (Table 2), and, in parallel, the 6-MITC 0-128μM treatment for 24h of PBL (Table 3).
Viability
The percentage of live cells measured at 24h and normalised to the viability in control cultures (considered to be 100%), was used to obtain the dose-response curve. The IC 50 value calculated by interpolation was 8.65μM for Jurkat cells, 16μM for HL-60 cells and 86.1μM for PBL, respectively ( Figure 1A, 1B, 1C).
The analysis at 48h and 72h allowed confirmation that an acceptable percentage of live Jurkat and HL-60 cells remained at up to 8μM, even after longer treatment times (Table 1 and Table 2).
Apoptosis
Already at 24h of treatment, Annexin V-PE/7-AAD double staining highlighted a statistically significant increase in Jurkat cells at all concentrations tested. More specifically, with respect to the control cultures at 2μM and 4μM, a population doubling was detected (16.1% vs 7.6% and 16.2% vs 7.6%), while a 3 and 4 times increase was detected at 8μM and 16μM, respectively (25.4% vs 7.6% and 30.3% vs 7.6%) (Table 1 and Figure 2A, 2B). A similar pro-apoptotic effect was observed on the HL-60 cells. In fact, the percentage of apoptotic cells increased in a statistically significant manner at a concentration of 4μM (7.1% vs 5.2% in controls) and at 8μM (12.7% vs 5.2% in controls), while doubling at a concentration of 16μM (12.6% vs 5.2% in controls) ( Table 2 and Figure 2A, 2B). The induction of apoptosis mediated by 6-MITC on tumour cells was both dose-and time-related. Indeed, a larger increase in the fraction of apoptotic cells was recorded after 48h of treatment than at 24h, while -in Jurkat cells -a 3-times increase was recorded at 4μM (15.4% vs 6.1% in controls) and a 6-times increase at 8μM (35.0% vs 6.1% in controls) ( Table 1 and Figure 2C), and -in HL-60 cells -a 5-times increase was recorded at 8μM (22.6% vs 4.8% in controls) ( Table 2 and Figure 2C). In addition, after 72h a further 7-times increase of apoptotic cells was recorded in Jurkat cells (31.6% vs 4.6% in controls) (Table 1 and Figure 2D) and an 8-times increase recorded in HL-60 cells at the highest concentration Data are presented as mean ± SEM of five independent experiments. Figure 2D). To further confirm the 6-MITC's pro-apoptotic effect, nuclear condensation and fragmentation were evaluated by fluorescence microscopy (Figure 3). In order to support the hypothesised selectivity of 6-MITC's action, we proceeded to similarly analyse its pro-apoptotic potential in PBL. The results showed a statistically significant increase in the percentage of apoptotic cells that only started from a concentration of 16μM (17.0% vs 10.6% in controls) and remained constant at 32μM (15.9% vs 10.6% in controls). At the highest concentration tested, 64μM, a reduction in apoptotic cells was observed in favour of the necrotic cell fraction, which nonetheless remained below 50% (Table 3 and Figure 2A, 2B). Comparing the results obtained in the different cell lines, it is evident that 6-MITC induces much stronger cytotoxicity on cancer cells than on healthy cells, through stimulation of an apoptotic mechanism (Figure 2A, 2B, 2C, 2D).
Necrosis
With regard to necrosis, it is important to underline that the results obtained in Jurkat cells further support the hypothesis of pro-apoptotic activity. In fact, at the 8μM concentration, increasing treatment time resulted in a decrease in the percentage of necrotic cells from 10 to 8 to 4 times, while in HL-60 cells the percentage increased (Table 1 and Table 2).
Evaluation of pro-apoptotic pathway triggered by 6-MITC on Jurkat cells and HL-60 cells/Guava Caspase-8 and Guava MitoPotential assay
In order to assess whether the 6-MITC-induced apoptosis was triggered by the extrinsic or the intrinsic pathway, tumour cells were treated for 24h, 48h, and 72h at concentrations of 4μM and 8μM (<IC 50 obtained in Jurkat and HL-60 cells).
The levels of cells with activated caspase-8 revealed that the apoptosis induced by 6-MITC was mediated exclusively by extrinsic pathway activation in both cell lines, while the intrinsic pathway did not seem at all involved. In fact, the percentage increase in apoptotic cells with respect to the control cultures measured by the Guava Caspase-8 Assay in Jurkat cells was statistically significant at both concentrations tested at 24h (9.5% and 24.0% vs 6.5%), 48h (14.0% and 31.5% vs 4.5%) and 72h (17.5% and 37.0% vs 5.5%) ( Figure 4A, 4B, 4C, 4D). This percentage increase in the treated versus control cultures was, moreover, fully comparable to that measured in the previously conducted Guava Nexin Assay. For example, after 72h treatment with 8μM the increase was 7 times ( Figure 2D and 4C), with a similar result recorded in HL-60 cells. The percentage of activated caspase-8 cells was also statistically higher at both concentrations tested at 24h (7.9% and 12.7% vs 4.5% in controls), 48h (7.5% and 27.5% vs 5% in controls) and 72h (8.5% and 48.5% vs 5.5% in controls) ( Figure 5A, 5B, 5C, 5D). Moreover, this percentage was fully comparable to that measured in the previously conducted Guava Nexin Assay, being 8 times higher in the treated versus control cultures after 72h at 8μM ( Figure 2D and 5C). In contrast, the fraction of cells with mitochondrial potential depolarisation in treated cultures matched that in control cultures, in both Jurkat and HL-60 cells ( Figure 4A, 4B, 4C, 4D; Figure 5A, 5B, 5C, 5D).
Effect of 6-MITC on cell cycle progression of Jurkat cells, HL-60 cells and PBL/Guava Cell cycle assay
In order to assess whether the apoptosis induced by 6-MITC was an independent event or subsequent to a cell cycle slowdown/block, Jurkat and HL-60 cells were treated with 4μM and 8μM concentrations for 24h, 48h and 72h. PI staining allowed us to highlight the percentage distribution of cells in the different phases of the cell cycle. In particular, after 24h, Jurkat cells demonstrated a statistically significant percentage reduction of cells in the S phase at both 4μM (11.62% vs 17.4% in controls) and 8μM (8.8% vs 17.4% in controls) ( Figure 6A, 6B).
After 48h of treatment, a statistically significant percentage reduction of cells in the S phase was only observed at the higher concentration tested (11.2% vs 13.7%) ( Figure 6C), while after 72h, no effect on the cell cycle was observed ( Figure 6D).
In HL-60 cells, 6-MITC induced no effect after 24h of treatment ( Figure 7A), while at 48h and 72h a statistically significant increase of cells in the G 0 /G 1 phase was observed at 4μM (70.2% and 69.4% vs 48.2 %) and 8μM (72.7% and 69.4% vs 48.2%) with respect to the control cultures but with no difference in percentage terms between the two treatment times ( Figure 7B, 7C, 7D). At the same time, a statistically significant 6-MITC-induced decrease in the S phase was observed at 48h and 72h at concentrations of 4μM (16.4% and 16.5% vs 33.4% in controls) and 8μM (17% and 14.6% vs 33.4% in controls) ( Figure 7B, 7C, 7D).
These results suggest that 6-MITC has the ability to slow down the Jurkat cell cycle and to induce a true block of the HL-60 cell cycle in G 0 /G 1 , in both cases leading to a subsequent decrease in S phase cells.
Similarly, the potential effects of ITC on the cell cycle of healthy lymphocytes were analysed, with no activity being observed ( Figure 8A, 8B).
Effect of 6-MITC on differentiation of HL-60 cells/analysis of cytodifferentiation
We evaluated 6-MITC's ability to induce cytodifferentiation in HL-60 cells following 24h, 48h and 72h treatment at concentrations of 4μM and 8μM. The results obtained showed that isothiocyanate induces no effect at either concentration after 24h and 48h of treatment (data not shown); however, after 72h of treatment at the higher concentration, it induced a statistically significant increase in differentiated cells of both macrophage and granulocyte phenotypes, more specifically, a 3-times increase in CD-15 positive cells and a 2-times increase in CD-14 positive cells ( Figure 9A, 9B).
Effect of 6-MITC on apoptotic and cell cycle proteins on Jurkat cells and HL-60 cells/analysis of cytochrome C release and cell cycle and apoptotic proteins by flow cytometry (FCM) and by Western immunoblotting (WB)
In order to assess whether the pro-apoptotic and cytostatic ability demonstrated by 6-MITC in Jurkat cells involved modulation of p53 protein, its levels were analysed following treatment at an 8μM concentration for 6h, 24h, 48h and 72h.
As shown in Table 4, the levels of p53 remained unchanged in treated cultures compared to control cultures at all treatment times (Table 4). WB analyses confirmed that 6-MITC's treatment did not affect p53 protein expression level ( Figure 10).
Moreover, in order to check the exclusive involvement of the extrinsic pathway, we also analysed any modulation of the BAX, BCL-2 and cytochrome c proteins in Jurkat and HL-60 cells. The BAX and cytochrome c levels in the treated samples proved perfectly comparable to those of the controls in both cell lines (Table 4 and Table 5). These data corroborate the hypothesis that the pro-apoptotic effect of 6-MITC does not correlate with a loss of mitochondrial transmembrane potential. In contrast, BCL-2 levels increased in the treated cultures with respect to the control cultures, resulting in the BAX/BCL-2 ratio dropping in both cell lines (Table 4 and Table 5).
Since 6-MITC was found to arrest the HL-60 cell cycle in the G 0 /G 1 phase, causing a subsequent decrease of cells in the S phase, we also evaluated its effects in this cell line on the expression of cyclin E2 and the cyclin D3, two proteins involved in the G 1 /S transition phase. As shown in Figure 12, the anti-cyclin E2-PE mean fluorescence intensity remained relatively constant after treatment with 6-MITC at 8μM for 24h, 48h and 72h, while the anticyclin D3-FITC mean fluorescence intensity increased at each treatment time, reaching statistical significance at 48h and 72h ( Figure 11A, 11B, 11C, 11D). WB analyses of Cyclin D3 protein expression level confirmed this trend ( Figure 12). (up) and mitochondrial membrane potential (down) analysis at 72h treatment at 0μM (left) and 8μM (right) (D). Active caspase-8 and altered mitochondrial membrane potential was evaluated by FCM as described in Methods. Each bar represents the mean ± SEM of five independent experiments. Data were analysed using repeated ANOVA followed by Bonferroni post-test. * p<0.05 vs control; ** p<0.01 vs control. Protein levels were evaluated by FCM as described in Methods. Data are presented as mean ± SEM of five independent experiments and analysed using the t-test for paired data. Protein levels were evaluated by FCM as described in Methods. Data are presented as mean ± SEM of five independent experiments and analysed using the t-test for paired data.
DISCUSSION
The aim of this study was to evaluate if 6-MITC elicits chemopreventive activity in leukaemia cell lines.
In recent years there has been an increasing interest in the chemopreventive potential of many naturally occurring substances. In particular, laboratory research and epidemiological studies have shown that the risk of developing various types of cancer may be reduced by the use of compounds that act with multiple mechanisms. Some molecules act early, preventing the activation of pro-carcinogens or favouring the elimination and detoxification of carcinogens through the modulation of biotransformation enzymes; others act on already transformed cells, stimulating apoptosis, arresting/ slowing their proliferation or inducing cytodifferentiation, which represent three fundamental mechanisms of chemoprevention [29].
In this context, a major role is exerted by ITCs, a broad group of highly reactive compounds characterised by a common sulphur-containing functional group (-N = C = S) and a variable alkyl or allyl portion. In particular, 6-MITC, the main ITC derived from the rhizome of Wasabia japonica, has recently stimulated the interest of researchers through its proven anti-inflammatory, antioxidant and neuroprotective properties that have led to its hypothesised use as a chemopreventive agent [23,[30][31][32].
In this study, we evaluated the cytotoxic, cytostatic and cytodifferentiating effects of 6-MITC on cancer cells and checked its selectivity of action by monitoring the same effects on healthy cells. The analysis of the specific mechanism of cell death (apoptosis and/or necrosis) demonstrated 6-MITC's ability to induce apoptosis in a dose-and time-dependent manner in both cell lines tested. In fact, at the highest concentration tested for the longest treatment time, the fraction of apoptotic cells increased 7 times in Jurkat cells and 8 times in HL-60 cells in treated cultures with respect to control cultures. Moreover, statistically significant induction of apoptosis occurred in Jurkat cells at 2μM and in HL-60 cells at 4μM, concentrations that are respectively 8 times and 4 times lower than those required to induce apoptosis in PBL. These results suggest that this ITC possesses an important chemopreventive potential in acting selectively on transformed cells and inducing low toxicity in non-transformed cells; this was confirmed by the observation of an IC 50 value in PBL more than 10 times higher than that in Jurkat cells and more than 5 times higher than that in HL-60 cells. It is, therefore, possible to define a range of concentrations in which 6-MITC acts selectively. The apoptotic pathway is generally classified as the intrinsic, mitochondrial, and the extrinsic, death receptor, pathways according to the way of activation. In the extrinsic pathway the activation of death recepror results in the cleavage of pro-caspase-8 and activation of caspase-8 [33]. An analysis of molecular pathways highlighted the interesting capacity of 6-MITC to trigger apoptosis through involvement of the extrinsic pathway, in contrast to many widely studied phytochemicals, such as Hemidesmus Indicus [33], or other ITCs, such as SFN [34,35], phenethyl isothiocyanate [3,36,37] and benzyl isothiocyanate [3,38,39], which all generally modulate the mitochondrial pathway. In fact, after 72h of treatment at 8μM, the increase in activated caspase-8 apoptotic cells in both cell lines perfectly matched that of the Annexin V-PE positive/7-AAD negative cells previously reported. These data suggest that the pro-apoptotic effect of 6-MITC was due exclusively to the involvement of the extrinsic pathway, since the apoptosis did not seem to correlate with a loss of mitochondrial transmembrane potential. This hypothesis is further corroborated by the comparable number of cells with a depolarised mitochondrial membrane potential observed in the treated and control cultures. BAX and BCL-2 are two proteins located on the mitochondrial membrane. More specifically, BAX exerts pro-apoptotic activity while BCL-2 is an anti-apoptotic protein that inhibits apoptosis and is overexpressed in cancer [40,41]. Most targeted cancer therapies are based on stimulating the expression of BAX protein and/or suppressing BCL-2 protein. Conversely, in this study the BAX and cytochrome c levels remained unaltered in both the control and treated cultures, while BCL-2 expression was up-regulated in the treated cultures, resulting in a reduced BAX/BCL-2 ratio. This effect could represent a possible attempt at resistance by the cancer cells through a compensatory mechanism in response to the apoptosis induced by the ITC, which is, however, able to induce it by triggering the extrinsic pathway.
6-MITC exhibited antiproliferative effects in both cell lines, as evidenced by the distribution of cells in the different phases of the cell cycle. In particular, it is able to limit Jurkat cell replication by slowing down the cell cycle, causing a resultant reduction in the percentage of S phase cells after 24h of treatment, statistically significant at both tested concentrations; this reduction remained observable after 48h only at the highest concentration tested, and was no longer visible after 72h. These results also suggest a cross-talk between apoptosis and cell cycle regulation. In fact, the different treatment times have allowed us to detect a statistically significant percentage of apoptotic cells at 24h, which continues to increase after 48h and 72h, while the decrease in S phase cells, statistically significant at 24h, gradually disappears as time progresses, so the apoptotic cells observed are either directly induced or those who exit from the cell cycle slowing-down.
6-MITC has also been shown to be able to induce a potent inhibitory effect on HL-60 cell proliferation, resulting in a blockage of the cell cycle's progression in the G 1 phase, statistically significant after 48h of treatment and remaining constant after 72h. At the same time, a statistically significant decrease in the S phase was observed. In human cells, cell cycle progression is controlled at three checkpoints -G 1 , S, G 2 /M -by a series of cyclins and cyclin-dependent kinase (CDK) complexes [41][42][43][44]. In particular the progression from G0/G1 phase to S phase is regulated by D-type cyclins and E-type cyclins. D-type cyclins acting precociously in the progression through the G1 phase while E-type cyclins become up regulated later during the transition [45]. In the current study, 6-MITC increased the expression of cyclin D3 but did not modulate cyclin E2 levels, A possible for 24h (A), 48h (B) and 72h (C) with 6-MITC 8μM. Cell lysates were immunoblotted with anticyclin D3 antibodies as reported in Methods. Results of scanning densitometry analysis performed on three independent autoradiographs are presented. Relative amounts, presented as means ± SEM, were normalized to the intensity of β-tubulin and reported as fold increase vs control. Data were analysed using the t-test for paired data. * p<0.05 vs control. explanation could be that 6-MITC blocks HL-60 cellcycle acting precociously on the G1/S transition and the potential critical factors for G 1 arrest is cyclin D3.
Recently Wang et al. [46] underpin that the overexpression of cyclins lead sometimes to apoptosis. So we hypothesize that cyclin D3 over-expression might be involved also in 6-MITC-induced apoptosis.
Moreover, the different treatment times also in this case allowed us to hypothesise that the cytotoxic and cytostatic effects exhibited by 6-MITC on HL-60 cells were connected. In fact 6-MITC stimulates apoptosis either as a direct action or indirectly through the blockage of the cell cycle. Further confirming its selectivity of action, is the fact that 6-MITC does not exercise any kind of activity on healthy cell proliferation.
In light of these demonstrated activities, p53 expression levels were analysed in Jurkat cells. p53, in fact, is a tumour suppressor gene that plays an important role in the cell cycle and apoptosis. In non-transformed cells, p53 levels are normally low and rise as a result of DNA damage or other injuries. Its elevation causes a slowdown/blocking of the cell cycle and/or apoptosis induction. However, most tumour cells are p53-mutated or p53-null and, consequently, proliferate indiscriminately and beyond the normal mechanisms of cell survival regulation [9,47], so making an investigation of this aspect particularly interesting. Indeed, ITC may be able to modulate the expression of p53 on p53-mutated cells (Jurkat) while, alternatively, exercising its effect without any involvement, as demonstrated in HL-60 cells that are p53-null. Data analysis showed no change in p53 levels at any treatment time, so supporting the hypothesis that ITC induces cytostatic and cytotoxic effects with an independent p53 mechanism.
A large number of malignant cells undergo mitosis, and these cells are poorly differentiated [25,27]. A chemopreventive agent could act as differentiation inducers, stimulating the differentiation of transformed and immature cells into normal and mature cells. By measuring the expression levels of CD-14 and CD-15 (membrane proteins characteristic of macrophages and granulocytes respectively), 6-MITC demonstrated an ability to induce cytodifferentiation of promyelocytic cells into both macrophage and granulocyte phenotypes after the longer treatment time.
Overall, the results obtained and summarised in the table shown in Figure 13 demonstrated that 6-MITC is able to modulate many of the molecular and cellular pathways constituting the main chemopreventive mechanisms.
Cell cultures Jurkat
Jurkat cells (acute T lymphoblastic leukaemia) were grown at 37°C and 5% CO 2 in RPMI-1640 supplemented with 10% FBS, 1% PS, and 1% L-GLU. To maintain exponential growth, the cultures were divided every third day in fresh medium.
The cell density of Jurkat did not exceed the critical value of 3x10 6 cells/ml of medium and, for every 6-MITC treatment concentration and time, were seeded at 3.75x10 5 cells/ml.
To reduce their spontaneous differentiation, the HL-60 cells were never allowed to exceed a density of 1.0x10 6 cells/ml and, for every 6-MITC's treatment concentration and time, were seeded at 1.25x10 5 cells/ml.
PBL
Authorization to the use of human blood samples (Buffy coat), for research purposes, has been obtained from AUSL Bologna IT, S. Orsola-Malpighi Hospital -PROT GEN n° 0051937, and informed consent was obtained by AUSL Bologna IT, S. Orsola-Malpighi Hospital from donors for the use of their blood for scientific research purposes.
PBL were isolated using density gradient centrifugation with Histopaque-1077 from the whole peripheral blood of 5 AVIS (Italian Voluntary Blood Donors Association) donors. The donors had the following characteristics: under the age of 35, healthy, non-smoker and with no known exposure to genotoxic chemicals or radiation. The PBL were cultured at 37°C and 5% CO 2 in RPMI-1640 supplemented with 1% PS, 15% FBS, 1% L-GLU, and 0.5% PHA. The cell density of PBL did not exceed the critical value of 1x10 6 cells/ml of medium and, for every 6-MITC treatment concentration and time, were seeded at 1.5x10 5 cells/ml.
FCM
All FCM analyses were performed using a Guava easyCyte 5HT flow cytometer equipped with a class IIIb laser operating at 488 nm (Merk Millipore, Darmstadt, Germany).
Guava ViaCount assay
The cellular density and percentage of viable cells were assessed by FCM and analysed using Guava ViaCount software. After treatment, Guava ViaCount Reagent was added to the cells to discriminate viable from dead cells; the reagent contains the dye propidium iodide (PI), which is only able to penetrate the altered membrane of necrotic cells, bind covalently to the DNA and emit red fluorescence. In contrast, cells with an integral membrane are impermeable to PI and, thus, emit low red fluorescence. The obtained results were expressed as total cells/ml and as the percentage of live cells in treated cultures compared to that in the control cultures. www.impactjournals.com/oncotarget
Guava nexin assay
The percentage of apoptotic cells was assessed by FCM and analysed using Guava Nexin software. After treatment, Guava Nexin Reagent was added to the cells: the reagent contains two dyes, 7-aminoactinomycin D (7-AAD) and Annexin-V-PE. As previously described for PI, 7-AAD allows the discrimination between live and dead cells, while Annexin-V-PE allows the identification of apoptotic cells by binding to phosphatidylserine and emitting yellow fluorescence. More specifically, live cells are negative to both 7-AAD and Annexin-V-PE, apoptotic cells are 7-AAD negative and Annexin-V-PE positive, and necrotic cells are positive to both 7-AAD and Annexin-V-PE. The obtained results were expressed as the percentage of apoptotic cells in treated cultures compared to that in the control cultures.
Guava caspase-8 assay
The percentage of cells with activated caspase-8 was assessed by FCM and analysed using Guava Caspase software. Guava Caspase-8 Reagent was added to the cells: the reagent contains two dyes, FLICA (an inhibitor of caspase-8) linked to FAM, and 7-AAD. As previously described, 7-AAD allows the discrimination between live and dead cells, while FLICA is cell permeable. Once inside the cell, FLICA binds covalently to the activated caspase-8 and emits green fluorescence. More specifically, live cells are negative to both 7-AAD and FLICA, cells with activated caspase-8 are 7-AAD negative and FLICA positive, and necrotic cells are positive to both 7-AAD and FLICA. The obtained results were expressed as the percentage of cells with activated caspase-8 in treated cultures compared to that in the control cultures.
Guava MitoPotential assay
The percentage of apoptotic cells with an altered mitochondrial membrane potential was assessed by FCM and analysed using Guava MitoPotential software. Cells were stained with the Guava MitoPotential Reagent that contains two dyes, JC-1 and 7-AAD. 7-AAD allows the discrimination between live and dead cells, as previously described, while JC-1 is a cell-permeant cationic dye that fluoresces either green or orange depending upon the mitochondrial membrane potential. More specifically, live cells (polarised cells) are 7-AAD negative and orange JC-1 positive, apoptotic cells (depolarised cells) are 7-AAD negative and green JC-1 positive, and necrotic cells are 7-AAD positive and green JC-1 positive. The obtained results were expressed as the percentage of apoptotic cells with an altered mitochondrial membrane potential in treated cultures compared to that in the control cultures.
Guava cell cycle assay
The percentage of cells in each stage of the cell cycle was assessed by FCM and analysed using Guava Cell Cycle software. After treatment, cells were fixed and permeabilised with ice-cold 70% ethanol and washed with PBS. The cultures were then suspended in Guava Cell Cycle Reagent that contains the dye PI. PI is able to penetrate the membrane of cells, bind covalently to DNA and emit red fluorescence. More specifically, cells initially in the G 0 /G 1 phase begin to synthesise DNA in the S phase, until complete duplication in the G 2 /M phase. For this reason, cells in the G 2 /M phase have a double fluorescence compared to those in the G 0 /G 1 phase, while cells in the S phase have an intermediate fluorescence.
The obtained results were expressed as the percentage of cells in each of the different stages of the cell cycle in treated cultures compared to those in the control cultures.
Analysis of cytodifferentiation
The percentage of CD-14 or CD-15 positive cells was assessed by FCM and analysed using Guava InCyte software. Upon conclusion of the treatment time, cells were washed with ice-cold PBS. Cells were then incubated with Anti-CD-14-FITC or Anti-CD-15-FITC and washed. The obtained results were expressed as the percentage of CD-14 and CD-15 positive cells in treated cultures compared to those in the control cultures.
Analysis of cytochrome C release
The mean fluorescence intensity value of cytochrome c present was analysed by FCM using Guava InCyte software. Cells were permeabilised with Digitonin (100μg/mL) and fixed in a 4% formaldehyde solution in PBS. Cells were then washed in PBS 1X, incubated in an incubation buffer (0.5g BSA in 100mL PBS 1X) and then incubated overnight at 4°C with anti-cytocrome c monoclonal antibody. At the end of incubation, cells were washed in PBS1X and incubated at room temperature with fluorescein isothiocyanate-labelled secondary antibody. The obtained results were expressed as the mean fluorescence intensity value of cells in treated cultures compared to that in the control cultures. Non-specific binding was excluded by isotype control.
Analysis of cell cycle and apoptotic protein
The mean fluorescence intensity value of proteins present was analysed by FCM using Guava InCyte software. Cells were fixed in a 4% formaldehyde solution in PBS and permeabilised with 90% cold methanol. Cells were then incubated with Anti-p53-PE, Anti-BCL-2-FITC, Anti-cyclin E2-PE, Anti-BAX and Anti-cyclin D3 antibodies. The cells (except those stained with BAX and cyclin D3) were washed and analysed. Cells stained with Anti-BAX and Anti-cyclin D3 were washed and incubated with anti-mouse IgG-FITC secondary antibody. The obtained results were expressed as the mean fluorescence intensity value of cells in treated cultures compared to that in the control cultures. Non-specific binding was excluded by isotype control. www.impactjournals.com/oncotarget
Analysis of apoptosis by fluorescence microscopy
Apoptosis-associated nuclear condensation and fragmentation were evaluated in untreated and treated Jurkat and HL-60 cells by fluorescence microscopy at 100x magnification. 1x10 6 Jurkat and HL-60 cells were loaded into cytospin chambers and centrifuged ad 450 rpm for 10 minutes. Cells were then fixed in formaldehyde 3.7%, washed in PBS pH 7.2, permeabilised in 0.15% triton X-100 and nuclei were stained with 0.5 μM Hoechst 33258 as reported by Henry et al. [48].
Analysis of cell cycle and apoptotic protein by WB
Jurkat and HL-60 cell lysates was obtained as previously reported [49], Samples were denatured prior to separation on 4%-20% Mini-PROTEAN TGX Precast Protein Gels. The proteins were transferred to a nitrocellulose membrane at 110 V for 90 min in Tris-glycine buffer. Membranes were then incubated in a blocking buffer containing 5% (w/v) BSA and incubated with anti-p53, anti-Cyclin D3 and anti-βtubulin, as internal normalizers, overnight at 4°C on an orbital shacker. The results were visualized by chemiluminescence using Clarity Western ECL reagent according to the manufacturer's protocol (BIO-RAD). Semiquantitative analysis of specific immunolabeled bands was performed using Image Lab 6.0 (BIO-RAD).
Statistical analysis
All results are expressed as mean ± standard error mean (SEM) of at least five independent experiments. For the statistical analysis of apoptosis, apoptosis pathways and cell cycle we used the Analysis of Variance for paired data (repeated ANOVA), followed by Bonferroni as the post-test. For statistical analyses of cytodifferentiation, protein levels and Western Blotting densitometry we used the t-test for paired data. All the statistical analyses were performed using Prism Software 6. | v3-fos-license |
2019-04-08T13:08:46.051Z | 2017-03-01T00:00:00.000 | 99368310 | {
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} | pes2o/s2orc | Transesterification of palm cooking oil using barium-containing titanates and their sodium doped derivatives
BaTiO3, Ba2NiTi5O13 and their Na-doped analogues were successfully synthesized via sol–gel and calcination methods. The surface properties of the prepared catalysts were characterized by scanning electron microscopy and nitrogen adsorption–desorption analysis while the basicity was determined by benzoic acid titration. The catalytic activity of the Ba-based catalysts was verified in transesterification of palm cooking oil with methanol. The optimization study was conducted by varying the oil:methanol ratio, catalyst loading, reaction temperature and duration. The highest methyl ester (ME) yield was ~95% for 5 wt/v% Na-Ba2NiTi5O13 at a methanol to oil ratio of 12:1 at 150 °C for 2 h.
Introduction
Biodiesel has attracted interest as a promising energy source to substitute the drastically depleted fossil fuels in recent decades [1]. The main content in biodiesel is fatty acid methyl ester (FAME), which is an environmentally friendly fuel with clean burn, easily made, biodegradable, non-toxic and free of sulfur and aromatic compounds [2]. Transesterification reaction is the most common method to convert vegetable oils or animal fat into FAMEs using methanol in the presence of suitable catalyst. Generally, heterogeneous catalysts are preferred over homogenous catalysts as it provides advantages such as simple separation process, good reusability and saponification prevention [3,4].
Metal oxides have been extensively studied as heterogeneous base catalysts in transesterification. The presence of M d? -O dion pairs in these oxides produces basic sites that are believed to be actively involved in the transesterification reaction [5]. Several attempts have been employed to further improve their catalytic properties and stability, such as doping [6,7], immobilizing or supporting [8] and mixed oxide systems [9,10]. For example, Mukenga et al. [8] have shown that the oil conversion for TiO 2 supported ZnO is higher than unsupported ZnO catalyst. Mar and Somsook [6] showed that the catalytic activity and stability of CaO catalyst can be enhanced by doping with KCl. Lee et al. [9] also proved that the binary MgO-ZnO catalyst exhibits superior physicochemical properties as compared to MgO or ZnO catalysts.
Barium oxide catalysts were reported to exhibit superior catalytic performance in transesterification. The catalytic reactivity of BaO is higher than CaO, SrO and MgO due to its high basicity [11][12][13][14][15][16]. The catalytic properties of BaO supported on Al 2 O 3 [17], SiO 2 [18] and SrO(Al 2 O 3 ) [19] towards the transesterification reaction have also been studied. The reactivity of the supported BaO catalysts can be related to its dispersion on the support, surface composition and surface properties. Thus far, the study on barium-based binary or tertiary oxides in transesterification is rarely reported. The only two recent studies were conducted by Sherstyuk et al. [20,21] [22] and Ba 2 NiTi 5 O 13 [23] particles via solgel and calcination method. As the continuation of the work, herewith, we report their catalytic study in the transesterification of palm cooking oil. The Na-doped Ba 2 NiTi 5 O 13 was also prepared for comparison purposes. The surface properties of un-doped and doped oxides are studied via SEM, BET and benzoic acid titration. The influence of sodium dopant on the surface properties, as well as the catalytic performance is also reported in this study.
Materials
All materials were used as purchased. Titanium tetraisopropoxide (Ti(OPr i ) 4
Preparation of catalysts
In general, the catalysts were prepared via sol-gel method followed by calcination. Detailed description on the preparation of BaTiO 3 , Na-doped BaTiO 3 and Ba 2 NiTi 5-O 13 has been reported elsewhere [22,23].
BaTiO 3 and sodium doped BaTiO 3 (Na-BaTiO 3 )
As much as 399.7 mg of Ba(OH) 2 .H 2 O was dissolved in 40 mL of distilled water heated in an oil bath at 50°C. Then, 8 mL of 0.044 M of Ti(OPr i ) 4 in isopropanol (molar ratio of Ba:Ti = 6:1) was then added. The mixture was stirred for 2 h under reflux. The product (in gel form) was separated via centrifugation and washed with distilled water. The BaTiO 3 catalyst was obtained by calcination of the gel at 900°C for an hour. The 0.5 mol% sodium doped derivatives was similarly prepared. However, the stipulated NaCO 3 was added to Ba(OH) 2 Typically, the Ba 2 NiTi 5 O 13 was synthesized via two-steps. As much as 5 9 10 -3 mol of Ni(CH 3 COO) 2 .4H 2 O was dissolved in a mixture of isopropanol/acetic acid. Then, 1.5 mL of 3.33 M Ti(OPr i ) 4 was added (at a mole ratio of Ni:Ti = 1:1). The mixture was stirred at room temperature for 24 h until an olive green solution was formed. As much as 50 mL of distilled water was then added before subjecting to partial freeze-drying process. The obtained product (as-prepared NiTiO 3 gel) was used in the subsequent step.
In a separate flask, 799.4 mg of Ba(OH) 2 .H 2 O was dissolved in 80 mL of distilled water and mixed with 32 mL of 3.0 M NaOH. The whole system was heated at 50°C while stirring to ensure the total dissolution. Then, 652.8 mg of the as-prepared gel obtained previously was added. This was followed by the addition of 16 mL of 0.044 M Ti(OPr i ) 4 in isopropanol. The mole ratio of Ba:Ni:Ti was fixed at 1:1:1.2. The mixture was stirred for another 8 h. The product (in gel form) was separated via centrifugation and washed with distilled water. The Ba 2-NiTi 5 O 13 catalyst was obtained by the calcination of gel product at 900°C for 8 h. The sodium doped derivatives (viz. 0.5 mol% Na) were similarly prepared. However, the stipulated NaCO 3 was added before addition of Ti(OPr i ) 4 in the second step. The obtained gel product was then calcined at 900°C for 5 h.
Characterizations
The surface structures and average size of the catalysts were determined by scanning electron microscope (SEM) equipped with electron energy dispersive X-ray spectroscopy (EDX) performed on a Leica Cambridge Stereoscan S360 operating at 15 kV. The surface area and porosity of the catalysts were examined using a Micromeritics ASAP 2000. Samples were degassed at 160°C prior to isotherm analysis. Branauer-Emmett-Teller (BET) isotherm was performed by adsorption of nitrogen at -196°C. The surface area was then calculated using BET equation. The basicity analyses of the respective catalysts were determined by benzoic acid titration using the following indicators: bromothymol blue (H -= 7.2), phenolphthalein (H -= 8.2-9.8), 2,4-dinitroaniline (H -= 15.0) and 4-nitroaniline (H -= 18.4) [24,25].
Transesterification reaction
Transesterification reaction was carried out by heating a mixture of palm cooking oil, methanol and the as-prepared catalyst in an autoclave at the desired temperature and duration. The BaTiO 3 catalyst was employed to study the reaction optimization for the series of catalysts investigated. The four parameters: reaction temperatures (80-180°C), duration (0.5-6.0 h), ratios of oil to methanol (1:3-1:15 v/v) and catalyst loading (0.5-10.0 wt/v% referred to the initial palm oil volume) were investigated to determine the optimum reaction conditions.
After the transesterification reaction, the mixture was allowed to cool down before separating the methyl ester (ME) by centrifugation. The products were weighed and analyzed using an Agilent 7890A gas chromatography (GC) fitted with a flame ionization detector (FID). The % of ME yield was calculated using the following equation [26]: Results and discussions
Surface characterizations
The typical morphological structures of un-doped and doped oxides are depicted in Fig. 1. All the samples show porous aggregates which were made up of distinct particles. These particles are mostly nearly spherical to cube in shape. Several studies [27,28] have shown that doping constrains the growth of oxide particles and thus smaller particles with higher surface area are obtained. However, this study shows otherwise, i.e., increment in the average particle size and a decrement in surface area and pore size upon Na doping. This may be attributed to the formation of a dense layer of Na secondary phase on the oxides and plugged the pores. XRD analyses (not shown) have confirmed the formation of Na 1.7 Ba 0.45 Ti 5.85 O 13 and NaBa 2-NiTi 5 O 13 on the respective outermost surface of the BaTiO 3 and Ba 2 NiTi 5 O 13 [22,23].
As seen in Table 2, BaTiO 3 and Ba 2 NiTi 5 O 13 exert a total basicity of 0.540 and 0.618 mmol g -1 , respectively. Most of the basic sites of these oxides are in the range of 7.2 \ H -\ 8.2 (weaker basic sites). It covers *54% of the total basic sites. The total basicity of the oxides increased almost twofold upon Na doping. This was 1.062 and 1.336 mmol g -1 for Na-BaTiO 3 and Na-Ba 2-NiTi 5 O 13, respectively. The percentage of medium basic sites in Na-Ba 2 NiTi 5 O 13 is higher as compared to Na-
Optimization of transesterification
The catalytic activities of BaTiO 3 at various reaction parameters were compared to obtain the optimum reaction conditions. As seen in Table 3, the percentage of ME yield increases with the increase in the ratio of oil:methanol up to 1:12 (v/v), and thereafter ME yield decreases. For instance, the ME yield for the oil:methanol ratio of 1:3 (v/ v) is 82.3%. This increases to 86.5 and 88.1% upon further increase in the oil:methanol volume ratio to 1:9 (v/v) and 1:12 (v/v) correspondingly. Further increase in the content of methanol to 1:15 (v/v) caused the ME yield to drop to 78.8%.
Palm oil transesterification is a reversible reaction (Eq. 2). Hence, forward reaction is favored in an excess amount of methanol, i.e., from 1:3 to 1:12 (v/v) oil:methanol. However, at high methanol ratio (in the 1:15 (v/ v) oil to methanol), the dilution effect becomes prominent. This caused a reduction in reaction rate, hence a lower ME yield is obtained [29]. In addition, large amount of methanol can also promote the solubility of glycerol in the oil and can cause difficulty in the separation of glycerol [30]. Therefore, the ratio of oil to methanol of 1:12 (v/v) is chosen for this transesterification study.
The catalyst loading is another factor that affects the ME yield. Typically, increasing the loading amount of catalysts may provide more catalytic sites for reactants to dock on. In this study, it is observed that the % of ME yield increases gradually from 0.5 to 10 wt/v% catalyst loading. The ME yield was 78.2, 84.5, 88.1 and 88.8% for the 0.5, 2.0, 5.0 and 10.0 wt/v% BaTiO 3 loading, respectively. The % yield for the 5 and 10 wt/v% catalyst is similar. Accordingly, the 5 wt/v% catalyst was chosen as the optimum amount for further studies.
An increment of ME yield from 76.0 to 88.1% is achieved via increasing the reaction temperature from 80 to 150°C. Beyond 150°C, the ME yield is constant. Generally, increasing temperature helps in homogenizing the reactants, making them more readily to react [31]. Hence, this may assist the transesterification to reach equilibrium faster, and thus affects the % of ME yield for transesterification.
As shown in Table 3, the % of ME yield increases from 74.8 to 88.1% when increasing the time duration from 0.5 to 2 h. After that, the reaction yield is more or less constant (*90-92%). Hence, it is envisaged that 2 h is sufficient to complete the reaction.
Transesterification of palm cooking oil
As shown in Table 2, BaTiO 3 demonstrated 88% of ME yield. Ba 2 NiTi 5 O 13 gave a higher ME value of 91% as compared to BaTiO 3 . These values increased 2-6% upon doping 0.5 wt% Na into the respective oxide catalysts. The catalytic activity of barium-containing catalysts follows the trend of decreasing order of Na-Ba 2 NiTi 5 O 13 [ Ba 2- In general, the catalytic properties are influenced by particle size, surface area, as well as the active sites of a catalyst. Small sized catalyst with high surface area and active sites will give rise to high catalytic performance. In this study, however, the sizes and surface areas seem to contribute to only minor extent towards transesterification. Instead, the catalytic performance of the series of un-doped and doped barium-containing titanate catalysts is believed to be closely related to their basic sites. For instance, the Na-Ba 2 NiTi 5 O 13 which the highest amount of medium basic sites displayed the highest catalytic activity. However, the Na-BaTiO 3 with the smallest average particle size gave lower catalytic activity even though it offers the highest surface area among the series. A similar finding was reported by Nascimento et al. [32]. As previously mentioned [22,23], a dense layer of Na secondary phase was formed onto the surface of barium-containing titanate catalysts in the Na-doped system. There are 50% more basic sites located on these Na secondary phase as compared to un-doped system. These sites are actively involved in the adsorption of methanol in which the O-H bonds of the methanol readily break into methoxides and proton [33]. The methoxides formed then reacted with the triglyceride to yield methyl esters and glycerols. The proposed mechanism is depicted in Fig. 2. Table 4 also shows a comparison of catalytic activity between this work and other barium-containing catalyst systems. As can be seen, the ME yield (88-95%) obtained in this work are comparable to the previous reported values. The highest ME yield achieved in the literature was 100% for the Ba 0.04 La 1.96 O 3 catalyst using rapeseed oil as the feed stock. Nevertheless, higher catalyst loading and temperature, as well as a longer reaction time were needed as compared to this study.
Conclusion
BaTiO 3 , Ba 2 NiTi 5 O 13 and their Na-doped analogues were successfully synthesized via sol-gel and calcination method. The prepared BaTiO 3 and Ba 2 NiTi 5 O 13 exert porous aggregated structure with specific surface area of 3-4 m 2 g -1 . They displayed a total basicity of 0.5-0.6 mmol g -1 with *81% of weak basic sites. Doping these oxides with Na increased their total basicity (i.e., to 1.1-1.3 mmol g -1 ) but decreased their surface area and pore size. This may be attributed to the formation of a dense 3 . The catalytic performance of these series of un-doped and doped barium-containing catalysts is believed to be closely related to their surface basic sites but not the total surface area. | v3-fos-license |
2021-05-08T00:03:05.278Z | 2021-02-24T00:00:00.000 | 233955037 | {
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} | pes2o/s2orc | Seed priming with brassinosteroids alleviates aluminum toxicity in rice via improving antioxidant defense system and suppressing aluminum uptake
Brassinosteroids (BRs) are growth-promoting hormones that exhibit high biological activities across various plant species. BRs shield plants against various abiotic stresses. In the present study, the effect of BRs against aluminum (Al) toxicity was investigated through seed priming with 24-epibrassinolide (0.01 μM) in two different rice cultivars. BRs application was found effective in confronting plants from Al toxicity (400 μM). The rice seeds primed with BRs showed enhancement in seed germination energy, germination percentage, root and shoot length, as well as fresh and dry weight under Al-absence and Al-stressed conditions as compared to water-priming. Especially under Al stress, BRs priming promoted the growth of rice seedlings more obviously. Al toxicity significantly increased the Al contents in seedling root and shoot, as well as the MDA concentration, H2O2 production, and the activities of antioxidative enzymes including ascorbate peroxidase, catalase, and peroxidase. Meanwhile, the photosynthetic pigments of seedling reduced under Al stress. When compared to sensitive cultivar (CY-927), these modifications were more obvious in the tolerant variety (YLY-689). Surprisingly, BRs were able to alleviate the Al injury by lowering MDA and H2O2 level and increasing antioxidant activities and photosynthetic pigments under Al stress. The results on antioxidant activities were further validated by gene expression study of SOD-Cu-Zn, SOD-Fe2, CATa, CATb, APX02, and APX08. It suggested that BRs were responsible for the mitigation of Al stress in rice seedlings by inducing antioxidant activities with an effective response to other seed growth parameters and reduced Al uptake under induced metal stress.
Introduction
Rice (Oryza sativa L.) is an important staple food of the developing world due to its high protein, vitamin, and mineral contents . Almost 85% of rice is cultivated in Asia, whereby China is the world's leading producer of rice. In southern China, rice is the main cereal crop to fulfil the nutritional requirements of underprivileged people (Ahmed et al. 2020;Huang et al. 2013). During past few years, soil properties have been changed due to rapid industrialization and excessive release of heavy metals (Ahmed et al. 2021b), such as aluminum (Al), cadmium (Cd), mercury (Hg), and lead (Pb), in environmental system (Ahmed et al. 2021c;Noman et al. 2020a;Noman et al. 2020b). In low pH soils, Al contamination has devastating impacts that catastrophically disturbed the agronomic traits, such as yield and quality, of cereal crops. It was estimated that almost 30-50% of soil is polluted with Al globally, whereby 21% of total arable land in China is Al-affected (Xu et al. 2012). Al has been found to alter the morphological (such as growth and biomass production) as well as physiological (such as oxidative stress) states of crop plants (Silva 2012). It boosts lipid peroxidation by inhibiting antioxidant enzymes including catalase, peroxidase, superoxide dismutase, and glutathione reductase, which ultimately trigger plant stress (Rout et al. 2001). It was discovered that Al toxicity can also be found in shoots that develop as a result of root system damage (Vitorello et al. 2005). Al toxicity has a number of negative effects in plants, including altered water balance, reduced stomatal conductance and photosynthetic activity, chlorosis, and necrosis of leaves (Ali et al. 2008a;Ma 2007). The excess amount of Al in soil or growth medium perturbs the entry of essential ions such as magnesium (Mg), potassium (K), calcium (Ca), manganese (Mn), and zinc (Zn) into the plant system, thereby disturbing the mineral balances and retarding the overall growth and development (Basit et al. 2021b;Manzoor et al. 2021;Mendonça et al. 2003). However, it raises proline levels, which acts as an osmoprotectant, membrane stabilizer, and ROS scavenger (Apel and Hirt 2004).
Brassinosteroids (BRs) are polyhydroxy steroidal phytohormones that have a strong ability to promote plant growth under diverse environmental conditions (Basit et al. 2021a;Latha and Vidya Vardhini 2018). 24-Epibrassinolide is the most biologically active BR compound, which is involved in a variety of developmental processes such as cell division, elongation, gene expression, and vascular differentiation and others (Basit et al. 2021a;Bergonci et al. 2014). BRs have been shown to help plants cope with a variety of stresses, including biotic and abiotic stresses (Bhandari and Nailwal 2020). Exogenously applied BRs improve the resistance mechanisms to low and high temperatures, drought, chilling, and various metal stresses. Similarly, BRs treatment improved tomato plant resistance to chromium stress by modulating physiological and molecular pathways (Jan et al. 2020). It has been discovered that BRs strengthen nitrogen fixation, secondary metabolites, and protection mechanisms in Cicer arietinum (Ali et al. 2005) and Brassica juncea under salinity and heavy metal stresses such as nickel (Ali et al. 2008b). It has also been documented that exogenous application of BRs improves the antioxidant system, photosynthesis, and growth characteristics of mung bean plants under aluminum stress (Ali et al. 2008a). Numerous studies have shown that BRs enhance plant growth under various heavy metal stresses by increasing chlorophyll contents, which play an important role in increasing photosynthetic capability, improving antioxidant system performance, boosting enzymatic activity, and upregulating stress-responsive genes [superoxide (SOD), peroxide (POD), catalase (CAT), glutathione reeducates (GR), and ascorbate peroxide (APX)] (Cominelli et al. 2008).
Hence, based on the hypothesis that BRs could boost the biomass of rice plants by reducing the uptake of Al and alleviating the oxidative stress under Al toxicity, we explored the impact of seed priming with BRs on rice seed germination, morphophysiological and biochemical parameters, nutrient acquisition, and Al uptake under Al toxicity as well as proposed the possible mechanism of Al stress mitigation in rice plants through seed priming with BRs in this study.
Brassinosteroids (BRs) preparation
24-Epibrassinolide was obtained from the Shanghai Aladdin Biochemical Technology Co., Ltd. It was liquefied in sufficient ethanol, and a stock solution of 10 −5 M was prepared by adding ddH 2 O with 0.05% Tween 20 for use as the priming reagent of BRs.
Plant materials and growth conditions
The seeds of two cultivars of Oryza sativa L. (cv. CY927 and YLY689) were obtained from the Zhejiang Nongke Seeds CO., LTD. Hangzhou, Zhejiang Province, China (Salah et al. 2015). Seeds were surface sterilized for 15 min with a 0.5% sodium hypochlorite (NaClO) solution and then washed several times with tap water before being washed three times with sterilized distilled water to remove any remaining disinfectant. Sterilized seeds were primed with 0.01-μM BRs at 15°C in the dark for 24 h. The seeds were then dried at room temperature to their original moisture content. The seeds primed with water (H 2 O) and BRs without Al stress were used as the control group (CK). After seed priming, germination tests were performed. Each treatment consisted of fifty seeds that were placed in a plastic germination box (12 cm × 18 cm) and repeated three times. The seeds were then incubated for 14 days at 25°C in a germination chamber with an 8/16 h light/ darkness period (Zhang et al. 2007). The 400-μM concentration of Al was supplied to seeds with nutrients solution. The nutrient solution was made up of the following ingredients: 0.5-μM potassium nitrate (KNO 3 ), 0.5-μM calcium nitrate (Ca(NO 3 ) 2 ), 0.5-μM magnesium sulfate MgSO 4 , 2.5-μM monopotassium phosphate (KH 2 PO 4 ), 2.5-μM ammonium chloride (NH 4 Cl), 100-μM ferric EDTA (Fe-K-EDTA), 30-μM boric acid (H 3 BO 3 ), 5-μM manganese monosulfate (MnSO 4 ), 1-μM copper sulfate (CuSO 4 ), 1-μM zinc sulfate (ZnSO 4 ), and 1-μ M ammonium heptamolybdate ((NH 4 ) 6 Mo 7 O 24 ) per 1000 mL ddH 2 O. The pH of the nutrients solution was adjusted to 5.0 with hydrochloric acid (HCl) and sodium hydroxide (NaOH). The Al concentration was determined using data from a primary experiment involving different Al concentrations of 0, 100, 200, 300, 400, 500, 600, 700, and 800 μM. Plant growth was slightly harmed at low aluminum concentrations (100-300 μM). Despite this, plant growth was significantly harmed by aluminum concentrations of 400 μM. Concentrations greater than 500 μM, on the other hand, were excessively toxic to the plant's growth.
Measurement of physiological parameters
For 14 days, the number of germinated seeds was counted every day. On the 5th day of germination, total germinated seeds were counted, and germination energy was calculated. On the 14th day, the germination percentage was determined. The following formulas were used to calculate the germination index (GI), mean germination time (MGT), and vigor index (VI) (Hu et al. 2005).
G t is the total calculated number of germinated seeds on day t, and T t is the time conforming to G t in days (Hu et al. 2005).
Experimental design and treatment pattern
The 2-week-old seedlings were treated with a 400-μM Al concentration after being primed with water (H 2 O) and 0.01-μM BRs. The experimental pattern was constituted through a completely randomized design (CRD), and the location of the pots within the growth chamber was changed every day. The plants were sampled 21 days after being treate d w i t h A l f o r 7 d a y s t o o b s e r v e a l t e r a t i o n s morphophysiological parameters.
Plant growth investigation
The plants were harvested with intact roots and immersed in a bucket filled with water to remove any traces of media contents. The plants were divided into roots and shoots, and the lengths of the roots and shoots were measured, followed by determination of their fresh weight using electronic weighing balance. The roots and shoots were dried in an oven at 80°C for 24 h and then weighed to determine their dry mass.
Measurement of photosynthetic pigments
Photosynthetic pigments, such as chlorophyll a and b and total chlorophyll, were investigated by following the methodology of Ahmed et al. (2021b). Briefly, fresh leaf tissues (0.2 g) were homogenized in 3 mL ethanol (95%, v/v). The supernatant was removed after centrifuging the homogenate at 5000 g for 10 min. 1 mL aliquot of the supernatant was added to 9 mL of ethanol (95% v/v). After that, the mixture was subjected to spectrophotometry to determine the absorbance at 665-and 649-nm wavelengths (Lichtenthaler and Wellburn 1983). To quantify chlorophyll pigments, the following equations were used: Chlorophyll a C a ð Þ ¼ 13:95 A665−6:88 A649 ð4Þ The pigment concentrations were calculated in milligrams per liter of plant extract.
Measurement of MDA contents and H 2 O 2 measurements
MDA concentration was examined in terms of production of 2-thiobarbituric acid (TBA) metabolites. Approximately 1.5 mL plant extract was homogenized in 2.5 mL of 5% TBA prepared in 5% trichloroacetic acid (TCA). The mixture was heated to 95°C for 15 min followed by incubation on ice. After that, the supernatant was centrifuged for 10 min at 5,000g. Finally, the absorbance of the supernatant was determined at 532 nm using the abovementioned unit of spectrophotometer. To reduce nonspecific turbidity, the absorbance value of reaction mixture at 600 nm was subtracted from the value at 532 nm (Rao and Sresty 2000). The MDA concentration was measured in nmol mg −1 protein. To measure hydrogen peroxide (H 2 O 2 ) concentration, the plant tissues were homogenized in phosphate buffer followed by centrifugation at 6000g. The supernatant was mixed with 0.1% titanium sulfate containing 20% (v/v) H 2 SO 4 followed by centrifugation. The intensity of yellow color was estimated calorimetrically at 410nm using abovementioned unit of UV-vis spectrophotometer (Velikova et al. 2000). H 2 O 2 concentration was calculated by using standard curve constructed using known concentrations of H 2 O 2 .
Enzymatic antioxidants activity assay
Fresh plant tissues were homogenized in 8 mL of 50 mM potassium phosphate buffer (containing 1-mM EDTANa 2 as well as 0.5% PVP W/V, pH 7.0) on ice. After that, centrifugation of the homogenate was performed at 12000rpm for 20 min at 4°С. After centrifugation, supernatant was collected in separate tube and stored at 80°С until further processing. The superoxide dismutase (SOD) activity was determined according to Giannopolitis and Ries (1977), by examining the capability of the enzyme to restrict the photochemical reduction of nitroblue tetrazolium chloride (NBT). The absorbance was measured at 560 nm, and one unit of SOD activity, represented as EU/mg protein, was shown to be the enzyme quantity required for inhibiting the rate of NBT photoreduction by up to 50% (Giannopolitis and Ries 1977). Peroxidase (POD) activity was examined according to the method of Zhang (1992) exhausting the elimination coefficient 25.5 mM −1 cm −1 . The homogenization of both root and shoot fresh tissues was done in phosphate buffer (50 mM and pH 7.8) followed by centrifugation at 8000g at 25°C. The OD of resulting mixture was measured at 470 nm. Catalase (CAT) activity was recorded as described by Aebi (1984) by the extermination constant of 39.4 mM −1 cm −1 . The absorbance was calculated at 240 nm, and CAT activity was expressed as EU/mg protein, whereby for the determination of ascorbate peroxidase (APX) activity, the decrease in absorbance at 290 nm for 3 min was observed, and APX activity was expressed as EU/mg protein (Nakano and Asada 1981).
RNA extraction and gene expression analysis
Antioxidant gene profiling was performed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Total RNA from plant samples was extracted using Trizol reagent as described by Sah et al. (2014). For cDNA synthesis, 1 μg of total RNA was reverse transcribed through PrimeScript™ RT reagent kit, and the resulting product was used as template for qRT-PCR. The qRT-PCR reaction was prepared in SYBR Premix Ex Taqkit (TaKaRa, Dalian, China), containing 10 mL 2× SYBR Premix Ex Taq buffer, 1-μg synthesized cDNA, and 10 μmol of each of genespecific primers in a final volume of 20 mL (Livak and Schmittgen 2001). OsActin was used as an internal control to normalize the data to measure the relative transcript abundance for target genes. Relative expressions of target genes were calculated using the 2-ΔΔCT method. Primers used for qRT-PCR are listed in Table S1.
Statistical analysis
Experimental data were analyzed using one-way analysis of variance treatments. The significance among means of different dataset was determined using least significant difference (Fisher's LSD) test with 95% confidence level using SPSS v16.0 (SPSS, Inc., Chicago, IL, USA). Principle component analysis (PCA) and agglomerative hierarchical clustering (AHC) were performed to classify two different rice cultivars used in the current study according to their vulnerability toward Al by using XLSTAT.
BRs promotes seed vigor and plant growth
The current study has demonstrated that Al toxicity (400 μM) caused a significant decrease in seed germination energy, germination percentage, vigor index, as well as germination index in both cultivars as compared to control; more reduction was observed in cultivar CY927. However, seed priming with BRs revealed a significant resistance against Al stress as compared to control (seed priming with water) in both cultivars. MGT was significantly enhanced in plants treated under Al stress as compared to untreated plants. However, seed priming with BRs reduced MGT in both cultivars (Table 1). More reduction was observed in cultivar CY927 as compared to cultivar YLY689. Current study demonstrated that 0.01-μM concentration of BRs significantly enhanced the GE, germination %, VI, and GI under metal toxicity as compared to the unprimed seeds in both cultivars (Table 1).
Al exposure reduced the morphological parameters of rice seedlings ( Fig. 1 and Fig. 2). Plants treated with Al toxicity caused a significant decrease in root and shoot length of rice plants as compared to untreated control plants. A significant reduction was observed in seedling fresh weight of CY927 cultivar after exposure to Al stress, whereby dry weight was significantly decreased in both cultivars under Al toxicity. More reduction was observed in cultivar CY927 as compared to cultivar YLY689. It was noticed that seed priming with BRs significantly improved the shoot/root length as well as fresh/dry weight as compared to control plants without BRs treatment under Al-spiked conditions (Table 2).
Seed priming with BRs increases photosynthetic pigments
The negative effect of Al stress on photosynthetic pigment concentration was observed in both rice cultivars (Fig. 3). The present study demonstrated that Al treatment alone resulted in a significant drop in chlorophyll pigments such as Chl a and b and total chlorophyll content as compared to control. Under Al stress, both cultivars showed a decrease in photosynthetic pigments. In comparison to YLY689, the decline was more obvious in CY927. Seed priming with 0.01-μM BRs mitigated Al toxicity in both genotypes to some extent. In the case of seed priming with BRs, the total chlorophyll pigment was increased by 37.2% on average across both cultivars. Seed priming with BRs improved Chl a and b and total chlorophyll concentrations in both cultivars when compared to respective controls in both treatments without and with Al stress. In both cultivars, plants treated with BRs alone showed more photosynthetic pigments than non-treated control plants (Fig. 3).
BRs supplementation reduces Al accumulation in rice seedling
Roots are the main part that interacts with heavy metals first, as well as the primary source of nutritional solution and heavy metal uptake. Al accumulation was found to be higher in roots than in shoots (Tables 3, 4). However, Al accumulation was noticeable in the YLY689 cultivar than in the CY927 cultivar. Seed priming with BRs reduced Al content by 10.90% and 6.140% in the CY927 and YLY689 cultivars, respectively. More interestingly, when exposed to Al toxicity, the concentrations of K + , Ca 2+ , Fe 2+ , and Mn 2+ in both roots and shoots reduced, whereas Zn 2+ concentration increased in both cultivars (Tables 3-4). Seed priming with BRs maintained the nutritional balance of both cultivars under Al stress and significantly improved the nutrients availability such as Mn 2+ and Fe 2+ inside shoots of cultivar CY927 and elevated K + , Ca 2+ , Mn 2+ , and Fe 2+ accessibility in shoots of YLY689 cultivar (Tables 3-4).
BRs ameliorates Al-induced oxidative stress
In both cultivars, the presence of Al raised MDA and H 2 O 2 contents when compared to the control. In comparison to the YLY689 cultivar, this rise was more pronounced in CY927. The application of BRs dramatically lowered MDA levels as well as H 2 O 2 generation in both cultivars (Fig. 4). MDA levels were found to be greater in shoots (64.70% and 55.40%) than that in roots (56% and 42%) in both CY927 and YLY689 cultivars under induced Al stress as compared to respective healthy controls, respectively. However, seed priming with BRs reduced the MDA levels in shoots (44.50% and 45%) and roots (26% and 39.70%) of both cultivars (CY927 and YLY689, respectively) as compared to corresponding controls under Al-spiked conditions.
Determination of antioxidant enzyme activities
Antioxidant activity was shown to be increased when Al was treated alone. A recent study found that stressed plants had Each value is demonstrating the mean of three repeats of every treatment. The similar letters inside a column specify that there was no significant difference at a 95% probability level at the p < 0.05 level, correspondingly Each value is demonstrating the mean of three repeats of every treatment. The similar letters inside a column specify that there was no significant difference at a 95% probability level at the p < 0.05 level, correspondingly higher levels of SOD, CAT, POD, and APX than control plants when exposed to 400-μM Al and that this effect was stronger in YLY689 than that in CY927 (Fig. 5). These activities were found to be more prevalent in roots than that in shoots. SOD activity was found to be 22.5% in CY927 and 43.6% in YLY689 cultivar shoots when Al was applied, whereby in roots of both cultivars, it was found in 47.7% and 58.2%, respectively. Both cultivars under combined treatment of Al and BRs showed 44.6% and 46.3% increase in shoot SOD activity and 57.4% and 58.2% increase in root SOD activity in contrast to Al-affected plants. Under independent treatment of Al, CAT activity in the shoots was found to be 45% in CY927 and 59% in YLY689. In roots, it was found to be 39% and 45%, respectively. The seed priming with BRs increased the CAT activity by 51% and 61% in shoots and by 46% and 58% in roots as compared to Al-stressed plants, respectively. Similarly, POD and APX levels were higher in the presence of Al alone, but this impact was more prevalent with BRs priming; however, POD activity was not increased significantly. However, 14.8% and 37.4% increase in POD activity was observed in shoots of CY927 and YLY689, whereby 30.2% and 46.8% boost in roots was observed in both cultivars after Al treatment, respectively (Fig. 5).
Determination of gene expression analysis
In both cultivars, there was a substantial difference in the expression of APX02 in both roots and shoots when compared to control. Under both independent Al stress and combined treatment of Al/BRs, the transcriptional level of APX02 was increased. The YLY689 rice cultivar had higher APX02 expression than the CY-927 rice cultivar (p < 0.01). Interestingly, the transcriptional level of APX02 in rice seedlings emerged from BRs primed seeds was found to be higher than plants under Al stress alone and supported the results of APX activity (Fig. 5). Similarly, the transcription level of APX08 was high in both roots and shoots of either cultivar when compared to control under independent and combined treatments of Al and BRs; however, the expression level of APX08 was found to be higher in the roots of YLY689 cultivar than that of CY927 cultivar (Fig. 6). Additionally, in contrast control condition, the expression levels of CATa and CATb were found to be higher in both roots and shoots of both cultivars under independent and combined treatments of Al and BRs. However, the rise was more pronounced in YLY689 cultivar than that in CY927 cultivar. Al stress also triggered the expression levels of CATa and CATb in both roots and shoots of both cultivars as compared to the non-treated control (Fig. 6). Under stressful conditions, both cultivars showed significant upregulation of SOD Cu-Zn and SOD-Fe2 genes, where YLY689 cultivar showed a greater increase. The transcriptional level of the SOD Cu-Zn and SOD-Fe 2 genes was found to be higher in roots than that in shoots in both cultivars. Regardless of Al stress, the transcriptional level of the SOD Cu-Zn gene was much higher in roots than in non-treated control (Fig. 6). In contrast to seeds primed with water, seedlings primed with BRs depicted higher expression of SOD Cu-Zn and SOD-Fe 2 genes. Under Al toxicity, it may be possible to modify the stress condition inside both cultivars by temporarily upregulating particular gene expression (Fig. 6). This data further confirms the BRs-based alterations in the concentrations of enzymatic antioxidants (Fig. 5). It was clearly demonstrated that BRs play a substantial role in stress tolerance in rice plants by modifying and regulating the transcriptional level of particular genes under induced Al stress. GP, they also had a negative relationship with SOD, POD, CAT, and APX in both cultivars (Fig. 7A, B). PCA analysis of both cultivars (CY927 and YLY689) demonstrated that YLY689 is a tolerant genotype, whereby CY927 is a sensitive genotype for Al stress. In CY927, the largest contribution of F1 (84.92) was observed, followed by F2 (10.20), with a total contribution of 95.12%, whereas in YLY689, the largest contribution of F1 (86.39) was observed, followed by F2 (10.69), with a total contribution of 97.09%. ACH results also confirmed the identical response of both varieties to distinct treatments (Fig. 8). It represented the close relationship between both cultivars (CY927 and YLY689) primed with BRs and primed with water under Al stress, as well as cultivars primed with water and BRs under normal condition. When compared to plants primed with water under Al toxicity, cultivars primed with BRs exhibited a close association with both controls (primed with water and BRs) (Fig. 8).
Discussion
Al is the 3rd most prevalent metal in the earth's crust, and while it is abundant, it is only minimally soluble, causing serious harm to biological systems (Bolt et al. 2020). Al is present in plant-accessible form at pH value less than 5.5, which can cause toxicity in plants, particularly the roots (Barcelo and Poschenrieder 2002). Because it has direct exposure to roots, Al toxicity affects shoot length, fresh weight, and dry weight (Table 2), causing cell elongation inhibition at an early stage and later causing damage to plant growth and development (Čiamporová 2002;Silambarasan et al. 2019). In addition to membrane permeability, Al promotes nutritional imbalance in plants by altering osmotic balance (Olivares et al. 2009). Similarly, Al treatment lowered the levels of K + , Ca 2+ , Fe 2+ , and Mn 2+ in both roots and shoots in the Each value is demonstrating the mean of three repeats of every treatment. Same letters are representing no significant differentiation at 95% probability level (p<0.05) (Tables 3-4). Al toxicity lowered K + , Ca 2+ , Fe 2+ , and Mn 2+ concentration in rice plants, with the effect being stronger in sensitive varieties than tolerant varieties. Similarly, Al inhibited the level of K + , Ca 2+ , Fe 2+ , and Mn 2+ in the roots and shoots of tomato and maize plants (Giannakoula et al. 2008;Simon et al. 1994). Consequently, photosynthetic pigments and associated processes were also disrupted (Fig. 3), resulting in a reduction in plant growth caused by Al exposure (Table 2). In comparison to seeds primed with water, seeds treated with BRs demonstrated a stronger shielding response to Al stress in rice seedlings (Tables 1-2). The concentration of Al in roots was higher than that in shoots (Tables 3-4). It could be due to the possibility that the roots are the main source of Al uptake, and the plants activate various mechanisms to minimize Al translocation from roots to shoots (Mendonça et al. 2003;Ali et al. 2008c). Current study demonstrated that Al absorption was dramatically reduced in both cultivars, particularly in those plants primed with BRs over those primed with water. This reduction was more noticeable in tolerant variety (cv. YLY689). Furthermore, seed priming with BRs significantly maintained the nutrient balance in both cultivars. The regulation of nutrient balance was more prominent in tolerant variety under Al stress. Consequently, both rice cultivars primed with BRs showed considerably increased biomass, root length, shoot length, fresh weight, and dry weight when compared to unprimed plants, where tolerant cultivar showed more pronounced rise in morphological parameters than sensitive cultivar (Tables 2-3). BRs seed priming further boosted photosynthetic pigments within both sensitive and tolerant varieties under Al toxicity (Fig. 3). This boost in photosynthetic pigments corresponds to better stress resistance behavior and alterations in plasma membrane as well as to activation of antioxidant enzymes under Al-stressed environment (Fig. 5) (Divi and Krishna 2009). Furthermore, BRs serve as a proton pump, enhancing water uptake; regulating gene suppression and activation, protein synthesis (Nawaz et al. 2017), and nucleic acid stimulation; and triggering antioxidant enzyme activity (Khripach et al. 2003). BRs-based modifications in plant behavior play a vital role in stimulating plant growth in stressful environments. According to a report, BRs relieve plants from stress by promoting plant growth, photosynthetic pigments, and water uptake (Rajewska et al. 2016). Similarly, the ameliorating roles of BRs in reducing cadmium toxicity in cowpea plants (Santos et al. 2018), salinity stress Rao 2001, 2003), zinc metal stress in B. juncea (Arora et al. 2010), heat stress (Wu et al. 2014), and heavy metal stress (Anuradha and Rao 2007;Vardhini 2016;Vázquez et al. 2013) are wellestablished.
The results acquired from present investigations showed antioxidant enzyme activities, such as CAT, APX, and SOD, which serve as a defense system during plant stress periods and are raised under Al stress (Fig. 5) because of elevation in ROS (Jones et al. 2006;Yamamoto et al. 2003b). These results coincide with previously reported studies that suggested increase in SOD, CAT, and APX levels in sensitive and tolerant maize after exposure to Al treatment (Liu et al. 2008;Yamamoto et al. 2003a;Yamamoto et al. 2003b). After Al exposure, the activity of SOD, POD, APX, and CAT in tea plants increased (Ghanati et al. 2005). BRs induced greater antioxidant activities in both sensitive and resistant cultivars in the present study (Fig. 5). Antioxidant activities, such as SOD, POD, and CAT, are critical, and they are boosted by BRs after Al exposure to lessen the toxicity caused by Al (Ali et al. 2008a). BRs have been discovered to improve antioxidant enzymatic activity in stress situations to ameliorate various stresses (Nawaz et al. 2017;Rattan et al. 2020) and to regulate plant normal behavior in a number of studies (Ali et al. 2008b;Sharma et al. 2007). Al stress damages the membrane, resulting in a decrease in hydrolytic enzyme activity and an increase in ROS activity (Fig. 4). An increase in ROS activity is thought to cause significant damage to the cellular structure as well as macromolecules (Halliwell 1999). Under a stress situation, the use of BRs reduced H 2 O 2 generation as well as MDA concentration (Fig. 4) to protect the plant's membrane from oxidative damage. It reduced the rate of superoxide radicle formation and boosted plant antioxidant activity (Anuradha and Rao 2007;Mazorra et al. 2002;Ogweno et al. 2008).
A more precise approximation of antioxidant genes to the behavior of antioxidant enzyme activities can be obtained by studying gene expression at the transcriptional level of plants during heavy metal stress. As a result, we did the expression profiling of a number of genes related to antioxidant activity in order to evaluate both enzymatic and transcriptional responses of both rice cultivars under normal as well as stressful conditions. Higher transcriptional levels of APX02 and APX08 were recorded in the present study (Yamamoto et al. 2001). Similarly, APX expression was triggered by high levels of H 2 O 2 in tobacco chloroplasts under heavy metal stress (Gupta et al. 1993). Moreover, significant upregulation of CAT genes (CATa and CATb) was also observed in both roots and shoots in both cultivars. Previously, no significant change in CAT gene expression was observed in leaves of Arabidopsis thaliana under varying light qualities (Cominelli et al. 2008). It could have happened because there were several allo-or isozymes present. Al, on the other hand, induces a rise in CAT gene expression as a result of protein breakdown, resulting in transcriptional upregulation. Under Al stress, gene expression of SOD-Cu-Zn and SOD-Fe 2 was increased, indicating that oxidative damage inside diverse cellular compartments was produced by Al toxicity. When compared to control, the pattern of gene regulation was different because its expression was more upregulated in roots than that in shoots in both cultivars. It is possible since the plant roots are the initial site of contact with the toxicity generated by Al stress. Furthermore, superoxide production, rather than lipoxygenase activity, appears to be the primary cause of oxidative stress in roots, despite H 2 O 2 being an important competitor in leaves. However, the knowledge regarding whether Fig. 8 Dendrogram of two different rice cultivars under various treatments obtained through agglomerative hierarchical clustering using ward's method on the basis of physiological traits H 2 O 2 is produced as a result of increased heavy metal content in the leaves or as a signal molecule from the roots is still elusive (Cominelli et al. 2008).
PCA is a multivariate method that is commonly used to categorize values based on biological state, quality, and origins. PCA is used to detect and categorize a big data collection into a small number of strongly associated variables (Aziz et al. 2018). Based on different treatments, agglomerative clustering hierarchy (ACH) revealed the interaction between different rice genotypes (Fig. 8). Using a combination of PCA and ACH, several treatments were used to separate the sensitive and tolerant genotypes and to show the association between various attributes based on physiological traits (Fig. 5). VI, F/W, D/W, SL, RL, GE, and GP were found to have a group and showed positive association with each other, but a negative relationship with MGT, H 2 O 2 , and MDA. On morphophysiological, biochemical, and molecular levels, this study adds to understanding the mechanistic response of both cultivars (CY927, YLY689) under Al stress, as well as seed priming with BRs under Al toxicity in metal-tolerant and metal-sensitive rice cultivars.
Conclusions
In the current study, Al has demonstrated phytotoxic effects on the physiological, antioxidant system, and molecular mechanism of rice seedlings, according to this study. Results of this study depicted that seed priming with BRs reduced the negative effects of Al on O. sativa seeds and improved both germination and early seedling growth in the presence of Al toxicity. The exposure of Al stress in rice plants boosted the antioxidant system (SOD, POD, CAT, and APX), which was substantially further activated by BRs. A transcriptional analysis of antioxidant genes in both cultivars demonstrated the similar pattern. As a result, it is possible that the improved resistance to Al in rice seedlings corresponded to the altered level of the antioxidant system. In this study, YLY689 was found to be a more resistant variety against Al stress than CY927. Furthermore, the use of BRs boosted resistance by improving plant growth, photosynthetic pigments, and other related processes in rice seedlings under Al stress. | v3-fos-license |
2018-12-06T22:15:39.798Z | 2015-09-30T00:00:00.000 | 55813190 | {
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} | pes2o/s2orc | Optimization of Eugenol Extraction from Clove Oil using Response Surface Methodology
The objective of this research was to obtain optimum condition of eugenol production from clove oil using a central composite design method. The main process occured in the eugenol production was saphonification and neutralization processes. In order to optimize these processes, the ratio of NaOH/clove oil and temperature were studied as design variables i.e. ratio of NaOH/clove oil=1:2.5-1:3.5 while temperature was varied between 40 and 60oC. The yield of eugenol was considered as the main response in of this experiment. The result showed that the optimum condition was achieved when the temperature and the ratio of NaOH/clove oil were 50oC and 2.75:1, respectively and the yield was 39.17%.
Introduction
Since the production of clove oil has reached 250 tons in 2011, Batang regency has been considered as one of the largest producers of clove oil in Indonesia (Widayat et al, 2011).The clove oil is mostly extracted from part of clove plants (Eugenia caryophyllata thunb) such as leaves, flower, and stem.The quality of cloves oil is determined by the content of phenol compound, especially eugenol (BSN, 2010) which is still considered as the main problem of clove oil in Indonesia.Currently, the clove oil still has high content of eugenol and it is higher than the National Indonesian Standard (SNI).It has been reported that most of clove oil at small medium entrepreneurship (SME) in Batang Regency have eugenol contents up to 80% (Widayat et al, 2014) .Besides eugenol, the clove oil is also determined by β-Caryophyllene, an impurities that decrease the clove oil quality (Widayat et al, 2014).
Figure 1.The molecule structure of eugenol However, besides it is resulted from side product of clove oil, eugenol has potential for industries.Eugenol or Phenol, 2-methoxy-4-(2-propenyl) (Figure 1) is the main component of clove oil and it is colourless, has spicy taste and special odour which therefore it was mostly used for fragrance and flavour industries.The eugenol is also mostly utilized in the perfume industries, flavor concentrates and in the pharmaceutical industries as an antiseptic and anesthetic drug.Oyedemi et al. (2009) reported that eugenol is efficient for the metabolic activity of bacteria Listeria monocytogenes, Streptococcus pyogenes, Escherichia coli and Proteus vulgaris.Furthermore, Cheng et al. (2008) has utilized this compound and sinamaldehid as an anti-fungal for the type of fungus and Laetiporus sulphureus, which is considerable used as activity inhibitory.Shelly et al. (2010) used eugenol derivative such as methyl eugenol to effectively increase the fertility of insects.Sadeghian et. al., (2008) had utilized eugenol derivatives to inhibit the activity of the enzyme 15-lipogenase which is involved in many diseases such as asthma and lung cancer.The results showed that these compounds could inhibit the performance of the enzyme 15-lipogenase.Furthermore, Chami et al. (2004) also have tested eugenol as anti fungus Candida albicans.Vidhya and Devaraj (2011) experienced eugenol for inducing lung cancer, which showed inhibitory phenomena in lung cancer.
The price of clove oil is determined by its purity, the clove oil with purity under 70% will have price of Rp 120.000,00/ kg, while the clove oil with purity 98 % have price of 500.000/kg.Therefore, extraction and separation of eugenol from clove oil is an essential step to obtain such valuable product.According to USP (United States Pharmacopeia) standard, eugenol product must have purity higher than 98 %.
Another aspect for increasing eugenol contents is by implementation of chelating agent to reduce the darkness.The chelating agents that mostly used are as citric acid and sodium EDTA (Marwati et al, 2005).Commonly, this process can increase eugenol contents up to 80%.Another process for separation of eugenol from clove oil is by using saponification and distillation as well as fractionation.In this method, sodium hydroxide will be reacted with clove oil and then was neutralized by adding sulphuric acid or hydrochloride acid.The eugenol product then is separated using decantation and distillation process.This process could increase eugenol contents up to 82,6 % (minimum) (Anny, 2002;Sukarsono et al, 2003).However, the optimization is still required to estimate the associated variables in eugenol production.Therefore, the objective of this research was to conduct optimization of eugenol production from clove oil using neutralization process and fractional distillation by using Response Surface Methodology (RSM).
Materials
The clove oil used in this study was obtained from essential oils cluster Batang District.The clove oil has eugenol contents of 80%.
The equipment used for saponification and distillation process is presented in Figure 2. The distillation was operated in vacuum pressure that obtained with vacuum pump.Distillation column used packing system.
Experimental Design
The experimental design used is Response Surface Methodology (RSM) as described in Table 1 with two variables: the operation temperature and the ratio of clove oil to sodium hydroxide (NaOH).Experiment process were performed on a laboratory scale and batch processes.The operating conditions used as follows:
Procedure
The experiments began with oil density measurement and specific volume of clove leaf oil.Prior to experiment, clove oil was purified using citric acid (Widayat et al, 2014).A 150 ml of clove oil and NaOH were added under specified ratios of variables.The saponification reaction occurred for 45 minutes, and then followed by decantation for 1 day.The formed soaps were then neutralized with hydrochloride acid and were reacted for 5 minutes.This process was continued by decantation for 15 minutes.The eugenl product ws purified by using distillation for 1.5 hours.The final product was collected and was measured for its volume and refractive index.The concentration of products was also analyzed by using GC and data was analyzed by using STATISTICA. Where: X 1 = Coded variables for ratio of oil to NaOH X 2 = Coded variables for temperature Y = Yield of eugenol (%)
Data Analysis
The responses of this experiment were density, viscosity and eugenol concentration.The eugenol concentration was analyzed by using gas chromatography at Malang Polytechnic Laboratory.The liquid product was analysed by using gas chromatography (HP 5890, with HP 608 column) equipped with FID detector.The operating condition used helium as gas carries with flow rate of 20 ml/minute, temperature of 100-200 o C with temperature gradient 5 o /minute and initial time 5 minute.Detector temperature was 275 o C. The product was analysed with internal standard methods.6 μL eugenol standard solutions was added with internal standard (benzyl alcohol) 6 μL and 1 ml of solvent.The solution injected on GC equipment and conducted in triple runs.The response of detector was calculated with sample area divided internal standard area.The yield of eugenol was calculated according to Eq 1.
Chromatography Analysis
Fig. 3 shows the chromatogram analysis of methanol(solvent), benzyl alcohol and eugenol with retention time of 3.39; 10.04 and 18.36 minute, respectively.According to this chromatogram, the eugenol contents in the product were varies between 75.10 to 96.00%.This result showed that the euogenol producton could achieve higher concentration of eugenol from clove oil. Figure 3a shows a chromatogram of eugenol and internal standard and Figure 3.b for sample product and both figures show similarities, although there was a small shift of retention time.
Response Surface Methods
The results of statistic analysis was performed by developing mathematical model, t test, analysis of variance, pareto analysis as well as model validation.The polynomial equation of multi linear regression was proposed by following form: Where: Y = predicted response β 1, β 2 = linear coefficient for 1, 2 variable β 11, β 22 = squared term coefficient for 1,2 variable The variable or coefficients were obtained by determining the multi linear regression by using STATISTICA software.Through this software, the coefficients were obtained and showed by Eq 3.
This equation represents correlation between yield of eugenol and realted parmeters to obtain the yield.However, this variable of X1 and X2 are still in coded variable.The real values of condition process must be determined inversely.
In Eq 3, it is shown that the values of coefficient for X 1 and X 2 are negative.This leads to the fact that if the ratio of oil to NaOH (X 1 ) and temperature (X 2 ) increase then the yield of eugenol will decrease (for X 1 and X 2 >0).In addition, the interaction between X1 and X2 and also quadratic variable are positive, which means that the increase of both variables will also increase of the eugenol yield.The complete statistical analysis of coefficient by using multivariate variables in Eq 3 is shown by Table 2. Variance of mathematical model was analyzed and the results presented in Table 3. Table 3 can also be used to determine whether the independent variables simultaneously significant effect on the dependent variable.The degree of confidence was 0.05 (Bos et al, 2005;Lazic, 2004)).The parameters have F value more than p for all parameters except for X1 quadratic variable (F value = 0.0113 p=0.9221) and interaction variable(F=0.1759and p= 0.2953).The results of variance analysis support the t-test in Table 2 for X1 and X2 variable (single variable).
It also shows that the mathematical model already in optimum condition (maximum / minimum) no stripes ascend and mathematical model can be used directly for optimization.Pareto diagram can be a help to assist determination of the most important parameter in the process (Lazic ,2004).Pareto diagram (Figure 4) show that a linear variable X2 has the smallest value.Therefore this variable can be neglected due to no having effect in the process.All histogram of variables don't cross the line p = 0.05.Pareto chart show quadratic variable of X22 which has a histogram near to line p = 0.05.This proofs that quadratic variable X22 has the most effect in the eugenol yield .This condition can be increased for obtaining the optimum condition.Figure 5 shows optimum condition in term of a surface response graph that consists of x and y axis as independent variable (X 1 and X 2 ) and z-axis as dependent variable or yield of eugenol (Y). Figure 5 has a minimum poin meaning that the process is already minimized.Mathematical model described by equations 3 was validated with experiments data (Figure 6). Figure 6 also shows that the mathematical model has lower significant regression value (R 2 =0.764).However, such value is still suficient to obtain the optimum condition.
In Table 4, the critical value of dimensionless numbers for each variable.Critical dimensionless value obtained for X1 (ratio clove oil to sodium hydroxide) is -0.0109 and X2 (temperature) 0.3095.X1 and X2 critical value is substituted to equation 6 to obtained yield of eugenol.Yield of eugenol in minimum condition is 39.17%.
Discussion
The clove oil has been purified and separated from its eugenol contents.Eugenol which is one of indicator for quality of clove oil has been considered also as an important product for food industries.The result shows that 75-96% eugenol can be extracted from cloe oil by using separation method of saphonification, and distillation.
The Surface Response Method (SRM) has been employed for optimization and analysis of production and purification of eugenol from clove oil by using neutralization and fractionation distillation.The minimum yield was obtained as 39.17% under X 1 (ratio clove oil to sodium hydroxide) is -0.0109 (equal to 2.75:1) and X 2 (temperature) of 0.3095 (equal to 55 o C).The regression coefficient for the mathematical model was achieved at R 2 = 0.7642.
The further research is required to increase the eugenol purity and yield.Conventional steam distillation is typically considered the best way to obtain essential oils including eugenol from clove oil.However, this method produces varying eugenol product qualities dependent upon the temperature, pressure and time used for distillation.An important point regarding steam distillation of eugenol oils is that the temperature involved in the process changes the molecular composition of the plant matter.However, in practice most essential oils including eugenol are obtained by distillation at rather elevated temperature, in order to optimize the yield.Other alternative were proposed to use process intensification through microwave, ultrasound or supercritical fluid extraction and distillation.However, their economical feasibility must also be considered.Further action is also required to investigate the eugenol risk to human in term of their use for food.
Conclusion
This study showed the distillation process of eugenol out of clove oil.The distillation method give much better improvements in term of its yield and purity of products.The optimum condition was achieved when the temperature 55oC and the ratio of clove oil/NaOH was 2.75 :1.
Further improvement is required to increase the yield by using process intensification through microwave, ultrasound or supercritical technology.
Figure 2. Vacuum Distillation equipment
Figure 5 .
Figure 5. Countour Graphics of optimization results material ratio and temperature
Table 1 .
Variable Central Composite Design for Experiment
Table 2 .
Regression analysis result
Table 4 .
Critical value results / optimization | v3-fos-license |
2020-08-20T10:03:32.008Z | 2020-08-12T00:00:00.000 | 221320815 | {
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} | pes2o/s2orc | Uniaxial Static Strain Promotes Osteoblast Proliferation and Bone Matrix Formation in Distraction Osteogenesis In Vitro
Objective We aimed at investigating the effects of uniaxial static strain on osteoblasts in distraction osteogenesis (DO). Methods To simulate the mechanical stimulation of osteoblasts during DO, 10% uniaxial static strain was applied to osteoblasts using a homemade multiunit cell stretching and compressing device. Before and after applying strain stimulation, the morphological changes of osteoblasts were observed by inverted phase-contrast microscopy, Coomassie blue staining, and immunofluorescence. Alkaline phosphatase (ALP) activity, mRNA levels (proliferating cell nuclear antigen [PCNA], ALP, Runx2, osteocalcin [OCN], collagen type I, hypoxia-inducible factor- [HIF-] 1α, and vascular endothelial growth factor [VEGF]), and protein levels (Runx2, OCN, collagen type I, HIF-1α, and VEGF) were evaluated by using ALP kit, real-time quantitative reverse transcription-polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Results After the mechanical stimulation, the cytoskeleton microfilaments were rearranged, and the cell growth direction of the osteoblasts became ordered, with their direction being at an angle of about 45° from the direction of strain. The proliferation of osteoblasts and the expression levels of mRNA and protein of ALP, Runx2, OCN, collagen type I, HIF-1α, and VEGF were significantly higher than in the nonstretch control groups. Conclusion Our homemade device can exert uniaxial static strain and promote the proliferation of osteoblasts and bone matrix formation. It can be used to simulate the mechanical stimulation of osteoblasts during DO.
Introduction
Distraction osteogenesis (DO), known as "endogenous bone tissue engineering," is a predictable and effective surgical technique to regenerate new bone in the gap between two bone segments to lengthen or widen the bone [1,2]. This technology was first reported by Italian scholar Codivilla in 1905 to treat a shortened femur [3], and then it was systematically developed for orthopedic surgery by Dr. Gabriel A. Ilizarov in the 1950s [4][5][6]. After many craniomaxillofacial animal models of DO were established, DO technology has gradually become a research hotspot in oral and maxillofacial surgery and plastic surgery [7,8]. Today, DO is being widely used to treat craniomaxillofacial congenital malformations or acquired large bone defects due to trauma, infections, and postresection of tumors.
The DO procedure requires the application of a gradual and controlled mechanical distraction force to the bone segments, which is crucial for new bone formation. However, the molecular mechanisms of DO remain unknown. Moreover, complications such as premature consolidation and incomplete callus formation [9] during DO still exist. All these questions have limited the application of DO in clinics. Therefore, there is a burning need to understand the underlying specific molecular mechanisms of DO, which can help us better regulate successful bone formation and reduce the complications during the DO process. Many previous studies have attempted to elucidate the mechanisms of DO, and various cell mechanical loading devices (such as FlexCell system [10] and four-point bending [11]) have been developed to simulate the mechanical environment of osteoblasts during DO. However, these studies were severely limited because the forces applied to osteoblasts were mainly fluid shear stress, equiaxial strain, multiaxial strain, centrifugal force, and cyclic reciprocating strain. These kinds of forces did not replicate the actual mechanical environment of osteoblasts during DO [12,13]. Actually, the mechanical stimulation of osteoblasts in DO involves mainly uniaxial static strain along the stretching direction [14,15] (Figure 1).
Our research group has successfully developed a multiunit cell stretching and compressing device (the patent number of invention: ZL200910164248, China) (Figure 2), which can exert uniaxial static strain to simulate the mechanical environment of DO. The mechanical stimulation can be transmitted to the cultured cells through the deformation of a silica gel membrane with good biocompatibility. Accordingly, 1%, 5%, 10%, and 15% uniaxial static strains were applied on the cultured osteoblasts by the device. The Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) assay showed that 10% uniaxial static strain is best able to promote the proliferation of osteoblasts. To further elucidate the underlying molecular mechanisms of new bone formation during the process of DO, 10% uniaxial static strain was applied on the osteoblasts by the device. The morphology changes, proliferation, and the synthesis and secretion of bone matrix by the osteoblasts were evaluated through real-time polymerase chain reaction (PCR), western blot, and enzyme-linked immunosorbent assay (ELISA).
Materials and Methods
2.1. Osteoblast Isolation and Culture. The Animal Care and Use Committee of Southern Medical University approved the procedures of the study. Osteoblasts were isolated and cultured in accordance with a previously published reference [16]. Briefly, the skull was isolated from 1-to 2 d old neonatal Sprague Dawley (SD) rats under aseptic conditions and then minced into fragments of about 0.5 mm 3 . The bone fragments were washed with phosphate-buffered saline (PBS) (containing 100 units/mL penicillin and 100 mg/mL streptomycin) three times and digested with 0.25% trypsin (Gibco, Life Technologies, Carlsbad, CA, USA) at 37°C for 30 min with shaking. Then, they were washed with serum-free Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) three times after the trypsin solution was removed. A total of 0.1% collagenase II (Gibco) in serum-free DMEM was added to the bone fragment, allowing digestion at 37°C for 30 min with shaking. The supernatant was centrifuged at 1,000 rpm for 5 min to obtain the cell pellet. The pellet was resuspended in serum-free DMEM and centrifuged; this process was repeated twice. Finally, the pellet was resuspended in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco), 100 units/mL penicillin, and 100 mg/mL streptomycin and then seeded in a 25 cm 2 culture flask at a density of 2 × 10 5 cells/mL. After incubating for 10 min at 37°C and 5% CO 2 (Thermo Scientific 311; Marietta, GA, USA), the culture medium and suspended osteoblasts were transferred to a new 25 cm 2 culture flask to remove the earlier adherent fibroblasts. Finally, the purified osteoblasts were incubated at 37°C and 5% CO 2 . The culture medium was refreshed every 2-3 d. The morphology and growth of the cells were observed and photographed using an inverted phase-contrast microscope. The cells were subcultured at a ratio of 1 : 3 upon reaching 80% confluence, and cells at the third passage were used for the following experiments.
Cell Proliferation Activity under Different Uniaxial Static
Strains. The schematic diagram and photograph of the device are shown in Figure 2. A screw connects the power take-off shaft of the stepping motor and the beam, and the beam is connected with the six cell culture units. The uniaxial static strain exerted by the stepping motor is transmitted to the silica gel membrane in the cell culture units and causes cell deformation. The displacement detector detects and controls the tensile and compression displacement, and the displacement indicator displays the displacement.
For our study, the osteoblast suspension (2 mL) was seeded on a silica gel membrane (cell seeding density: 2 × 10 4 cells/mL) coated with collagen type I from rat tail (Sigma-Aldrich) in a cell culture unit and further incubated for 24 h at 37°C and 5% CO 2 , allowing for osteoblast attachment. Then, an additional 10 mL of DMEM supplemented with 10% FBS was replenished. Subsequently, 1%, 5%, 10%, and 15% uniaxial static strains were applied to the osteoblasts 24 h later using the multiunit cell stretching and compressing device. The culture medium was refreshed every 2-3 d. Control groups (nonstretch groups) were cultured under the same conditions, except for the application of mechanical stimulation. The proliferation activity of cells was analyzed using a CCK-8 (Dojindo Laboratories,) assay at days 1, 3, 5, and 7 of the experiment. Briefly, the adherent osteoblasts of the stretch and nonstretch groups were collected from the silica gel and transferred to a culture plate. CCK-8 solution was added to each well (n = 4). The cells were then incubated at 37°C and 5% CO2 for 2 h. Optical density was measured at 450 nm using a multiscan spectrometer (Varioskan Flash; Uniaxial static strain BioMed Research International Thermo Electron Corporation). Each experiment was repeated three times independently.
Osteoblast Morphology.
According to the CCK-8 assay, 10% uniaxial static strain is best able to promote the proliferation of osteoblasts. Therefore, in the following experiments, we used the homemade device to apply a 10% uniaxial static strain to simulate the mechanical environment of osteoblasts during DO in vivo. Inverted phase-contrast microscopy, Coomassie blue staining, and immunofluorescence were used to observe the changes in morphology and growth direction of osteoblasts. After culturing for 3 d, the osteoblasts of the stretch and nonstretch groups were washed with PBS and fixed with 4% paraformaldehyde for 20 min. Next, they were rinsed three times with deionized water and stained with Coomassie bright blue staining solution (2 g/L) (Sigma-Aldrich) for 60 min at room temperature. The cells were washed three times with deionized water, and methanol-glacial acetic solution was added to stop the staining reaction until the cytoplasm was slightly clear under an inverted phase-contrast microscope. Finally, the osteoblast morphology was observed and photographed with an inverted phase-contrast microscopy. The immunofluorescence procedure was as follows. First, after culturing for 3 d, the osteoblasts of the stretch and nonstretch groups were washed with PBS and fixed with 3.7% paraformaldehyde in PBS for 30 min at room temperature. Next, the cells were rinsed three times with 0.1% Triton X-100 (Sigma-Aldrich) in PBS. Thereafter, 1 mL of diluted (1 : 50) Actin-Tracker Green solution (Sigma-Aldrich) was added to the osteoblasts on the silica gel membrane and incubated for 40 min at room temperature in the dark. The osteoblasts were washed and 1 mL of 4 ′ , 6-diamidino-2phenylindole (DAPI) staining solution (Sigma-Aldrich) was added to cover the osteoblasts and maintained for 5 min at room temperature. After aspirating the DAPI staining solution and washing, the osteoblasts were observed and photographed under a fluorescence microscope.
ALP Activity
Assay. The ALP activity of osteoblasts was detected using an ALP kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) after culturing for 1, 3, and 5 d. Briefly, the adherent osteoblasts of the stretch and nonstretch groups were separated and collected from the silica gel by trypsin digestion (2,000 rpm for 1 min). An aliquot (100 μL) of cell lysate containing 0.05% Triton X-100 was used to lyse the osteoblasts at 4°C for 12 h. A 20 μL sample and 100 μL of substrate buffer (containing p-nitrophenyl phosphate) were added to a 96-well culture plate, shaken for 1 min, and incubated at 37°C for 15 min, followed by addition of 80 μL of reaction termination liquid and shaking for 1 min. Finally, the absorbance value was measured at 3 BioMed Research International 520 nm wavelength, and the resulting nitrophenol level was calculated following the instructions in the ALP kit. Referring to the normal standard, total intracellular protein content was detected using the bicinchoninic acid (BCA) Protein Assay Kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). The relative ALP activity was calculated by dividing the amount of nitrophenol by the corresponding total protein.
Quantitative
Real-Time Reverse Transcription-PCR (RT-PCR). RT-PCR was performed to evaluate the expression levels of proliferating cell nuclear antigen (PCNA), ALP, Runx2, osteocalcin (OCN), collagen type I, hypoxiainducible factor-(HIF-) 1α, and vascular endothelial growth factor (VEGF). Briefly, after culturing for 1, 3, and 5 d, the silica gel membranes with the attached osteoblasts from the stretch and nonstretch groups were transferred to a Petri dish, and the total mRNA was extracted using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA).
Reverse transcription was performed to synthesize cDNA from the purified RNA using Oligo (dT) primers (Promega, San Luis Obispo, CA, USA) and SuperScript III reverse transcriptase (Invitrogen). Finally, cDNA was subjected to real-time PCR (Applied Biosystems 7300; Applied Biosystems Foster City, CA, USA) using the SYBR Green probe (PerfeCTa SYBR Green FastMix, ROX; Quanta Biosciences, Gaithersburg, MD, USA) with custom-designed primers (Takara Bio, Dalian, China; Table 1).
Expression of the target gene was first normalized to that of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the same sample (△Ct = threshold cycle change). Then, the target gene of the experimental groups was normalized to the average baseline expression of the gene measured for the control groups (△△Ct). The 2 -△△Ct method was used to convert the normalized gene expression levels in accordance with a previously published reference [17].
2.6. Western Blot. Western blot was performed to evaluate the protein levels expressed by the osteoblasts, including Runx2, collagen type I, HIF-1α, and GAPDH. Briefly, the adherent osteoblasts were scraped from the silica gel membrane and centrifuged at 2,000 rpm for 1 min. After lysing the cells in lysis buffer for 15 min on ice, cell debris was removed by centrifugation at 14,000 rpm for 15 min at 4°C. The supernatant was collected and stored at -80°C until use. The protein content was determined using the Bradford method, and the samples were diluted to 1 μg/μL with the lysate. Equivalent amounts of protein were separated with 5% stacking gel and 10% separating gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) for 90 min at 120 V using a Trans-Blot SD electrophoresis apparatus (Bio-Rad). The PVDF membranes were blocked with 5% skimmed milk for 1 h at room temperature and incubated with primary antibodies (including Runx2, collagen type I, HIF-1α, and GAPDH from mouse) (R&D Systems, Minneapolis, MN, USA) at 4°C overnight, then washed three times with 0.1% Tween-Tris-buffered saline (TBS) (TTBS, pH 7.5), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1 : 2,000) (R&D Systems) for 1 h at 37°C. Finally, the specific protein bands were visualized with an Immuno-Star Western C kit on a ChemiDoc XRS+ System (Bio-Rad) using BCA as a standard.
2.7. ELISA Tests. Protein levels of OCN and VEGF secreted by the osteoblasts were analyzed using an ELISA kit (R&D Systems). Briefly, after culturing for 1, 3, and 5 d, the culture supernatant of the stretch and nonstretch groups was collected, centrifuged at 3,000 rpm for 20 min at 4°C, and then stored at -20°C for use. First, we added 50 μL sample dilutions at a dilution ratio of 1 : 5 to the wells. The plates were sealed and incubated at 37°C for 30 min. Then, we aspirated . Differences were assessed by one-way analysis of variance (ANOVA) and were considered statistically significant at P < 0:05. The data are indicated with * for P < 0:05. All the experiments were repeated at least three times.
Results
3.1. CCK-8 Assay. As shown in Figure 3, the osteoblasts in the five groups kept growing during the experiment. However, osteoblasts grew the fastest under 10% uniaxial static strain at 3, 5, and 7 d when compared to other groups.
In Vitro Culture and Morphological Observation of
Osteoblasts. The osteoblasts of the nonstretch control groups were mostly short fusiform, triangular, or star-shaped and in a disorderly array (Figures 4(a), 4(c), and 4(d)). After being subjected to 10% uniaxial static strain for 3 d, the osteoblasts became elongated and appeared as long spindles under the inverted phase-contrast microscope. The growth direction gradually developed from random disorder to cells aligned in the same direction, at an angle of about 45°from the direc-tion of strain (Figure 4(b)). Coomassie blue staining and immunofluorescence staining images showed that the osteoblasts were still slightly randomly oriented (Figures 4(d) and 4(f)). The orientation of osteoblasts in images D and F was not so regular as that in image B. However, the orientations of the osteoblasts had a trend to arrange regularly in images D and F.
3.3. ALP Activity. As a key enzyme related to bone metabolism, ALP plays an important role in bone formation and mineralization. The changes in ALP activity can reflect the differentiation ability of osteoblasts. The results ( Figure 5) show that after the application of 10% uniaxial static strain, the ALP activity was higher than the control group at the three time points. The ALP activity of the experimental group was the highest on Day 1 and then gradually slowed at Days 3 and 5, while, for the control group, the activity was almost the same. The results indicated that 10% uniaxial static strain promoted the secretion of ALP by osteoblasts.
3.4. RT-PCR Assay. The expression levels of PCNA, ALP, Runx2, OCN, collagen type I, HIF-1α, and VEGF of osteoblasts from the experimental and control groups were measured at Days 1, 3, and 5 ( Figure 6). The expression levels of PCNA of the experimental group were the highest at Day 1 and then decreased gradually, while in the control group, the level slowly increased. Moreover, the experimental group showed significantly higher levels at all three time points compared with the control group. This suggested that 10% uniaxial static strain was able to promote the proliferation of osteoblasts.
ALP, Runx2, OCN, and collagen type I are important osteogenesis-related markers. HIF-1α and VEGF are important angiopoiesis-related factors. The results of mRNA expression of ALP were inconsistent with the ALP activity detected by the ALP kit. Specifically, after the application of Figure 3: CCK-8 assay after application of 0% (control), 1%, 5%, 10%, and 15% uniaxial static strain to osteoblasts using the device. * * * P < 0:001 when compared with other groups.
BioMed Research International
10% uniaxial static strain, the mRNA expression levels of ALP in the osteoblasts were higher than in the control group at Days 1, 3, and 5. The highest level was on Day 1 and it gradually decreased at Days 3 and 5, while the results for the control group were almost unchanged at the three time points.
Similarly, the mRNA levels of Runx2, OCN, collagen type I, HIF-1α, and VEGF showed a significantly higher expression after the application of 10% uniaxial static strain at Days 1, 3, and 5 than those of the control group. The mRNA levels reached the highest value on Day 1 and decreased gradually, while those of the control group slowly increased from Day 1 to Day 5.
Western
Blot. The expression amounts of the proteins Runx2, collagen type I, and HIF-1α were measured at Days 1, 3, and 5 ( Figure 7). The amounts of protein of Runx2 and collagen type I peaked at Day 1 and decreased gradually, while the amount of protein of HIF 1α increased gradually and peaked at Day 5. The variation trends of Runx2 and collagen type I were consistent with the results of the mRNA expression levels detected by RT-PCR, while the variation trend of HIF 1α was opposite to the result of RT-PCR.
3.6. ELISA Tests. As shown in Figure 8, the expression levels of OCN and VEGF were measured at 1, 3, and 5 d after the BioMed Research International application of 10% uniaxial static strain. The results of the experimental group decreased slightly, while that of the control group slowly increased from Day 1 to Day 5. The amounts of OCN and VEGF proteins in the experimental groups were significantly higher than those in the control groups. The variation trends were consistent with the results of mRNA expression levels detected by RT-PCR.
Discussion
Biological living tissues have potential biological plasticity. Slow and sustained mechanical force can stimulate cell proliferation, increase biosynthetic function, and promote tissue metabolism, thus leading to tissue regeneration. As Ilizarov proposed in the "law of tension-stress" of DO, gradual and continuous distraction can stimulate and maintain bone regeneration and growth [18]. However, the underlying specific molecular mechanisms of DO have not been fully understood. To better understand the mechanisms of DO, the first thing to do is create a similar mechanical microenvironment with DO in vivo. Therefore, our team developed a multiunit cell stretching device. Compared to previous studies, our experimental results demonstrate that this device can exert uniaxial static strain on osteoblasts, which can be used to better simulate the actual mechanical stimulation of osteoblasts in the DO process. Appropriate mechanical stimulation is particularly important and can promote osteoblast growth and maintenance of function, whereas excessive or inadequate force will have no effect or cause damage to the structure and function of osteoblasts. In this study, we found that a 10% uniaxial static strain is the best for the proliferation of osteoblasts. Our result is similar to or a little different from those in other studies [19,20]. Bhatt et al. [21] found that a 9% strain is the best for osteoblast proliferation using a Flexcell system, while Gao et al. [22] found that 12% cyclic stretch is best for osteogenesis-related gene expression in MG-63 cells. The differences may be that other factors, such as the type of strain, magnitude, frequency, and duration, may influence the mechanical response of osteoblasts.
Osteoblast is a kind of mechanosensory cell; it has a variety of mechanoreceptors, such as the extracellular matrix (ECM) and integrin, focal adhesions, and cytoskeleton, special structures of cell membranes, cadherin and ephrin, primary cilia, ion channels, and connexins [23,24]. When subjected to a mechanical force, the mechanoreceptors of osteoblasts can feel the stimulation signal quickly, causing changes in the osteoblast skeleton, such as microfilament. In our experiment, we can see that when subjected to mechanical force, the osteoblasts became longer as observed under an inverted phase-contrast microscope. The cell growth direction gradually developed from random disorder to cells aligned in the same direction, at almost 45°angle with the direction of strain. However, the orientation of osteoblasts in images D and F was not so regular as that in image B, which were still slightly randomly oriented. The reason might be that the duration of mechanical stimulation is not enough. The dyeing process of the osteoblasts may also have some influences. Wang et al. [25] applied 10% periodic strain to epithelial cells for 30 min and found that the angle between cell arrangement and the strain direction was 70°. The duration and magnitude of mechanical stimulation might have influenced the angle between cell arrangement and the strain direction. Our results indicate that osteoblasts can respond to uniaxial static strain, leading to changes in cytoskeletal microfilaments and cell growth direction. Microfilament forms a functional complex with related proteins, integrin, and ECM. It can maintain normal cell morphology and respond to external mechanical stimulation. It also participates in the transduction of multiple signals and in material transport inside and outside the cell. Wang et al. [25] demonstrated that signal transduction of mechanical stimulation relies on the integrity of the microfilaments.
Mechanical signals can be gradually transmitted to the intracellular area and nucleus; these signals mediate the cellular functions for osteoblast proliferation, which was verified by the mRNA expression level of PCNA. In this study, it was found that the proliferation of the experimental group was higher than that of the control group, and this result was in agreement with the results reported by most researchers [26,27]. The mechanism of promotion of cell proliferation by strain may be that strain can enhance the mitosis of osteoblasts by increasing the expression of the proto-oncogene c-fos [28].
In the present study, we also observed changes in the expression levels of ALP, OCN, collagen type I, and Runx2 after application of mechanical stimuli to osteoblasts. These proteins act synergistically to regulate the normal function of osteoblasts and affect the entire process of bone metabolism. ALP and OCN are usually considered as early and late markers of bone matrix formation, respectively [29,30]. Collagen type I is the main matrix of the bone secreted by osteoblasts. It plays an important role in the process of adhesion, proliferation, and bone matrix mineralization of osteoblasts [31]. Runx2, also known as Cbfα1, is an important transcription factor in osteogenic differentiation. It can bind to osteoblast-specific cis-acting elements and promote the transcription and translation of ALP, OCN, and collagen type I, thereby promoting the deposition of bone matrix. The 9 BioMed Research International variation trends in the gene expression levels were consistent with those of the protein amounts. That is, the expression levels of the genes and the proteins (ALP, Runx2, OCN, and collagen type I) were enhanced after applying 10% uniaxial static strain, but subsequent increases in expression were gradually reduced. Kaspar et al. [32] applied a 1,000 με (0.1%), 1 Hz periodic reciprocating tensile strain to human osteoblasts using a four-point bending device, and they found that the ALP activity and OCN secretion were significantly reduced. Kaspar et al. [32] found that 17% periodic intermittent equiaxial strain through the Flexcell system can promote the ALP activity of osteoblast-like cells (HT-3). Kanno et al. [33] found that uniaxial cyclic strain (15% tensile stress, 0.5 Hz) promotes ALP activity and levels of OCN and Runx2 of osteoblast-like MC3T3-E1cells. When Fong et al. applied 10% continuous equiaxial strain to cranial osteoblasts of neonatal rats, the expression of collagen type I increased [27]. There are different views on whether strain can promote the expression of osteogenic-related genes in osteoblasts, probably due to the differences of osteoblast sources and cell-loading devices. Furthermore, the type, magnitude, frequency, and duration of the mechanical stimulation may also influence the mechanical response of osteoblasts. The mechanism of strain-induced osteogenesis enhancement may be that the transcription factor FosB or the cell signaling pathway of Wnt/β-catenin and Ras-extracellular signal-regulated kinase (ERK) 1/2-mitogen-activated protein kinase (MAPK) was activated by the strain [33][34][35]. In previous studies, a periodic reciprocating tensile strain of 0.1% 1 Hz significantly reduced the ALP activity and OCN secretion of osteoblasts [32], probably because the mechanical stimulation was insufficient to initiate osteogenesisrelated signaling pathways.
In DO, angiogenesis runs through the whole process of new bone formation [36]. Blood vessels not only act as a transport conduit system for oxygen and nutrients for the new bone but they also promote bone formation by regulating the interaction between bone-related cells and vascular endothelial cells [37][38][39]. Therefore, exploring the molecular mechanisms of vascularization and finding strategies and methods to accelerate and optimize the DO process are very important in expanding the clinical application of DO. Hypoxic conditions and VEGF are essential conditions for angiogenesis during bone formation. Osteoblasts can participate in the process of vascularization of bone tissue by secreting cytokines such as HIF-1α and VEGF [40].
HIF-1α is an important transcription factor that regulates oxygen homeostasis. Under hypoxic conditions, HIF-1α expressed in osteoblasts can relocate to the nucleus and bind to HIF-1β to form active HIF-1, which can bind to the hypoxia-responsive element (HRE) on the target gene, further activating hypoxia-related genes (such as VEGF and angiopoietin) and promote angiogenesis [41]. HIF-1α may also directly bind to the upstream site of the osteoprotegerin (OPG) gene to increase the gene's expression and then promote bone formation [42]. VEGF plays an important role in angiogenesis and osteogenesis. It can be regulated by many factors, such as growth factors, hormones, transcription factors, and mechanical stimulation. Hypoxia and mechanical stress can promote the expression of osteoblast VEGF as VEGF is a downstream factor of the HIF-1α pathway. VEGF can interact with VEGF receptors on vascular endothelial cells; this process activates tyrosine kinases and other downstream signaling molecules, induces proliferation of vascular endothelial cells, and increases capillary permeability, all of which provide the conditions for vascular endothelial cell migration and angiogenesis [43]. The neovessel not only delivers the oxygen and nutrients needed for the formation of bone tissue but vascular endothelial cells also release osteogenic factors, such as bone morphogenetic protein-(BMP) 2 or BMP-4, to accelerate osteoblast differentiation and maturity [44].
In this experiment, the expression levels of VEGF and HIF-1α increased after the application of 10% uniaxial static strain. The variation trend of the mRNA expression levels of VEGF was consistent with the result of the protein contents, while the variation trend of the mRNA expression levels of HIF 1α was opposite to the result of the protein contents.
The results are consistent with those of Fong et al. [27], who found that 10% sustained equiaxial strain increased the VEGF expression level in neonatal rat cranial osteoblasts. Similar results were obtained by Kim et al. [45] who found that VEGF secretion of bone marrow stromal cells increased after application of 0.04% equiaxial static strain. Kim et al. [45] also found that the expression of HIF-1α was elevated when skeletal muscle was subjected to strain. Petersen et al.
found that the expression of VEGF and HIF-1α increased in tendon fibroblasts after applying cyclic reciprocating uniaxial tension [46]. Chang et al. [47] applied isometric cyclic tension (20%, 1 Hz) to rat vascular smooth muscle cells by using negative pressure on the elastic basement membrane and found that HIF-1α mRNA and protein expression increased, and that this process may be regulated by the p42/p44 MAPK kinase (MAPKK) pathway. Chang et al. [47] concluded that static strain-induced VEGF expression may be observed by regulating MAPK (ERK and p38). These findings demonstrate that static strain can promote the expression levels of VEGF and HIF-1α in osteoblasts, which is important for providing both blood supply and nutrition for new bone formation during DO.
Conclusion
In the present study, we applied 10% uniaxial static strain to osteoblasts using a homemade multiunit cell stretching and compressing device and investigated the biological effects by observing osteoblast morphology, as well as performing ALP activity assay, RT-PCR, western blot, and ELISA. The results showed that the cytoskeleton microfilaments were rearranged and the cell growth direction of the osteoblasts became ordered. Moreover, uniaxial static strain promoted the proliferation, osteogenesis, and angiogenesis of osteoblasts. This study provides a basis for further elucidation of the molecular mechanisms of DO and provides a possible way to control the DO process in the clinic by regulating the above osteogenesis-and angiogenesis-related factors. The relationships between the above factors in related signaling pathways will be investigated in further studies to elucidate the molecular mechanisms of DO.
Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper. | v3-fos-license |
2020-01-29T16:29:41.391Z | 2020-01-29T00:00:00.000 | 210938675 | {
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} | pes2o/s2orc | Activation of pH-Sensing Receptor OGR1 (GPR68) Induces ER Stress Via the IRE1α/JNK Pathway in an Intestinal Epithelial Cell Model
Proton-sensing ovarian cancer G-protein coupled receptor (OGR1) plays an important role in pH homeostasis. Acidosis occurs at sites of intestinal inflammation and can induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), an evolutionary mechanism that enables cells to cope with stressful conditions. ER stress activates autophagy, and both play important roles in gut homeostasis and contribute to the pathogenesis of inflammatory bowel disease (IBD). Using a human intestinal epithelial cell model, we investigated whether our previously observed protective effects of OGR1 deficiency in experimental colitis are associated with a differential regulation of ER stress, the UPR and autophagy. Caco-2 cells stably overexpressing OGR1 were subjected to an acidic pH shift. pH-dependent OGR1-mediated signalling led to a significant upregulation in the ER stress markers, binding immunoglobulin protein (BiP) and phospho-inositol required 1α (IRE1α), which was reversed by a novel OGR1 inhibitor and a c-Jun N-terminal kinase (JNK) inhibitor. Proton-activated OGR1-mediated signalling failed to induce apoptosis, but triggered accumulation of total microtubule-associated protein 1 A/1B-light chain 3, suggesting blockage of late stage autophagy. Our results show novel functions for OGR1 in the regulation of ER stress through the IRE1α-JNK signalling pathway, as well as blockage of autophagosomal degradation. OGR1 inhibition might represent a novel therapeutic approach in IBD.
Results
OGR1 induces ER stress under acidic conditions. In order to investigate the role of the pH-sensing OGR1 receptor in the induction of ER stress, OGR1-overexpressing Caco-2 and VC Caco-2 cells, were subjected to an acidic pH shift for 24 h. The stress inducer tunicamycin induced protein expression of the ER stress marker BiP in a dose dependent manner in VC Caco-2 cells and Caco-2 cells overexpressing OGR1 ( 1C) and p-IRE1α to total IRE1α (Fig. 1D) is presented. BiP mRNA expression also significantly increased under acidic conditions in Caco-2 overexpressing OGR1 compared to VC cells (Fig. 1E). Interestingly, at acidic pH the expression of BiP and phosphorylation of IRE1α were markedly reduced in OGR1-overexpressing Caco-2 cells in the presence of the OGR1 inhibitor ( Fig. 1F and Supplementary Figure 3), suggesting that ER stress is induced by proton-activated OGR1 signalling. In OGR1 overexpressing Caco-2 cells, pH-dependent OGR1 signalling triggered the splicing of XBP1, which was prevented in the presence of the OGR1 inhibitor (Fig. 1G,H, and Supplementary Figure 4), confirming the role of OGR1 in the induction of ER stress. OGR1 induces ER stress via IRE1α/JNK signalling. Next, we sought to identify the signalling factors involved in acidic pH-induced OGR1-mediated ER stress. Acidic pH induced BiP expression and JNK phosphorylation in OGR1-overexpressing Caco-2 cells compared to VC cells ( Fig. 2A and Supplementary Figure 5). Importantly, BiP expression and JNK phosphorylation were prevented in the presence of the OGR1 inhibitor ( Fig. 2A and Supplementary Figure 5). Strikingly, in OGR1-overexpressing cells the expression of JNK was increased in the presence of the OGR1 inhibitor. This result suggests a compensatory mechanism that would trigger JNK expression following blockade of JNK phosphorylation. Of note, acidic pH failed to induce cleavage of ATF6 ( Fig . ER stress is induced by acidosis activated OGR1-mediated signalling. Caco-2 cells were subjected to different pH medium, following 4-6 h incubation in pH 7.6 serum free medium. (A) Vector control Caco-2 (VC) and OGR1 overexpressing Caco-2 cells where treated with tunicamycin at the indicated concentrations for 24 h. Total protein was isolated and Western blotting was performed. The results are representative of two independent experiments. (B) After 24 h pH shift, total protein was isolated and Western blotting was performed. The results are representative of three independent experiments. (C) Densitometry after normalization of BiP to β-actin and (D) p-IRE1α to total IRE1α. Statistical analysis was performed using oneway ANOVA followed by Tukey's post-test. Data are presented as means ± SE of three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (E) After 24 h pH shift, total RNA was isolated and mRNA expression was investigated by qPCR. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-test. Data are presented as means ± SE of three independent experiments (*p < 0.05; **p < 0.01). (F) A specific small molecule OGR1 inhibitor (10 µM) was tested and the cells were subjected to low pH for 24 h, following 4-6 h incubation in pH 7.6 serum free medium. After 24 h pH shift, total protein was isolated and Western blot performed. Results are representative of two independent experiments. (G) Cells were treated as described in (F), then total RNA was extracted and analysed for expression of XBP1 (XBP1u) and spliced XBP1 (XBP1s) by conventional PCR. Results are representative of three independent experiments. Acidosis activated OGR1-mediated signalling does not induce apoptosis. Since IRE1α/JNK signalling has been shown to trigger apoptosis by inhibiting Bcl-2, we investigated the impact of OGR1 activation on the induction of apoptosis. VC and OGR1-overexpressing cells where subjected to an acidic pH shift in the presence or absence of the OGR1 inhibitor for 24 h. Annexin V and PI staining followed by FACS analysis revealed that the population of apoptotic cells was not affected by the acidic pH shift in OGR1-overexpressing cells ( Fig. 3A-C). Furthermore, cleavage of caspase 3 and poly (ADP-ribose) polymerase (PARP) were investigated by immunoblotting. Under the condition that BiP was upregulated on activation of OGR1, neither cleaved caspase 3 nor cleaved PARP was observed ( Fig. 3D and Supplementary Figure 9), confirming that apoptosis was not induced in OGR1-overexpressing cells.
Acidosis activated OGR1-mediated signalling blocks autophagy. ER stress has been linked to the blockage of autophagy. Therefore, we sought to investigate the role of OGR1 in autophagy. VC and OGR1-overexpressing Caco-2 cells were subjected to an acidic pH shift for 24 h and protein levels of LC3-I and LC3-II were investigated by immunoblotting. Acidic pH reduced the conversion of LC3-I into LC3-II, and blocked autophagosome degradation, evidenced by the accumulation of total LC3 in OGR1-overexpressing Caco-2 cells compared to VC cells ( Fig. 4A and Supplementary Figure 10). We confirmed these results using immunofluorescence microscopy. OGR1-overexpressing cells subjected to an acidic pH shift showed increased LC3 staining, which was reversed in the presence of the OGR1 inhibitor. On the other hand, no changes were observed in the VC under different pH conditions with or without OGR1 inhibitor ( Fig. 4B-D). These results suggested that autophagy is blocked by proton-activated OGR1 signalling.
Discussion
Our results show that proton-activated OGR1-mediated signalling triggers the expression of the ER stress marker BiP together with the phosphorylation of IRE1α and splicing of XBP1 in a human intestinal epithelial cell line stably overexpressing OGR1. Furthermore, we found that activation of OGR1 triggers the IRE1α-JNK signalling pathway, but not the other branches involved in the UPR, namely PERK or ATF6. Acidosis and activation of the UPR in intestinal epithelial cells are closely linked to the development of intestinal inflammation (Fig. 5). Our results provide confirmatory evidence of a crucial role for OGR1-mediated IRE1α/JNK activation in the induction of ER stress under low pH conditions, which might underlie the reported impact of OGR1 in the development of IBD 18,19 . In our previous studies, we observed significant and pH-dependent OGR1-mediated signalling, including IP3/Ca 2+ /ERK signalling and enhanced SRF transcription under acidic pH conditions (pH = 6.8) 17 .
The link between acidic activation of GPCRs and MAPKs has been long established. Several reports have demonstrated that GPCRs can induce intracellular signal transduction through ERK1/2 and MAPK pathways 53,54 . Acidic OGR1 stimulation has been shown to trigger IL-6 expression through ERK1/2 and p38 activation in human airway smooth muscle cells 30 . Proton-dependent Ca 2+ release from intracellular stores has been shown to trigger the MEK/ERK1/2 pathway, thereby linking acidification with cell proliferation 2 . Recent reports have shown that ER stress triggers apoptosis via the activation of the IRE1α-JNK signalling pathway 55,56 . Surprisingly, we did not detect apoptotic processes following acidic activation of the IRE1α-JNK pathway, suggesting that OGR1-mediated IRE1α-JNK signalling may therefore promote cell survival together with OGR1 inflammatory signalling in intestinal epithelial cells. Interestingly, the pro-apoptotic role of JNK has been suggested to be strongly influenced by the parallel activation of cell survival pathways and the strength of the apoptotic response. Several reports indicate that while the sustained activation of JNK is associated with apoptosis, the acute and transient activation of JNK is crucial for cell proliferation and survival [57][58][59] . In this regard, several studies have also suggested that two functionally distinct phases of JNK signalling are involved in the ER stress response, an early phase that promotes survival and a late phase associated with cell death. Brown et al. showed that early JNK activation in ER-stressed cells triggers the expression of several apoptosis inhibitors early in the ER stress response. Using MEFs from IREα-and TRAF2-deficient mice, these authors showed that the early JNK activation requires both IRE1α and TRAF2 60 .
Additionally, acidic activation of OGR1 has been suggested to enhance survival in osteoclasts through the induction of PKC activation, which may affect the phosphorylation of pro-or anti-apoptotic proteins, or stimulate ERK1/2 signalling 61,62 . Although the role of PKC in autophagy regulation is still controversial, several studies have suggested that PKC is involved in the suppression of autophagy 63 . In HEK293 cells stably expressing LC3, activation of PKC significantly attenuated autophagy induced by starvation or rapamycin through the phosphorylation of LC3, while inhibition of PKC with pharmacological inhibitors increased autophagy 64 . PKC has also been shown to mediate cisplatin nephrotoxicity in vivo by suppressing autophagy 49 . Moreover, PKC has also been suggested to block autophagy in pancreatic ductal carcinoma cells through the activation of tissue transglutaminase 2 65,66 .
Acidic activation of OGR1 triggers the activation of JNK-mediated ER stress, which suggests a role of IRE1-JNK signalling in controlling autophagy 71 . Strikingly, our results show an increase in total LC3 Caco-2 cells were subjected to different pH medium, following 4-6 h incubation in pH 7.6 serum free medium. After 24 h pH shift, total protein was isolated and Western blotting was performed. Autophagy was measured by variations in the ratio of LC3-II/ LC3-I and the total amount of LC3 (LC3-I plus LC3-II) relative to GAPDH. Results are representative of two independent experiments. (B,C) Caco-2 cells were subjected to different pH medium, with or without OGR1 inhibitor (10 µM, following 4-6 h incubation in pH 7.6 serum free medium.) After 24 h pH shift, cells were fixed in 4% paraformaldehyde and stained with an anti-LC3 antibody. Cells were analysed by immunofluorescence microscopy and images were acquired under a confocal laser microscope. Results are representative of three independent experiments. Scale bars indicate 50 µm. (D) Quantification of the ratio of LC3/DAPI is presented. Changes in LC3 accumulation were calculated relative to DAPI staining from at least 4 areas. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-test. Data are presented as means ± SE of three independent experiments (*p < 0.05; **p < 0.01). pH conditions: High pH 7.5-7.8; Normal pH 7.2-7.4; Low pH 6.6-6.8.
Scientific RepoRtS |
(2020) 10:1438 | https://doi.org/10.1038/s41598-020-57657-9 www.nature.com/scientificreports www.nature.com/scientificreports/ accumulation, but not in LC3-I to LC3-II conversion in OGR1 overexpressing cells following acidic pH shift, indicating an OGR1/IRE1/JNK-mediated blockage of the final stages of autophagy. The role of IRE1α-JNK in regulating autophagy remains a matter of controversy. Notably, JNK has been shown to play a role in autophagy suppression in neurons 72 . Conversely, the activation of ER stress triggered both apoptosis and autophagy through the IRE1/JNK/beclin-1 axis in breast cancer cells 73 . Another study showed that IRE1α upregulated autophagy under ER stress independently of XBP1 signalling 71 . Recently, phosphorylation of the anti-apoptotic protein BCL-2 by IRE1α was linked to the initiation of autophagy through the modulation of the activity of Beclin-1 74 , an essential component of the autophagy machinery 72,75,76 . JNK has been shown to participate in the expression of MAP1LC3 following TNF stimulation in vascular smooth muscle cells 77 . Inhibition of the JNK pathway blocked ceramide-induced autophagy and up-regulation of LC3 expression 78 . Xie et al. reported that JNK plays a crucial role in bufalin-induced autophagy in HT-29 and Caco-2 cells 79 .
In our hands, acidic activation of OGR1 in an OGR1-overexpressing cell model increased accumulation of LC3, but not the conversion of LC3-I into LC3-II, pointing to a blockage of late stage autophagy. Of note, our results suggest that partial activation of OGR1 under normal pH conditions is able to block late-stage autophagy in OGR1 overexpressing cells, and this effect is enhanced when OGR1 is fully activated at low pH. Interestingly, ROS-induced JNK activation induces both autophagy and apoptosis in cancer cells 80 . Taken together, our results suggest that acidic activation of OGR1 triggers opposite pathways leading to cell survival as well as the blockage of the late stages of autophagy. It is plausible that acidic activation of OGR1 initiates autophagy through IRE1α-JNK signalling together with parallel signals that block autophagosomal degradation, thereby contributing to the pro-survival and pro-inflammatory effects of OGR1.
Further investigations are required to elucidate the exact mechanisms of OGR1/IRE1/JNK-mediated blockage of the late stages of autophagy. Taken together, our results indicate that OGR1 may have novel functions in the regulation of ER stress and autophagy and could represent a novel therapeutic target of IBD. www.nature.com/scientificreports www.nature.com/scientificreports/ Methods Reagents. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA), including Tunicamycin (T7765) and Staurosporine (S6942), unless otherwise stated. A specific c-Jun N-terminal kinase (JNK) inhibitor (SP600125) was purchased from Calbiochem (La Jolla, CA). The OGR1 inhibitor was kindly provided by Takeda Pharmaceuticals San Diego, USA. All cell culture reagents were obtained from Thermo Fisher (Allschwil, Switzerland), unless otherwise specified.
Cell culture and pH shift. Caco-2 cells (LGC Promochem, Molsheim, Switzerland) and derived clones
stably overexpressing OGR1 were cultured in a humidified atmosphere with 5% CO 2 at 37 °C in Dulbecco's Modified Eagle's Medium (DMEM) with GlutaMAX (Invitrogen, Carlsbad, CA USA) supplemented with 400 µg/ ml geneticin (G418)-selective antibiotic (Invitrogen) and 10% fetal bovine serum (Invitrogen). Construction of the hu-OGR1-pcDNA3.1 + plasmid, clone generation, selection and characterization has been previously described 17 . pH treatment. pH shift experiments were carried out in serum-free RPMI-1640 medium supplemented with 2 mM GlutaMAX and 20 mM HEPES (all from Invitrogen). For pH adjustment of the RPMI medium, the appropriate quantities of NaOH or HCl were added, and the medium was allowed to equilibrate in the 5% CO 2 incubator at 37 °C for at least 36 h before it was used. Caco-2 cells were seeded and cultured for 24-48 hours before the pH shift was performed. Cells were starved for 4-6 h in serum free RPMI medium, pH 7.6, and then subjected to an acidic pH shift for 24 h. No. A11032, Invitrogen) for 1 h and DAPI (Sigma-Aldrich) for 5 min before mounting with anti-fade medium (Dako, Glostrup, Denmark). Cells were analysed by a Leica SP5 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany). Fluorescence images were processed using Leica confocal software (LAS-AF Lite, Leica Microsystems). Quantification of LC3/DAPI was performed using ImageJ software [National Institutes of Health] 81 using the software's colour threshold tool, which calculates the area of positive staining. The resulting value was normalised to quantification of nucleus staining and represents the positively stained area normalised to cell numbers present in the given area.
Annexin V staining. Externalization of phosphatidylserine in apoptotic cells was detected with Annexin
V and dead cells were stained with propidium iodide (PI), using the Dead Cell Apoptosis Kit (Annexin V FITC and PI, Cat. No. V13242, Thermo Fischer Scientific), according to the manufacturer's instructions. After 10 min incubation at room temperature in the dark, cells were washed in PBS and resuspended in the binding buffer. Single-cell suspensions were analysed by FACS-Canto II flow cytometry (BD Biosciences, Allschwil, Switzerland) using FlowJo software.
RNA extraction and real-time quantitative PCR (qPCR). Total RNA was isolated using the RNeasy
Mini Kit (Qiagen, Hombrechtikon, Switzerland) according to the manufacturers' instructions. For removal of residual DNA, a DNase treatment was performed, according to the manufacturer's instructions, for 15 min at room temperature. For reverse transcription, the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used following the manufacturer's instructions. Determination of mRNA expression was performed by qPCR on a 7900HT real-time PCR system (Applied Biosystems) under the following cycling conditions: 20 s at 95 °C, then 45 cycles of 95 °C for 1 s, and 60 °C for 20 s with the TaqMan Fast Universal Master Mix. Samples were analysed as triplicates. Relative mRNA expression was determined the by the ΔΔCt method, which calculates the quantity of the target sequences relative to the endogenous control β-actin and a reference sample. TaqMan Gene Expression Assays (all from Applied Biosystems), used in this study were human BiP (Hs 00268858-S1) and human β-actin Vic TAMRA (4310881E). (2020) 10:1438 | https://doi.org/10.1038/s41598-020-57657-9 www.nature.com/scientificreports www.nature.com/scientificreports/ XBP1 splicing assay. XBP1 splicing was measured by specific primers flanking the splicing site yielding PCR product sizes of 152 and 126 bp for unspliced XBP1 and spliced XBP1 mRNA, respectively. Primers (forward 5′-CCTGGTTGCTGAAGAGGAGG-3′, reverse 5′-CCATGGGGAGATGTTCTGGAG-3′) were used. PCR was carried out at 95 °C for 15 min, then 40 cycles at 94 °C for 30 sec, 56.5 °C for 30 sec, and 72 °C for 1 min. The size difference between the spliced and the unspliced XBP1 is 26 nucleotides. These products were resolved on 3.5% agarose gels. Band intensity of XBP1s and XBP1u was determined using ImageJ and the ratio of XBP1s/XBP1u was quantified.
Statistical analysis. Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA). Data are presented as means ± SE and statistical significance was determined using the Kruskal-Wallis test. p < 0.05 was considered significant. Where indicated, one-way ANOVA was performed, followed by Tukey's post hoc test.
Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. | v3-fos-license |
2018-12-12T06:05:10.466Z | 2009-04-30T00:00:00.000 | 54718100 | {
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} | pes2o/s2orc | Effect of Antioxidant Preservative on Cold Protection Ability of Low Grade Riverine Buffalo ( Bubalus bubalis ) Bull Spermatozoa
An experiment was conducted to investigate the effect of Butylated Hydroxy Anisole (BHA), Butylated Hydroxy Toluene (BHT), Pentoxifylline (PTX), Theophylline (TPY) and Theobromine (TBR) on cold protection ability of Murrah buffalo semen at room (22-25°C) and refrigerated temperature (4-7°C). Each semen sample was divided into six parts of equal volume and sperm concentration; the first was kept as a control and the remaining five were treated with BHA, BHT, PTX, TPY or TBR. Sperm motility, abnormal spermatozoa, live-dead count, hypo-osmotic swelling and acrosomal integrity were studied at room and refrigerated temperature for various incubation periods viz.; 0, 4, 8, 12 and 24 h at room and 0, 12, 24, 36, 48, 60 and 72 h at refrigerated temperature. Significant improvement in sperm motility, live-dead count, hypo-osmotic swelling and acrosomal integrity were observed in BHT, PTX and TPY fortified extender at room and refrigerated temperature for various incubation periods. From the present study it could be concluded that cold protection ability of buffalo semen can be improved through the addition of BHT followed by PTX and TPY. (
INTRODUCTION
Buffaloes in the Asian continent play an important role as a producer of milk, draught power, dung and other value added products.Their contribution to the national milk grid in India is around 50% in spite of the fact that the population of buffaloes is less than half of the total cattle population.Reproduction is the central trait in animal production throughout the world.High reproduction capacity increases economic efficiency in milk and meat production.Buffalo semen is known for its poor quality and freezability which is well documented in the literature (Roy et al., 1962;Sengupta, 1963).Motility as well as fertility of spermatozoa can be improved by incorporating various motility enhancing agents like pentoxifylline (PTX) (Yovich et al., 1984;Sikka et al., 1995), or antioxidants (Irvine, 1996;Perinaud et al., 1997).PTX increases cAMP level by a methylxanthine inhibition of phosphodiesterase and thus improves motility, capacitation and acrosome reaction (Yovich et al., 1984;Sikka et al., 1995).This increase in cAMP causes activation of protein kinase and phosphorylation of endogenous protein.Motility of spermatozoa is increased due to utilization of increased energy production by accelerating glycolysis and TCA cycle activity (Haesungcharern and Chulavatnatol, 1973).
The antioxidants check the chemical breakdown of substrate resulting from oxidation.Antioxidant preservatives neutralize the free radicals that initiate and help propagate these reactions.The maintenance of sperm membrane phospholipids together with the susceptibility to peroxidation depends on adequate antioxidant properties, which reduce the risk of damage to spermatozoa and probably their lack of survival during storage (Strzeżek et al., 1999;Strzeżek, 2002).Thus, a deficiency of these fractions can affect the overall protection of the spermatozoa from oxidative damage, which can have a negative effect on sperm motility and fertilization.
Among methyl xanthines, caffeine, PTX, Theobromine (TBR) and Theophylline (TPY) have been used.Methyl xanthine supplementation has resulted in better seminal characteristics in fresh and cryopreserved spermatozoa viz., Toluene (BHT), Pentoxifylline (PTX), Theophylline (TPY) and Theobromine (TBR) on cold protection ability of Murrah buffalo semen at room (22-25°C) and refrigerated temperature (4-7°C).Each semen sample was divided into six parts of equal volume and sperm concentration; the first was kept as a control and the remaining five were treated with BHA, BHT, PTX, TPY or TBR.Sperm motility, abnormal spermatozoa, live-dead count, hypo-osmotic swelling and acrosomal integrity were studied at room and refrigerated temperature for various incubation periods viz.; 0, 4, 8, 12 and 24 h at room and 0, 12,24,36,48,60 and 72 h at refrigerated temperature.Significant improvement in sperm motility, live-dead count, hypo-osmotic swelling and acrosomal integrity were observed in BHT, PTX and TPY fortified extender at room and refrigerated temperature for various incubation periods.From the present study it could be concluded that cold protection ability of buffalo semen can be improved through the addition of BHT followed by PTX and TPY.(Key Words : Buffalo Semen, Sperm Motility, Non-Eosinohilic, Butylated Hydroxy Anisole, Butylated Hydroxy Toluene, Pentoxifylline, Theophylline, Theobromine) motility and curvilinear velocity (Chauhan et al., 1983).The addition of methyl xanthine to sperm suspension seems to improve sperm function leading to better sperm fertilizing ability (Chauhan et al., 1983).
Therefore, this study was an endeavour towards the improvement of chilled buffalo semen by using Butylated hydroxy anisole (BHA), Butylated hydroxy toluene (BHT), PTX, TPY and TBR as sperm motility promoting factors.
MATERIALS AND METHODS
Four healthy Murrah buffalo bulls, 2.5 to 5.0 years of age with body weight varying from 300 to 700 kg and maintained under identical nutrition and management conditions at the National Dairy Research Institute, Karnal, India, were selected randomly from the herd for the study.Vaccination and deworming were done regularly as per the farm schedule.
Semen collection and evaluation
The bulls were washed before taking to the site of collection.Semen was collected (Walton, 1945) using an artificial vagina (AV) of 12 inches size "Danish Model" with smooth lining, over a male dummy bull once a week.On each collection, two ejaculates were taken in succession, (Foster et al., 1970) and each ejaculate was preceded by a period of sexual preparation consisting of at least two false mounts separated by about one minute restraint.The second ejaculate was collected about 15 minutes after the first.Immediately after collection, each ejaculate was placed in a water bath at 30°C and examined for various physical attributes viz.volume, mass activity, sperm motility and sperm concentration.
Semen volume was collected in a 15 ml graduated metal free glass tube (0.1 ml accuracy).Mass motility was assessed just after the semen collection.Gross swirl rating (GSR) of undiluted semen was performed within one min of collection.Two 10 μl aliquots of undiluted semen were placed separately on a warmed slide located on a stage warmer (37°C) and scored on a scale of 0-4 using a 10X objective lens on a phase contrast microscope (Nikon Eclipse E600, Tokyo, Japan).Motility was expressed qualitatively on a motility scale (0-4) as described by Matharoo et al. (1985).Semen with +3 or below grade was used for further processing.Manual progressive motility and percentage motile spermatozoa were determined by placing 100 μl of undiluted semen into pre-warmed tubes containing 900 μl of Tris buffer and mixing.Twenty microliters of diluted semen was placed on a warmed glass slide (37°C) and allowed to spread uniformly under the cover slip.Strength of motility rating was scored using 200× magnification with a phase contrast microscope (Nikon Eclipse E600, Tokyo, Japan) equipped with a 37°C-heated stage.Percent progressive motility (0-100%) was measured at five representative areas of the slide.The average of the five scores for each category was recorded.If the difference between two consecutive counts exceeds 10 percent, two new counts were performed.
Experimental design
A total of 9 double ejaculates was taken from 4 Murrah buffalo bulls (MU) making a total of 72 ejaculates.Every ejaculate was divided into six parts, each containing an equal volume and number of sperm, before room and refrigerated temperature incubation, and subjected to the following treatments: Each semen sample was diluted with PBE at 30 million sperm/ml after adding the aforementioned sperm motility enhancers.
Live-dead count : Semen samples were kept at 37°C for 30 min before analysis.Forty microlitre (μl) of neat semen was mixed in a micro-centrifuge tube with 400 μl eosinnigrosine staining solution.The suspension was kept for one minute at room temperature (27°C).Then, a 12 μl droplet was transferred by pipette to a labelled microscope slide (pre-warmed to 37°C) where it was smeared.Duplicate smears were made from each sample, allowed to air dry at room temperature and examined directly.About 200 sperm were assessed at a magnification of 1,000× under oil immersion with a high-resolution 100× bright field objective (not phase contrast).Sperm that were white (unstained) were classified as non-eosinophilic and those that showed any pink or red colouration were classified as dead, with the sole exception of sperm with a slight pink or red appearance restricted to the neck region ('leaky necks'), which were assessed as eosinophilic.
Morphological abnormality : The same slide which was made for eosin-nigrosine staining was used for calculating morphological abnormality.A drop of oil was applied to the cover-slip and the semen was examined at 1,000× under a DIC (differential interference contrast) microscope.If the preparation was too thick, examination was difficult because many sperm heads were laid on their edges rather than flat.Each cell, even in thin preparations, was usually not totally in one focal plane and it was necessary therefore to focus up and down slightly on each cell.About 200 spermatozoa were counted in different fields and the percentage of abnormal spermatozoa was calculated as follows: ( ) Hypo-osmotic swelling test (HOST) : The hypo-osmotic swelling test was performed according to the methods described by Correa and Zavos (1994).Sperm tail curling was recorded as an effect of swelling due to influx of water.A total of about 200 spermatozoa were counted in different fields at 400× magnification under a DIC microscope.The total proportion of swollen spermatozoa was calculated by dividing the number of reacted cells by the total spermatozoa counted in the same area and multiplying the figure by 100.The proportion of swollen spermatozoa from a control sample was subtracted from this value.
These spermatozoa were classified into four different classes according to the presence of the following swelling patterns (Takahashi et al., 1990), namely, A, No swelling, no membrane reaction; B, Swelling of tip of tail; C, Different type of hairpin-like swelling pattern or swelling of mid-piece and D, Complete tail swelling.Spermatozoa displaying B, C or D were considered positive for the HOST test.
Acrosome integrity : Staining was carried out as described by Hancock (1952).A thin smear of extended semen was prepared on a non-greasy, clean and dry slide.The smear was air-dried at room temperature for at least 10 minutes in a current of warm air.The smear was fixed by immersion in buffered formal saline (10 percent) for 15 minutes, then washed in running tap water for 15-20 minutes and dried.Again the slide was immersed in buffered Giemsa solution for 90 minutes, rinsed briefly in distilled water and dried.The dried smears were studied at 1,000× under a light microscope using oil immersion without a cover glass.Each time about 200 spermatozoa were counted for acrosomal status after staining.
Statistical analysis
The data were subjected to analysis of variance to study the effect of treatments on different physical and morphological attributes of semen during various intervals of storage.The following statistical models were used for ANOVA as described by Snedecor and Cochran (1989) and the significant difference between two parameters was evaluated through LSD.
Where, Y ijk = k th observation of j th stage or interval of preservation, μ = Overall mean, I j = the effect of j th stage or interval of preservation, (EI) ij = The effect of (ij th ) additive-interval or stage interaction, e ijk = The Random Error, NID (0, σ 2 e) Descriptive statistics (SAS 8.2) were performed on the data to determine normality.
Performance of additives at room temperature (22-25°C)
A comparative study of performance of various additives at room temperature was carried out and the data are shown in Table 1 to 3. Sperm motility (per cent) : Analysis of data revealed significant (p<0.05)differences in individual motility between additives at various hours of incubation in buffalo bull semen.
Sperm motility (%) was higher in BHT, PTX, TPY and TBR than in BHA and the control group at 0 hour in MU bull semen (Table 1).For the remaining periods, motility (%) was significantly (p<0.01)greater in BHT, PTX and TPY than in the Control (C), BHA and TBR for MU bull semen.Overall, results showed a significant deterioration in motility after each preservation stage.Motility was found to be better preserved in extender fortified with BHT, PTX and TPY.
Non-eosinophilic count (per cent) : Non-eosinophilic count was significantly (p<0.05)higher in BHT, PTX and TPY than in TBR, BHA and C groups at 0 h in cattle bull semen (Table 1).
TPY gave a significantly (p<0.05)better result compared to BHA, PTX and TBR in terms of noneosinophilic count (%) at 0 h in MU bull semen (Table 1).BHT, PTX and TPY supported the sperm non-eosinophilic count significantly (p<0.05)better than all other additives in MU bull semen after 24 h of incubation.
Overall, results showed a significant deterioration in non-eosinophilic count after each preservation stage.Damage to spermatozoa was found to be least in extender fortified with BHT, PTX and TPY.
Sperm abnormalities (per cent) : Tail abnormality (per cent) was the most prominent out of various types of abnormalities in Murrah bull semen (Table 2).There was no difference in various additives in head abnormality (per cent) for most of the periods, except for 24 h in MU bull semen.All additives resulted in significantly (p>0.05) less head abnormality (per cent) than the control group at 0 h.Head abnormality in BHT, PTX and TPY was significantly (p<0.01)less than the control in MU bull semen after 24 hours of incubation.No difference in mid-piece abnormality (per cent) was observed between additives at any incubation period, except for BHT and TPX in MU which were significantly (p<0.05) less than the control after 24 h of incubation.The tail abnormality (per cent) was significantly (p<0.01)less in BHT, PTX and TPY in MU bull semen.As tail abnormality (per cent) constituted the major portion of total abnormality (per cent), so the same trend was visible in total abnormality (per cent) as in the tail abnormality (per cent).
Overall, the results showed a significant increase in spermatozoal tail and total abnormality after each preservation stage, however, the head and mid-piece abnormalities were largely unaffected.Tail and total abnormality was found to be least in extender fortified with BHT, PTX and TPY.
Acrosomal Integrity (per cent) : Initially, acrosomal integrity (per cent) in BHA, BHT, PTX, TPY and TBR was significantly (p<0.01)higher than the control when used as semen additive in PBE for the extension of SW, KF and MU semen (Table 3).
BHT, PTX and TPY addition resulted in significantly (p<0.05)better acrosome integrity than for the control, BHA and TBR addition in bovine bull semen after 24 h of incubation.Overall, results showed a significant deterioration in intact acrosome (per cent) after each preservation stage.From the results, it was evident that spermatozoa acrosome integrity was best preserved in extender fortified with BHT, PTX and TPY after 24 h of storage.
Hypo osmotic swelling test (per cent) : There was no difference in HOST (%) value of different additives at 0 h in MU bull semen (Table 3).BHT, PTX and TPY were significantly (p<0.01)better than C, BHA and TBR in MU bull semen.
Overall, results showed a significant deterioration in plasma membrane integrity (per cent) after each preservation stage.The plasma membrane integrity as measured by HOST was found to be better preserved in extender fortified with BHT, PTX and TPY.
Performance of additives at refrigerator temperature (4-7°C)
A comparative study of performance of various additives at refrigerator temperature was carried out and the data are shown in Table 4 to 6.
Sperm motility (per cent) : Initially sperm motility (per cent) of MU bull semen was significantly (p<0.05)higher in PBE fortified with BHT, PTX and TPY than in BHA, TBR and control groups at refrigerator temperature (Table 4).BHT, PTX and TPY were significantly (p<0.05)better than BHA, TBR and control groups in MU bull semen after 72 h of incubation.
Overall, the results showed a significant deterioration in motility after each preservation stage.Motility was found to be better preserved in extender fortified with BHT, PTX and TPY.
Non-eosinophilic count (per cent) : No difference in any of the additives for non-eosinophilic count was manifested in MU bull semen at 0 h of refrigerator temperature (Table 4).BHT, PTX and TPY supplemented groups were significantly (p<0.05)superior to the control group in MU bull semen after 12 h of incubation at refrigerator temperature.Subsequently for all periods of incubation, non-eosinophilic count was significantly (p<0.01)higher in BHT, PTX and TPY supplemented groups than in BHA, TBR and control groups in MU bull semen.
Overall the results indicated that PBE fortified with BHT, PTX and TPY could support the preservation of livability of spermatozoa to the maximum extent in storage at refrigerator temperature (4 to 7°C).
Sperm abnormalities (per cent) : Tail abnormality was the most prominent in MU bull semen (Table 5).
There was no significant (p>0.05)difference between head and mid-piece abnormality of any of the additives for any period of incubation at the refrigerator temperature under study.The tail abnormality was significantly (p<0.05)lower in PBE fortified with BHT, PTX and TPY than in control groups up to 72 h of incubation at refrigerator temperature in MU bull semen.As the tail abnormality constituted the major portion of total abnormality, so the same trend was found in total abnormality.Overall, the results showed a significant increase in spermatozoal tail and total abnormality after each preservation stage at refrigerator temperature (4 to 7°C), however, the head and mid-piece abnormalities were largely unaffected.Tail and total abnormality was found to be significantly (p<0.05) less in extender fortified with BHT, PTX and TPY.
Acrosomal Integrity (per cent) : Initially there was no significant difference among any of the additives in acrosomal integrity for MU bull semen (Table 6).But this trend was changed subsequently when BHT, PTX and TPY were significantly (p<0.05)better than control, BHA and TBR groups in MU bull semen after 72 h of incubation at refrigerated temperature.
Overall, the results showed a significant deterioration in the acrosome integrity up to 48 h, thereafter there was not much degradation of integrity.From the results, it was evident that spermatozoal acrosome integrity was best preserved in PBE fortified with BHT, PTX and TPY in MU bull semen after 72 h of storage.
Hypo osmotic swelling test (per cent) : There was no difference for HOST (per cent) among the various additives initially in MU bull semen (Table 6).
But this trend was changed between periods of incubation where reaction of sperm to hypotonic medium was significantly higher in PBE fortified with BHT, PTX and TPY than control, BHA or TBR fortified groups with variable degree of significance (p<0.01 to 0.05).
Overall, the results showed a significant deterioration in the plasma membrane integrity up to 48 h, thereafter there was not much degradation of integrity.After 72 h of storage, it was evident that the plasma membrane integrity as measured by HOST was best preserved in PBE fortified with BHT, PTX or TPY in MU bull semen.
DISCUSSION
Mammalian spermatozoa are extremely sensitive to oxidative damage (Lucy, 1972).Lipid peroxidation plays an important role in spermatozoon ageing, shortening life-span and affecting the preservation of semen for artificial insemination (Alvarez and Storey, 1982).Maintenance of sperm membrane phospholipids and susceptibility to peroxidation depends on adequate antioxidant, which reduces the risk of damage to spermatozoa and increases their survival chances during storage (Strzeżek et al., 1999;Strzeżek, 2002).Thus, a deficiency of these fractions can affect the overall protection of the spermatozoa from oxidative damage, which can have a negative effect on sperm motility and fertilizing ability.The process of peroxidation induces structural changes, particularly in the acrosomal region of the sperm cell, a fast irreversible loss of motility, deep change in metabolism and a high rate of intracellular component release (John and Mann, 1977a).In vitro studies have shown BHA and BHT to act as free radical scavengers which protect cell membrane against lipid peroxidation (Beconi et al., 1993).During the process of freezing, spermatozoa have to undergo cold shock which increases their susceptibility to lipid peroxidation (John and Mann, 1977b;John et al., 1979;Pursel and Park, 1985).Stimulatory effects of methyl xanthine on capacitation and acrosome reaction have also been demonstrated.Overall, the addition of methyl xanthine to sperm suspension seems to improve sperm function leading to better sperm fertilizing ability (Chauhan et al., 1983).Similar results were also observed in the present study while fortifying the PBS for dilution of bovine semen with PTX and TPY.However, desirable results could not be achieved by fortification of PBE with BHA and TBR.Killian et al. (1989) hypothesized that BHT serves as a scavenger of free oxygen radicals, associated with the diluent and sperm, to minimize damage to the sperm motility apparatus and membranes, and which also may affect motility indirectly.Graham and Hammerstedt (1992) reported that BHT with no egg yolk present reduced sperm motility, but addition of egg yolk in BHT-treated sperm improved motility, and lipid vesicles in milk and egg yolk (Hu et al., 2006) interacted synergistically with BHT to protect spermatozoa from cold shock.Anderson et al. (1994) reported that the use of BHT improved viability of frozen and thawed sperm and inactivated lipid containing viruses.In the present study also, BHT fortification in PBE for extension of bovine bull semen has improved the viability of sperm.
Methyl xanthine supplementation resulted in better seminal characteristics in fresh and cryopreserved spermatozoa viz., motility and curvilinear velocity (Chauhan et al., 1983).Fayed and Hattab (1991) observed that supplementation of TPY (0.5 ng/ml) improved the keeping quality of chilled semen up to 5 days.PTX may be added to boost the motility of the sperm (Aitken et al., 1993).PTX increases the duration of activity of spermatozoa by increasing the level of cyclic adenosine monophosphate (cAMP) or by reducing the decomposition of cAMP (Perry and Higgs, 1998) by stimulating the enzyme adenylate cyclase (AC stimulator).Thus, in the present study, improved motility was observed in bovine bull semen after fortification of PBE with BHT, PTX and TPY.
Sperm abnormalities (per cent) are one of the most significant indicators of subsequent fertility in a bull (Saacke, 1990).Disturbance in spermatogenesis gives rise to morphological abnormalities.The relationship between sperm morphology and fertility has been evaluated in several studies.Abnormal sperm morphology has been correlated with reduced fertility in cattle (Sekoni and Gustafsson, 1987;Barth and Oko, 1989;Correa et al., 1997;Thundathil et al., 2000) and buffalo (Sengupta and Bhela, 1988).Bull fertility depends upon morphologically normal spermatozoa being present in the ejaculate (Tharwat, 1998) and is hardly affected if abnormal spermatozoa do not exceed 15-20 per cent (Pant et al., 2002).In particular, the occurrence of abnormal sperm head morphology is associated with lower fertility in the bull (Saacke and White, 1972;Sekoni and Gustafsson, 1987).However, a number of other studies have shown no correlation between sperm morphology and fertility (Bratton et al., 1956;Linford et al., 1976) with clear associations between normal bull sperm morphology and fertility continuing to remain elusive (Johnson, 1997).In the present study the increase of abnormality (per cent) is mainly in the tail which suggests that osmolality change in the media may be responsible for this increase as suggested by Joshi et al. (2006).The additives responsible for resisting change in the morphology of spermatozoa are BHT, PTX and TPY.
The addition of methyl xanthine to sperm suspension seems to improve sperm function leading to better sperm fertility (Chauhan et al., 1983).These authors found that when BHT-treated ram sperm were subjected to cold shock, acrosome damage was reduced, and the percentage of motile sperm was higher than for untreated sperm.Similarly, in the present study, methyl xanthine inclusion in the PBE for extension of bovine bull semen resulted in reduced acrosome damage.
Antioxidant preservatives (like BHA, BHT and methyl xanthines) are used to stop auto-oxidation that causes a chain reaction in the unsaturated fatty acids in oils and lipid, and help in slowing down the oxidation of fats and oils.Oxygen reacts preferentially with antioxidants rather than oxidizing fats or oils, thereby protecting them from spoilage.BHA is a synthetic analogue of vitamin E and acts by reducing oxygen radicals and interrupting the propagation of oxidation processes.However, in the present study, BHA has not shown its motility enhancer role.
BHT is an organic soluble molecule which modifies the properties of lipid bilayers and membrane of sperm cells (Hammerstedt et al., 1978).BHT readily incorporates into sperm membranes and prevents membrane damage after exposure to cold (Anderson et al., 1994).Use of spin labels and electron spin resonance techniques suggests that BHT acts on membranes to increase fluidity and to render them less susceptible to cold shock (Anderson et al., 1994).These reports explain the superiority of BHT as an additive over others as evidenced from the present results.Thus, future prospects for use of fortified PBE for the preservation of MU bull semen could be recommended as also supported from the results of acrosome integrity testing.Similarly, Hammerstedt et al. (1978) found that sperm from bulls and rams treated with 0.5 mM BHT were protected from membrane damage during cold shock.Addition of 0.5 mM BHT to whole milk extender during semen processing did not affect bull non-return rates (Anderson et al., 1994).
PTX, TPY and TBR are methylxanthine phosphodiesterase inhibitors which reduce super oxide anions responsible for DNA apoptosis when used in a concentration of 3.6 mM (Maxwell et al., 2002).In the present study, TBR was not able to perform well as a semen additive, unlike PTX and TPY.Similarly, Vega (1997) reported that addition of PTX (6.0 mM) prolonged the viability of post-thaw bovine semen.Apart from modulation of sperm function, a protective effect on sperm membrane by PTX has been demonstrated (Vega, 1997).This effect may be ascribed to neutralization of reactive oxygen species (ROS) and reduction of lipid peroxidation (Vega, 1997).The use of BHT improved viability of frozen and thawed sperm and inactivated lipid containing viruses (Anderson et al., 1994).In the present study, BHT, PTX and TPY being oxygen radical scavengers, supported the preservation of viability of spermatozoa, in agreement with other studies.So, further studies should be carried out to standardize the optimum concentration of TBR to be incorporated in PBE for extension of Murrah bull semen in order to obtain its maximum benefit.
Table 1 .
Effect of additives on sperm motility* (%) and non-eosinophilic count* (%) of Murrah bull semen at different hours of preservation at room temperature
Table 2 .
Effect of additives on various types of abnormalities* (%) at different hours of preservation at room temperature of Murrah bull Mean±SE.Means bearing different superscripts within same row differ significantly ( abcd p<0.05,AB p<0.01).
Table 3 .
Effect of additives on acrosome integrity* (%) and HOST* (%) of bovine semen (N = 12) at different hours of preservation at
Table 5 .
Effect of additives on various types of abnormalities (%) at different hours of preservation at refrigerator temperature (4-7°C) of * Mean±SE.Means bearing different superscripts within same row differ significantly ( abcd p<0.05,AB p<0.01).
Table 4 .
Effect of additives on sperm motility* (%) and non-eosinophilic count (%) of Murrah bull semen at different hours of Mean±SE.Means bearing different superscripts within same row differ significantly ( abc p<0.05,ABC p<0.01). | v3-fos-license |
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} | pes2o/s2orc | Binding of amelogenin to MMP-9 and their co-expression in developing mouse teeth
Amelogenin is the most abundant matrix protein in enamel. Proper amelogenin processing by proteinases is necessary for its biological functions during amelogenesis. Matrix metalloproteinase 9 (MMP-9) is responsible for the turnover of matrix components. The relationship between MMP-9 and amelogenin during tooth development remains unknown. We tested the hypothesis that MMP-9 binds to amelogenin and they are co-expressed in ameloblasts during amelogenesis. We evaluated the distribution of both proteins in the mouse teeth using immunohistochemistry and confocal microscopy. At postnatal day 2, the spatial distribution of amelogenin and MMP-9 was co-localized in preameloblasts, secretory ameloblasts, enamel matrix and odontoblasts. At the late stages of mouse tooth development, expression patterns of amelogenin and MMP-9 were similar to that seen in postnatal day 2. Their co-expression was further confirmed by RT-PCR, Western blot and enzymatic zymography analyses in enamel organ epithelial and odontoblast-like cells. Immunoprecipitation assay revealed that MMP-9 binds to amelogenin. The MMP-9 cleavage sites in amelogenin proteins across species were found using bio-informative software program. Analyses of these data suggest that MMP-9 may be involved in controlling amelogenin processing and enamel formation.
Introduction
Dental enamel is formed by ameloblasts originally derived from embryonic oral epithelium. The differentiation of dental epithelium into functional ameloblasts occurs in spatial-temporal patterns during tooth development and these ameloblasts synthesize and secrete enamel matrix proteins. Amelogenin is the most abundant enamel matrix protein, accounting for about 90 % of total enamel organic matrix. This gene was cloned and characterized from the teeth of bovine, porcine, rat, mouse and human (Snead et al. 1983;Takagi et al. 1984;Gibson et al. 1991a;Nakahori et al. 1991;Salido et al. 1992;Bonass et al. 1994;Hu et al. 1996;Diekwisch et al. 1997). It is synthesized in a distinct time frame during amelogenesis. In mice, amelogenin gene expression was identified in ameloblasts and enamel matrix from embryonic day 15 to postnatal day 14 (Snead et al. 1988;Couwenhoven and Snead 1994;Hu et al. 2001;Iacob and Veis 2006). Rat amelogenin expression was also characterized in teeth from embryonic Junsheng Feng and Jennifer S. McDaniel contributed equally to this work. day 18.5 to postnatal day 15 as well as the continuously erupting incisors at the later stages (Fong et al. 1996;Inage et al. 1996;Bleicher et al. 1999). In developing hamster teeth, amelogenin expression was identified in pre-ameloblasts, secretory, transition, and early maturation stage ameloblasts and in the enamel matrix (Karg et al. 1997). A similar expression pattern was observed in pig teeth except for ameloblasts after the transition stage (Wakida et al. 1999). Originally, amelogenin was thought to be expressed solely by ameloblasts during tooth development (Snead et al. 1988;Inai et al. 1991;Karg et al. 1997;Hu et al. 2001). Recently, it has been found that this gene is also expressed in odontoblasts and other tissues (Veis et al. 2000;Oida et al. 2002;Nagano et al. 2003;Papagerakis et al. 2003;Iacob and Veis 2006;Haze et al. 2007). Amelogenin plays a critical role in the structural organization of enamel as well as in mineralization (Deutsch et al. 1995;Bartlett and Simmer 1999). Amelogenin gene mutations in humans and mice cause amelogenesis imperfecta (AI), one of the most common enamel genetic diseases (Lagerström et al. 1991;Aldred et al. 1992;Gibson et al. 2001;Wright et al. 2011).
Large-molecular-mass amelogenin proteins as well as small-molecular-mass amelogenin polypeptides have been isolated and characterized in different species teeth during the process of development and mineralization (Bronckers et al. 1995;Boabaid et al. 2004). The biochemistry and biological roles of these different fragments in enamel formation and biomineralization have been studied (Gibson et al. 1991b;Le et al. 2007;Warotayanont et al. 2008;Nakayama et al. 2010;Pugach et al. 2010). It is known that amelogenin requires processing by proteinases to activate functional domains (Fincham and Moradian-Oldak 1993). For instance, matrix metalloproteinase 20 (MMP-20) and kallikren 4 (Klk4) are expressed in ameloblasts during amelogenesis and are capable of catalyzing enamel matrix proteins including amelogenin (Li et al. 1999;Ryu et al. 1999;Bourd-Boittin et al. 2004;Sun et al. 2010;Uskoković et al. 2011). These two proteinases are critical for enamel matrix processing. MMP-20 and Klk4 gene mutations in humans and mice cause hypomaturation AI (Caterina et al. 2002;Hart et al. 2004;Kim et al. 2005;Simmer et al. 2009).
Matrix metalloproteinase 9 (MMP-9), also known as gelatinase B or type IV collagenase, belongs to a member of the MMP gene family and is expressed in ameloblasts and odontoblasts as well as other dental cells during tooth development (Tjäderhane et al. 1998;Sahlberg et al. 1999;Randall and Hall 2002;Goldberg et al. 2003;Palosaari et al. 2003;Yoshiba et al. 2003;Takahashi et al. 2006;Paiva et al. 2009;Gomes et al. 2011). This enzyme is involved in bone resorption and degradation of the basement membrane during tooth development as well as extracellular matrix (ECM) turnover in association with tooth eruption (Linsuwanont et al. 2002;Basi et al. 2011). MMP-9 has a broad range of substrate specificity including native collagenous and non-collagenous proteins as well as non-structural ECM components (Vu et al. 1998;Kridel et al. 2001;Somerville et al. 2003;Nagase et al. 2006). However, the entire assortment of substrates for the MMPs has not been fully elucidated. MMP inhibitors were used to treat mouse tooth germs, resulting in impairment of murine amelogenesis (Fanchon et al. 2004;Bourd-Boittin et al. 2005). Furthermore, bone deficiency was observed and osteoblast apopotosis was increased in MMP-9 knock out mice (Vu et al. 1998). Our previous study showed that abnormal tooth morphology, immature ameloblast differentiation, and loss of ameloblast polarization occur in MMP-9 null mice (Yuan et al. 2009, unpublished data). Although expression of both amelogenin and MMP-9 during tooth development was described, their relationship in tooth formation has not been observed. In the present study, we were interested in the interaction between amelogenin and MMP-9 as well as comparing the spatialtemporal distribution of both proteins during mouse tooth development. We hypothesized that interactions between amelogenin and MMP-9 as well as developmental changes in the spatial distribution of these two proteins might exist. To test these hypotheses, we designed experiments to examine the binding of MMP-9 to amelogenin. We further analyzed the distribution of amelogenin and MMP-9 at the same stages of mouse tooth development. Our studies indicated that MMP-9 binds to amelogenin and they are co-expressed in ameloblasts in developing mouse teeth. Furthermore, computational data analysis indicated that potential cleavage sites of MMP-9 exist in amelogenin across different species.
Animals and tissue preparation
All experimental procedures involving the use of animals were approved by the University of Texas Health Science Center at San Antonio (UTHSCSA), TX. ICR mice were purchased from Harlan-Laboratory Animals Inc. (Indianapolis, IN, USA). The MMP-9 knock out mice were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA). For developmental studies, mice with litters of embryonic stage E18.5 and postnatal days 2, 5 and 7 were sacrificed. Mouse tissues were dissected and fixed in 4 % paraformaldehyde overnight. After demineralization in 15 % EDTA, samples were dehydrated in increasing concentrations of ethanol, embedded in paraffin, sectioned, and prepared for immunohistochemistry analysis.
RNA preparation and reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from mouse teeth and MO6-G3 and EOE-3M cell lines by using RNA STAT-60 kit (Tel-Test, Inc. Friendswood, TX, USA), treated with DNase I (Promega, Madison, WI, USA), and purified with the RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA). RNA concentration was determined at an optical density of OD 260 . The RNA was transcribed into cDNA by Super-Script II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). For PCR analysis, specific primers of mouse amelogenin and MMP-9 were synthesized as follows: mouse amelogenin, forward 5 0 -TGAAGTGGTACCAGAGCA-3 0 and reverse 5 0 -ACAGGGATGATTTGGTGGTG-3 0 ; MMP-9, forward 5 0 -CAGACCAAGAGGGTTTTCTT-3 0 and reverse CTTGTTCACCTCATTTTGGA-3 0 . The PCR reaction was first denatured at 95°C for 5 min, and then carried out at 95°C for 30 s, at 55-60°C for 30 s and at 72 o C for 60 s for 35 cycles and with a final 10 min extension at 72°C. Five ll of PCR products were analyzed using agarose gels and ethidium bromide staining. Corrective DNA was verified by DNA sequencing.
Expression and purification of recombinant proteins
The full-length mouse amelogenin gene was amplified by PCR using mouse tooth cDNA as a template with primers adding Xho I and Not I sites for directional ligation into the expression vector pGEX-6P1 with Xho I and Not I sites (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and named pGST196 (amino acids ). The pGST196 expression and purification was performed according to the manufacturer's instruction (Amersham Pharmacia Biotech). The recombinant, untagged murine amelogenin (rM179, amino acids ) was subcloned into pET11a vector with Ndel and BamH I sites (Novagen, Madison, WI, USA) and termed pET179. This pET179 vector was kindly provided by Dr. Simmer (The Department of Biological and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI, USA). A recombinant amelogenin protein was expressed and purified as described previously (Simmer et al. 1994). Recombinant mutant MMP-9 (rMut-MMP-9) construct was generated as described previously (Xu et al. 2005). Briefly, to obtain rMut-MMP-9 with intact ligand-binding properties but without catalytic activities, the active site Glu 402 of proMMP-9 was substituted for alanine residue to generate MMP-9 E402A (Morgunova et al. 1999). The point mutation was introduced into the coding DNA by overlap-extension PCR using the following primer pairs: forward, 5 0 -GTGGCGGCGCATGCGTTCGGCCACGCG-3 0 , and reverse, 5 0 -CGCGTGGCCGAACGCATGCGCCG CCAC-3 0 . The expression construct for MMP-9 in pRS-ETA vector served as a template in PCR reaction buffer that includes 5 ll of 109 reaction buffer, 100 ng of template DNA, 125 ng of each primer, 25 lM dNTP mixtures, and 2.5 units Pfu Turbo DNA polymerase. The cycles included 95°C for 2 min, then 95°C for 30 s, 55°C for 1 min and 72°C for 10 min for 12 cycles. Subsequently, 1 ll of the DpnI restriction enzyme was added for 1 h at 37°C to digest the parental DNA. The pRESTA vector containing histidine tagged rMut-MMP-9 gene was transformed into E. coli BL21 (DE3) competent cells and expressed and purified. A recombinant mouse MMP-9 protein was purchased from R&D Systems Inc. (Minneapolis, MN, USA; Catalog No. 909-MM).
Glutathione fusion protein (GST) pull down assay
For probing protein-protein interactions, either the full length recombinant amelogenin protein tagged with GST (pGST196) or GST protein was incubated with the recombinant mutant MMP-9 protein in the lysis buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA) overnight at 4°C with end-over-end mixing. After the reaction, the glutathione agarose beads were added for further incubation. The samples were then centrifuged and the supernatant was removed. After extensive washes, the beads were mixed with an equal volume of 29 SDS-PAGE gel-loading buffer and then boiled for 4 min followed by SDS-PAGE and Western Blot analyses.
Western blot analysis
The proteins were loaded onto a 10 % SDS-PAGE gel and transferred to a trans-blot membrane (Bio-Rad Laboratory, Inc., Hercules, CA, USA). Western blotting assay was carried out as described earlier (Chen et al. 2008). For observation of co-expression of amelogenin and MMP-9, the double-labeled immunostaining was carried out using two types of primary antibodies along with two types of fluorescent secondary antibodies. The tissue section was blocked with normal donkey serum (Sigma, St. Louis, MO, USA) for 60 min at room temperature and then incubated with one type of the primary goat polyclonal anti-amelogenin antibody, C-19, which was recognized by the donkey anti-goat secondary antibody conjugated with Alexa Fluo Ò 488 (Molecular Probes, Eugene, OR, USA) followed by incubation with a rabbit polyclonal antibody against MMP-9, H-129, recognized by the donkey anti-rabbit secondary antibody conjugated with Alexa Fluo Ò 568. The immunostained section using the two types of antibodies was observed under the same parameters in a Nikon inverted microscope and quantitated by means of NIS-GIEMENTS software. For each experiment, all slides were simultaneously processed for a specific antibody, so that homogeneity in the staining procedure was ensured between the samples. After the capture of these images at the same magnification, the threshold was set and maintained for each slide in the experiment. The optical density was calculated by use of the morphometric analysis within the software package. Hoechst was used for nucleus staining and either normal rabbit or goat immunoglobulin (IgG) served as a negative control (Dakocytomation).
Confocal microscopic evaluation and image acquisition
Sections were evaluated with a Nikon Eclipse 90i C1si laser scanning confocal microscope (Nikon Instruments, Melville, NY, USA) with a 40X/1.30 N.A. oil immersion objective. This evaluation included the selection of a laser gain setting used to image Na v 1.6-immunofluorescence to avoid saturated pixels so to allow a full dynamic range of Na v 1.6-immunofluorescence pixel intensity and to select laser gain settings used to image caspr staining.
Protein sequencing and data analyses A database search was performed at the National Center for Biotechnology Information website (http://www.ncbi.nlm. nih.gov/blast) using the BLAST program. The amelogenin nucleotides and derived amino acids were aligned with those from different species using the Gene Runner software program (http://www.generunner).
Expression of amelogenin and MMP-9 in mouse teeth
To determine the expression of the two genes in ameloblasts, we systematically evaluated the expression of amelogenin and MMP-9 in developing mouse teeth using immunohistochemistry assays. Amelogenin epitopes were highly localized in the pre-ameloblasts, secretory ameloblasts and enamel matrix at the postnatal day 2 (PN2) in mouse molars (Fig. 1b-d). MMP-9 expression overlapped with amelogenin in these regions and its signal was also present in the dental pulp cells, alveolar bone mesenchymal cells and stratum intermedium ( Fig. 1e-g). At PN 5, amelogenin protein in secretory ameloblasts and enamel layer was strongly expressed, but low levels of expression were observed in odontoblasts and stratum intermedium ( Fig. 2b-d). The spatial distribution pattern of MMP-9 was similar to amelogenin besides its expression in alveolar bone mesenchyme (Fig. 2f-h). Similar to PN 5, at PN7, amelogenin was highly expressed in secretory ameloblasts and enamel in developing molars, whereas its signal was weakly detected in odontoblasts (Fig. 3b-d). Compared to amelogenin, the MMP-9 gene was widely expressed although MMP-9 was also present in ameloblasts and odontoblasts ( Fig. 3f-h).
To further study the co-expression of the two proteins in ameloblasts, their distribution profiles in developing mouse teeth were examined using double labeling immunohistochemistry. Amelogenin protein in secretory ameloblasts was strongly expressed, and the signal intensity per cell was higher in enamel matrix (Fig. 4b, f). This protein in secretory ameloblasts was mostly located in the supranuclear area (towards the enamel), but was also present in the infranuclear region (away from the enamel). Amelogenin expression was also detected in odontoblasts, but its expression level in odontoblasts was much lower than ameloblasts and the enamel layer. MMP-9 signal coincided with the expression of amelogenin in ameloblasts, enamel layer as well as odontoblasts (Fig. 4c, g). However, the MMP-9 expression pattern was widely detected in other tissues besides ameloblasts, enamel matrix and odontoblasts. To further confirm their co-localization, tissue sections from PN4 were examined using the confocal microscope. The data showed that their co-expression was seen mostly within the cytoplasm of the ameloblasts (Fig. 4j). Fig. 1 Amelogenin and MMP-9 expression patterns at postnatal day 2 in developing teeth. b Amelogenin expression was observed in preameloblasts, ameloblasts, enamel matrix and odontoblasts at postnatal day 2 in mouse molars. c, d were higher magnification of the boxed area in b. a Pre-immune IgG at the same concentration as antiamelogenin antibody was used as a negative control. e Mouse molar was stained with anti-MMP-9 antibody. f, g show higher magnification of the boxed area in e. MMP-9 was widely expressed in bone mesenchymal cells, stratum intermedium, dental pulp, odontoblasts, ameloblasts and enamel matrix. i The section was stained with preimmune IgG as a negative control. h A tissue section from a MMP-9 knock out mouse was incubated with anti-MMP-9 antibody as control. Am ameloblasts, D dentin, E enamel, Od odontoblasts, Ob bone mesenchymal cells, Si stratum intermedium Expression of amelogenin and MMP-9 in mouse enamel organ epithelial and odontoblast-like cells
D
To assess amelogenin and MMP-9 expression in EOE-3M and MO6-G3 cells, RT-PCR analysis was performed using specific primers for amelogenin and MMP-9. Figure 5a showed that amelogenin and MMP-9 RNAs were identified in mouse tooth tissues, EOE-3M and MO6-G3 cells. Furthermore, their protein expression was detected by Western blot and enzymatic zymography assays, respectively (Fig. 5b, c). Immunohistochemical analysis demonstrated that those proteins were expressed within the cytoplasm and cellular branches in EOE-3M and MO6-G3 cells (Fig. 4d-o).
MMP-9 binds to amelogenin in vitro
To determine binding of amelogenin to MMP-9, the recombinant amelogenin proteins from pET179 and pGST196 vectors were expressed in E.coli BL21 (DE3) cells. The crude extracts from sonicated cells were analyzed by SDS-PAGE to verify protein expression (Fig. 6a). The SDS-PAGE gel showed that the recombinant proteins, pET179 and pGST196, appeared at about 25 and 52 kDa, respectively. There was little sign of amelogenin expression in the un-induced cells. Following purification with glutathione beads of the pGST196 fusion protein, the recombinant amelogenin was resolved to near homogeneity as analyzed by SDS-PAGE gel (Fig. 6b) and further confirmed by Western blot analysis (Fig. 6c). For in vitro binding analysis, beads bearing either GST or pGST196 fusion protein were mixed with recombinant mutant MMP-9 protein. After binding and washing, bound proteins were eluted from the beads. The eluted proteins were separated by a SDS-PAGE gel and electrotransferred to a trans-blot membrane for Western blotting assay. These results showed that beads bearing GST-amelogenin protein pulled down MMP-9, whereas beads bearing GST alone failed to bind to MMP-9 protein (Fig. 6d, e). This result indicates that the amelogenin binds MMP-9 in vitro.
Prediction of MMP-9 cleavage site(s) in amelogenin
Using computer software program, we searched for potential cleavage site(s) of MMP-9 in amelogenin across different species. The results show that the MMP-9 scissile bonds were found in amelogenin proteins across different species (Table 1) Fig. 3 Amelogenin and MMP-9 expression patterns at postnatal day 7 in developing teeth. Both amelogenin and MMP-9 were expressed in ameloblasts, enamel matrix and odontoblasts. b. Tissue section was stained with anti-amelogenin antibody. c, d showed higher magnification of the boxed area in b. Expression of amelogenin was detected in ameloblasts and the enamel matrix. Also, low levels of amelogenin were found in odontoblasts. a Negative control. f Immunostaining indicated that MMP-9 was widely expressed in other cells besides ameloblasts, odontoblasts and the enamel matrix. g, h showed higher magnification of the boxed area in f. e Tissue section was incubated with pre-immune IgG as a negative control. Am ameloblasts, D dentin, E enamel, Od odontoblasts Fig. 4 Co-expression of amelogenin and MMP-9 in developing teeth. Amelogenin expression was detected in pre-ameloblasts, ameloblasts, enamel matrix and odontoblasts. However, the highest level of expression was in secretory ameloblasts and the enamel layer (b, f, i). Amelogenin protein was mostly located within the supra-nuclear area (towards the enamel) in secretory ameloblasts. MMP-9 was also expressed in these areas overlapped with amelogenin (c, g, i). d, h The cells were stained with Hoechst for the nucleus. i Image was merged. Bar = 10 lM. j Confocal micrographs of collapsed z-projection images (consisting of 20 z-sections with spacing increments of 1 mM) show amelogenin (green color) and MMP-9 (red color) staining relationships within ameloblastic cells and enamel matrix of a tooth section from a postnatal day 4. a 0 , b 0 . Higher magnification of the boxed area in j-a, b: amelogenin expression was weakly detected in pre-ameloblasts at the cervical loop (Cp) region, but MMP-9 expression (red color) was seen within the cytoplasm in preameloblasts. Co-expression of amelogenin and MMP-9 proteins was visible within the cytoplasm of ameloblasts and in the enamel layer. High expression of amelogenin was observed in secretory ameloblasts (Am) and enamel matrix (E)
Discussion
Although amelogenin and MMP-9 expression during tooth development was described (Snead et al. 1988;Diekwisch et al. 1997;Sahlberg et al. 1999;Linsuwanont et al. 2002;Goldberg et al. 2003), their co-expression patterns during mouse tooth development and the interaction between the two proteins have not been investigated. In the present study, we investigated the spatial distribution of amelogenin and MMP-9 during mouse tooth development using immunohistochemistry assays. Also, we tested the interaction between amelogenin and MMP-9. Our results showed that the spatial distribution of amelogenin and MMP-9 is co-localized in ameloblasts, enamel matrix and odontoblasts during mouse tooth development although their expression levels and temporal expression patterns sometimes varied during tooth morphogenesis. Furthermore, amelogenin is able to bind MMP-9 in vitro and the MMP-9 cleavage sites exist in amelogenin proteins across different species. Amelogenin expression was identified in the enamel organ of mouse molars as early as the embryonic day 15 (Couwenhoven and Snead 1994) and its expression was still visible in maturation stage ameloblasts at postnatal day 14 in mouse molars (Hu et al. 2001) whereas MMP-9 signal was found both in the dental epithelium and the mesenchyme at the bud stage (embryonic day 12) of developing mouse teeth (Sahlberg et al. 1999;Goldberg et al. 2003;Yoshiba et al. 2003). At the postnatal stages of tooth formation, expression of MMP-9 and other MMP family members were present in the differentiating ameloblasts (Yoshiba et al. 2003;Fanchon et al. 2004;Bourd-Boittin et al. 2005;Paiva et al. 2009). We observed co-expression of both amelogenin and MMP-9 in presecretory ameloblasts, secretory ameloblasts and odontoblasts as well as stratum intermedium at postnatal days 2-7 in mouse molars. Amelogenin accumulation increases with the gradient of the ameloblast differentiation and reaches an apparent plateau at the secretory stage. For the MMP-9 gene, its expression overlapped with amelogenin, in addition to the osteogenic mesenchyme and dental pulp cells. At postnatal days 5 and 7, the MMP-9 expression profile was similar to that of PN 2. Low amelogenin expression levels were present in odontoblasts and stratum intermedium cells through the postnatal stages of tooth development. This evidence was further verified in mouse enamel organ epithelial (EOE-3M) and odontoblast-like (MO6-G3) cells. Previous studies indicated that amelogenin expression was not detected in odontoblasts and other cell types (Snead et al. 1988;Inai et al. 1991Inai et al. , 1996Bleicher et al. 1999;Hu et al. 2001). However, recent studies have shown that amelogenin expression is present in odontoblastic cells using in situ hybridization, immunohistochemistry and RT-PCR analyses (Oida et al. 2002;Papagerakis et al. 2003;Iacob and Veis 2006). Our results are in agreement with previous studies by other groups (Oida et al. 2002;Papagerakis et al. 2003;Iacob and Veis 2006). However, the biological roles of amelogenin in odontoblasts and stratum -9 antibody (h, i, n, q). Negative control was shown in d, e, i and k. Both amelogenin and MMP-9 signals were detected within the cytoplasm and cellular branches in EOE-3M and MO6-G3 cells intermedium cells are not known. Besides co-expression of amelogenin and MMP-9 in ameloblast and odontoblasts, we further found that MMP-9 is able to interact with amelogenin, suggesting that amelogenin is a novel partner of MMP-9.
The amelogenin protein comprises about 90 % of the enamel matrix proteins and has a range of molecular masses due to alternative splicing and proteolytic cleavage. Proteolytic processing of nascent amelogenin molecules serves to generate proper amelogenin fragments as enamel development proceeds (Fincham and Moradian-Oldak 1993;Bartlett and Simmer 1999). Studies have revealed that the NH 2 -terminal and COOH-terminal domains of amelogenin proteins are essential for proper enamel formation (Gibson et al. 1991b;Le et al. 2007;Warotayanont et al. 2008;Nakayama et al. 2010;Pugach et al. 2010). In contrast, inappropriate processing of amelogenin by proteinases causes enamel defects (Caterina et al. 2002;Hart et al. 2004;Kim et al. 2005;Simmer et al. 2009).
MMP-9 is a member of the MMP family and has a broad range of substrates and mediates extracellular matrix remodeling (Kridel et al. 2001;Somerville et al. 2003;Lund et al. 2011). It is involved in normal physiological processes including bone remodeling and tooth eruption (Linsuwanont et al. 2002;Basi et al. 2011) and in pathological processes like periodontal disease (Rai et al. 2008; Silva et al. 2008), dental caries (Chaussain-Miller et al. 2006;Shimada et al. 2009) and cancer invasion (Patel et al. 2011). In MMP-9 null mice, impairment of ossification and vascularization of the skeletal growth plates was observed (Vu et al. 1998). Skeletal growth plates of MMP-9 null mice in a culture system showed a delayed release of an angiogenic activator, establishing a role for this enzyme in controlling angiogenesis. Engsig et al. (2000) demonstrated that MMP-9 is essential for the recruitment of osteoclasts into developing bone. Our study found that mice lacking MMP-9 gene exhibit abnormal tooth morphology, immature ameloblast differentiation, loss of ameloblast polarization and delayed tooth eruption as well as an increased amelogenin expression during amelogenesis compared to the wild-type mice (Yuan et al. 2009 and unpublished data). Amelogenin has been suggested as a possible substrate for MMP-9 due to the reciprocal concentrations of amelogenin and MMP-9 observed during tooth formation (Fanchon et al. 2004;Bourd-Boittin et al. 2005). Fanchon et al. used two MMP inhibitors, Marimastat, a general MMP inhibitor, or CT 1166 , a more selective inhibitor of MMP-2 and MMP-9, and found that when mouse tooth germs were treated with either Marimastat or CT 1166 , gelatinase activity was inhibited, resulting in disturbance of murine amelogenesis. This suggests that MMP-9 is involved in amelogenin processing and enamel development. However, although MMP-9 interacts with amelogenin in vitro and there are potential MMP-9 cleavage sites in amelogenin genes across species, whether MMP-9, like MMP20 and KlK4, is capable of catalyzing amelogenin and its role during amelegenesis needs to be further investigated.
Conflict of interest
The author(s) declared no potential conflicts of interest with respect to the authorship and/or publication of this article. extracts. c Expression of purified pET179 (lane 1) and pGST196 (lane 2) amelogenin proteins was confirmed by Western blotting analysis using goat polyclonal anti-amelogenin antibody (C-19, Santa Cruz Biotechnology Inc.). d, e Either 5 lg of GSTamelogenin fusion protein (pGST196) or 5 lg of GST protein alone was mixed with the recombinant mutant MMP-9 protein in binding buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA) and followed by adding 20 ll of glutathione-agarose beads for further incubation. After the binding reaction, the beads were washed twice with binding buffer and once with washing buffer. Beads were boiled in 19 SDS loading dye and the samples were divided into equal aliquots, and the eluted proteins were separated by SDS-PAGE and electrotransferred to the trans-blot membranes. Western blot assay was carried out using polyclonal anti-GST antibody (Amersham Pharmacia Biotech) (d) and polyclonal anti-MMP-9 antibody (Santa Cruz Biotechnology Inc.) (e) P proline, Hy hydrophobic amino acids, S serine, T threonine, G glycine, Y tyrosine, I isoleucine, N asparagine, X any amino acids. Scissile bonds are shown with ; Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. | v3-fos-license |
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} | pes2o/s2orc | 4,15-Dimethyl-7,12-diazoniatricyclo[10.4.0.02,7]hexadeca-1(12),2,4,6,13,15-hexaene dibromide monohydrate
The crystal structure of the viologen 4,4′-dimethyl-2,2′-dipyridyl-N,N′-tetramethylene dibromide monohydrate is presented, along with details of an improved synthesis and NMR spectroscopic analysis.
The title compound, C 16 H 20 N 2 2+ Á2Br À ÁH 2 O (1) is a member of the class of compounds called viologens. Viologens are quaternary salts of dipyridyls and are especially useful as redox indicators as a result of their large negative oneelectron reduction potentials. Compound 1 consists of a dication composed of a pair of 4-methylpyridine rings mutually joined at the 2-position, with a dihedral angle between the pyridine rings of 62.35 (4) . In addition, the rings are tethered via the pyridine nitrogen atoms by a tetramethylene bridge. Charge balance is provided by a pair of bromide anions, which are hydrogen bonded to a single water molecule [D OÁ Á ÁBr = 3.3670 (15) and 3.3856 (15) Å ]. The crystal structure of 1, details of an improved synthesis, and a full analysis of its NMR spectra are presented.
Chemical context
The title compound (1) is a member of the class of compounds called viologens. Viologens are quaternary salts of dipyridyls, which have proven useful as redox indicators as a result of their large negative one-electron reduction potentials (Anderson & Patel, 1984). The herbicides, paraquat, and diquat are viologens. We found that the literature synthesis of 4,4 0 -dimethyl-2,2 0 -dipyridyl-N,N 0 -tetramethylene dibromide, i.e., 1 could be improved by a change in the solvent. We report details of our improved synthesis of 1 along with the crystal structure and a full analysis of its NMR spectra. give general directions for the syntheses of a series of bridged dimethyl 2,2 0 -dipyridyl salts. Our attempts to make the title compound by their directions failed; only a salt of the starting dipyridyl was recovered. Homer & Tomlinson (1960) noted that HBr is formed by dehydrohalogenation of the dibromide. We think that the conditions used by Anderson & Patel (1984), i.e., refluxing ISSN 2056-9890 o-dichlorobenzene, b.p. 453 K, produced a good deal of HBr, which protonated the dipyridyl, rendering it unreactive. Carrying out the reaction in refluxing xylene (mixed isomers, b.p. ca 413 K) does not produce HBr, but the reaction is slow; after five h, about 50% of the starting dipyridyl was recovered. The quaternization of tertiary amines is known as the Menschutkin reaction (Menschutkin, 1890). The velocity of this reaction shows a strong dependence on solvent (Abraham & Grellier, 1976), with about a 65,000-fold increase from hexane to DMSO. The addition of nitrobenzene to the solvent gave satisfactory yields of the product in a reasonable time (see Synthesis and crystallization section).
Structural commentary
The molecular structure of 1 is shown in Fig. 1. It consists of a dication composed of a pair of 4-methylpyridine rings mutually joined at their 2-positions, with a dihedral angle between the pyridine rings of 62.35 (4) . In addition, the rings are tethered via the pyridine nitrogen atoms by a tetramethylene bridge. There are no unusual bond lengths or angles. As a result of the two bridges between the pyridine rings, 1 occurs as two optical isomers, and therefore provides an example of atropisomerism (Eliel et al., 1994;Alkorta et al., 2012;Mancinelli et al., 2020). Crystals of 1, however, were centrosymmetric, with space group P2 1 /n, and are thus strictly racemic. Charge balance is provided by a pair of bromide anions, which are hydrogen bonded to a single water molecule of crystallization [D OÁ Á ÁBr = 3.3670 (15) and 3.3856 (15) Å ] (Table 1).
Supramolecular features
Aside from the hydrogen bonds between the water molecule and bromide anions, the only other notable intermolecular contacts are interactions of type C-HÁ Á ÁBr (Fig. 2 A view of 1 showing the atom-labeling scheme. Displacement ellipsoids are drawn at the 50% probability level. Hydrogen bonds between water and Br À are shown as dashed lines.
Figure 2
A packing plot of 1 viewed down the crystallographic a axis. Hydrogen bonds between water and Br À are shown as dashed lines, while weaker C-HÁ Á ÁBr interactions are shown as dotted lines. standard van der Waals radii of C, H, and Br (Bondi, 1964) are 1.2, 1.7, and 1.85 Å , respectively. The percentages of atomÁ Á Áatom contact types between asymmetric units were obtained from Hirshfeld-surface fingerprint plots (Figs. S1 and S2 in the supporting information; Spackman & McKinnon, 2002;McKinnon et al., 2004) using CrystalExplorer 17.5 (Turner et al., 2017), and are presented in Table 2.
Database survey
The most similar structures to 1 in the Cambridge Structural Database (CSD, V5.41, update of November 2019; Groom et al., 2016) are BIYTEL, BIYTUB, BIYTOV, BIYVAJ, and BIYTIP (Sanchez et al., 2019). BIYTEL has a trimethylene bridge, BIYTUB has a dimethylene bridge, BIYTOV has a trimethylene bridge but lacks the 4-Me substituents, BIYVAJ has a trimethylene bridge but 5-Me groups instead of 4-Me, and BIYTIP has a dimethylene bridge but is a methanol solvate. CSD entry TMEPYR (Derry & Hamor, 1970) contains a tetramethylene bridge, but lacks 4-Me subsituents. CSD entries DIQUAT (Derry & Hamor, 1969) and DQUATB (Sullivan & Williams, 1976), have dimethylene bridges but also lack the 4-Me substituents. Atomic coordinates for TMEPYR, DIQUAT and DQUATB are, however, not present in the CSD. CSD entry PICGAM (Talele et al., 2018) has a -CH 2 C 6 H 4 CH 2 -linker and is an acetonitrile solvate. These crystal structures have Br À anions for charge balance and (unless otherwise stated) include water of crystallization. The tetramethylene bridge is present in CSD entries HIJGAI (Hofbauer et al., 1996), YOBWAN , and YUFCOR , but these crystal structures feature complex organometallic anions rather than bromide and are not hydrates. The dihedral angle between the two pyridine rings in each structure is strongly dependent on the length of the bridging tether. These range between 15.78-19.01 for dimethylene, 49.40-53.96 for trimethylene, and 63.87-67.15 for tetramethylene [cf. 62.35 (4) in 1]. In PICGAM, the dihedral angle is 72.64 , presumably as a result of the increased rigidity of the tether.
Table 2
Percentage of atomÁ Á Áatom contacts between asymmetric units in 1.
Contact percentages were derived from Hirshfeld-surface fingerprint plots (Spackman & McKinnon, 2002;McKinnon et al., 2004) using CrystalExplorer 17.5 (Turner et al., 2017). Reciprocal contacts are included in the totals. The sum of all percentages in the table is 100.1% due to accumulation of rounding errors.
Figure 3
Analysis of 2-D NMR spectra: (a) HSQC and HMBC resonance assignments, (b) COSY resonance assignments. Peaks marked by an asterisk correspond to water or multiple quantum artifacts. 1-D traces are shown to the left and top of the figure.
methyl group exchange with deuterons in a base-catalyzed reaction (Zoltewicz & Jacobson, 1978). Our NMR sample, which also showed exchange, was neutral. Exchange was prevented by adjusting the 'pH' to $1 with DCl. This exchange with solvent deuterium led to some deuterium couplings with both protons and carbon and hence multiplicities in the NMR spectra, which were initially puzzling. Calder et al. (1967) discuss the effect of the length of the bridging group on the NMR spectra and the mobility of the structures.
There are eight resonance signals in the 1 H NMR spectrum recorded in D 2 O, including one on the downfield shoulder of the residual water resonance. All but one of the signals are of equal intensity and the one at 2.68 ppm is about three times larger. The 13 C NMR spectrum shows eight signals (C1-C8), two of which (C2 and C4) are barely separated. Quantitative measurement using inverse-gated decoupling with a long recycle delay (60 s) shows that the carbon signals are of equal intensity. The 1-D 13 C DEPT (Distortionless Enhancement by Polarization Transfer) and 2-D multiplicity-edited 1 H-13 C HSQC (Heteronuclear Single Quantum Coherence) establish a ratio of 3:2:1 for CH, CH 2 , and CH 3 , respectively. Further analysis of 2-D 1 H COSY (Correlation Spectroscopy) and 2-D 1 H-13 C HMBC (Heteronuclear Multiple-Bond Correlation spectroscopy) spectra led to the NMR assignments summarized in Table 3. A selective HMBC focusing on the C2/C4 region was recorded for unambiguous assignments of multiple-bond 1 H-13 C correlations related to these two carbons. These details together with the 2-D 1 H-15 N HMBC, which reveals stronger H2/N9 and H4/N9 cross-peaks than H1/ N9, clearly establish a symmetric three-ring molecular structure, as shown in Fig. 3, in full agreement with the crystal structure (Fig. 1).
The stereospecific assignment of the methylene protons was achieved by a systematic recording of 1-D selective NOESY (Nuclear Overhauser Effect Spectroscopy) and COSY spectra. A stronger NOE was observed between the proton at 4.73 ppm and H1, and thus this resonance was assigned to H6A while the geminal one at 4.03 ppm to H6B. The 1-D selective homonuclear decoupling 1 H NMR spectra led to the extraction of J-coupling constants between these methylene protons (Table 3). A large 3 J coupling exists between H6B and H7B (11.3 Hz), followed by a sizable 3 J coupling between H6A and H7A (6.1 Hz). As a result of the complexity of the spectra, the 3 J(H 6A H 7B ) and 3 J(H 6B H 7A ) could not be determined, but were estimated to be less than 2 Hz. Also, the 11.1 Hz coupling between H7A and H7B was tentatively assigned to the geminal coupling rather than the one across the C7-C7 0 bond.
All NMR spectra were recorded on a Bruker Ascend 700 MHz spectrometer equipped with a TXO cryoprobe at 298 K. Spectra were indirectly referenced to the deuterium lock frequency, set to 4.7 ppm.
Synthesis and crystallization
The starting materials were standard commercial samples of 95-98% purity. 4,4 0 -Dimethyl-2,2 0 -dipyridyl (0.92 g, 5 mmol) and 1,4-dibromobutane (0.6 mL, 1.08g, 5 mmol) were added to a mixture of 5 mL each of xylene (mixed isomers, b.p. ca 413 K) and nitrobenzene (b.p. 483 K). The mixture was refluxed for about 5 h, during which time a heavy precipitate formed. After cooling, the crude material was filtered and washed with acetone to yield 1.1 g of a tan-colored powder. Paper electrophoresis of this material at pH 7.5 showed (via UV) a small amount of starting material at R p ca zero and product at R p À2.2 (R p is movement relative to picric acid). Crystallization from methanol-acetone gave 0.5-0.6 g (ca 50%) of reddish crystals, m.p. 528-530 K [lit. 528-533 K; | v3-fos-license |
2020-05-28T09:14:28.089Z | 2020-05-01T00:00:00.000 | 218912120 | {
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} | pes2o/s2orc | A Facile Fabrication of Biodegradable and Biocompatible Cross-Linked Gelatin as Screen Printing Substrates
This study focuses on preparation and valuation of the biodegradable, native, and modified gelatin film as screen-printing substrates. Modified gelatin film was prepared by crosslinking with various crosslinking agents and the electrode array was designed by screen-printing. It was observed that the swelling ratio of C-2, crosslinked with glutaraldehyde and EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide) was found to be lower (3.98%) than that of C-1 (crosslinked with only glutaraldehyde) (8.77%) and C-0 (without crosslinking) (28.15%). The obtained results indicate that the swelling ratios of both C-1 and C-2 were found to be lower than that of C-0 (control one without crosslinking). The Young’s modulus for C-1 and C-2 was found to be 8.55 ± 0.57 and 23.72 ± 2.04 kPa, respectively. Hence, it was conveyed that the mechanical strength of C-2 was found to be two times higher than that of C-l, suggesting that the mechanical strength was enhanced upon dual crosslinking in this study also. The adhesion study indicates that silver ink adhesion on the gelation surface is better than that of carbon ink. In addition, the electrical response of C-2 with a screen-printed electrode (SPE) was found to be the same as the commercial polycarbonate (PC) substrate. The result of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay suggested that the silver SPE on C-2 was non-cytotoxic toward L929 fibroblast cells proliferation. The results indicated that C-2 gelatin is a promising material to act as a screen-printing substrate with excellent biodegradable and biocompatible properties.
Introduction
In order to study the electrical activity of biological cells, electrode arrays can provide useful information. In general, electrode arrays have been fabricated on hard substrates such as silicon [1], glass [2], and plastics [3]. However, the reliable communication between a biological cell and an electrode would be seriously affected by the mechanical mismatch between the soft biological tissues
Chemicals and Reagents
Type A gelatin, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and glutaraldehyde were purchased from Sigma (St. Louis, MO, USA). Polycarbonate (PC) substrate was obtained from Jan Yan Print Int'l Corp (Taoyuan City, Taiwan) and used as received without any modification. All the chemicals used in this study were of reagent grade.
Preparation of Gelatin Film
Gelatin was dissolved in deionized (DI) water at 60 • C to prepare 15 w/v % gelatin solution. The gelatin solution was poured into a Petri dish and then air-dried at room temperature for 24 h. Three types of gelatin samples were prepared in this study: (1) gelatin film without any crosslinking (C-0, no crosslinking); (2) gelatin film crosslinked with 2% glutaraldehyde (pH 4.8) for 24 h (C-1, single crosslinking); and (3) gelatin film crosslinked with 0.50% EDC/0.18% NHS (pH 6.4) for 24 h followed by crosslinking with 2% glutaraldehyde for 24 h (C-2, dual crosslinking). Finally, these resultant gelatin films were washed repeatedly with DI water to remove any traces of reacting agents and then air-dried in an oven at 40 • C overnight. All the prepared gelatin films were stored in a vacuum desiccator at room temperature. The morphology of the gelatin film was examined by a scanning electron microscope (SEM, Hitachi-4700, HORIBA, Kyoto, Japan). The gelatin film samples were sputter-coated with gold prior to SEM examination.
Swelling Test of Gelatin Film
In order to obtain the swelling film, the gelatin film was immersed into phosphate-buffered saline (PBS) at room temperature. At predetermined time intervals (1,2,3,4,5,10,20,30, and 60 min), the film was removed from PBS and the film area was immediately measured (A 1 ). The swelling ratio was calculated by using Equation (1) with A 0 as the surface area of gelatin film before immersing in PBS. Each measurement experiment was repeated three times and expressed as average ±SD.
Mechanical Strength Test of Gelatin Film
The mechanical property tests were performed according to the ASTM D882 standard test method [39]. Gelatin films were cut into 1 cm × 6 cm rectangular shape and soaked in 0.1 M PBS (pH 7.4) for 24 h. The mechanical properties of these soaked gelatin films were calculated and recorded automatically by using a mechanical testing machine (Tinius Olsen, Horsham, PA, USA) at a crosshead speed of 10 mm/min.
Fabrication of Screen-Printed Electrode (SPE) on Gelatin
Carbon ink (SC-1010, ITK) and silver ink (NT-6307-2, PERM TOP) were printed onto the crosslinked gelatin and polycarbonate (PC) substrates by using a screen-printing machine (NSP-1A, Yulishih Industrial, New Taipei City 235, Taiwan) equipped with a 200 threads per inch polyester screen and polyurethane (PU). The size of all substrates was 1 × 3 cm 2 . The printed carbon-SPE and silver-SPE were dried at 60 • C for 30 min and 120 • C for 60 min, respectively.
Adhesion Test of the SPE
The adhesion strength of the screen-printed electrodes was evaluated by using a tape test according to ASTM D 3359-95 [40] to evaluate the effect of the carbon and silver ink adhesion to the crosslinked gelatin film substrate. The extent of adhesion between the inks and the substrate was analyzed by measuring the fraction of detached area after the test. The adhesion was evaluated by comparison with description and illustration in the ASTM D3359 manual. An evaluation scale (5B to 0B) was provided, where 5B indicates the best and 0B indicates the poorest.
Cyclic Voltammetry (CV) Measurement
The CV measurement was carried out using an IM6-eX electrochemical workstation (ZAHNER Zennium IM6, ZAHNER-elektrik GmbH & Co. KG, Kronach, Germany). The three-electrode system consisted of the screen-printed electrode as a working electrode, an Ag/AgCl wire as a reference electrode, and a platinum wire as a counter electrode. The CV scanning was performed at a scan rate of 100 mV/s with 0.1 mM, pH 7.2 potassium ferricyanide (K 3 Fe(CN) 6 ) as the redox probe.
Cell Biocompatibility Assay
The biocompatibility test of gelatin film was performed according to ISO 10993 [41] by MTT assay using L929 fibroblast cells. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was used to evaluate the cell viability based on the mitochondrial conversion of the tetrazolium salt into a purple colored formazan product at an absorbance of 570 nm. The mouse fibroblast cell line L929 was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. Each sample was placed into one well in a 24-well plate and L929 cells were seeded on each well at 2 × 10 4 cells/well. After 1, 2 or 3 days incubation, the original medium in each well was replaced with 100 µL MTT solution (5 mg/mL), and then the wells were incubated for 4 h at 37 • C in 5% CO 2 incubator to enable the formation of formazan crystals. After removing the solution, dimethyl sulfoxide (DMSO) was added to all the wells and mixed thoroughly to dissolve the dark blue crystals. After a few minutes in order to ensure that all crystals were dissolved, the plates were read at 570 nm on a multi-well scanning ELISA reader (Thermo Scientific, Waltham, MA, USA).
Statistics
All the data were expressed as mean ± standard deviation (SD). The data were compared by one-way analysis of variance (ANOVA) to evaluate differences among the groups. A difference with p < 0.05 was considered statistically significant.
Morphology of Gelatin Film
Optical photographs and SEM images of PC, gelatin without any crosslinking (C-0), the single crosslinked gelatin film (C-1), and the dual crosslinked gelatin film (C-2) are presented in Figure 1. The optical photographs showed that the crosslinked gelatin films became yellow, suggesting the formation of a successful crosslinking structure. The SEM surface image showed that all gelatin films had a smooth surface, and moreover, the cross-section of C-1 and C-2 gelatin films showed a finer scale microstructure. This indicates that the crosslinking could effectively increase the compactness of the gelatin film [28], and such a smooth and compact gelatin surface is appropriate for screen-printing. Figure 2 shows the swelling ratio measured at different time intervals for the C-0, C-1, and C-2 films. The swelling ratio for C-0 was increased drastically and reached saturation in 20 min with the swelling ratio of 28.15% and remained constant up to 60 min. Similarly, for C-1 the swelling ratio increased with time and attained 8.77% at 60 min, whereas for the dual-crosslinked gelatin film (C-2), the swelling ratio reached saturation in 5 min with the swelling ratio of 3.98% and remained constant up to 60 min, which is lower than C-0 and C-1. Gelation could adsorb water molecules as it is hydrophilic in nature. Upon incorporation of glutaraldehyde, the swelling property of the gelatin film was found to decrease possibly due to the increase in hydrophobicity of the matrix [42]. Another reason that could be attributed to this phenomenon was the increase in the crosslinking density between the glutaraldehyde and gelatin [43]. When glutaraldehyde was added to gelatin, the reaction between the amine (NH2) group of gelatin and the carbonyl (C=O) groups of glutaraldehyde would occur leading to the formation of a gelatin hydrogel network [27]. EDC/NHS crosslinking of gelatin film along with glutaraldehyde further reduced the swelling behavior of the gelatin film which could be possibly due to high crosslinking at longer duration (48 h). This is also possible from the production of short-range molecular crosslinks since reaction of EDC/NHS with gelatin matrices brought gelatin films more low-swelling structure [44]. In general, the degree of swelling was reduced for the polymer with high crosslinking and hence among all, the dual-crosslinked gelation film possessed a small rate of swelling indicating the low water adsorption capacity and increased hardness of the material [45]. Figure 2 shows the swelling ratio measured at different time intervals for the C-0, C-1, and C-2 films. The swelling ratio for C-0 was increased drastically and reached saturation in 20 min with the swelling ratio of 28.15% and remained constant up to 60 min. Similarly, for C-1 the swelling ratio increased with time and attained 8.77% at 60 min, whereas for the dual-crosslinked gelatin film (C-2), the swelling ratio reached saturation in 5 min with the swelling ratio of 3.98% and remained constant up to 60 min, which is lower than C-0 and C-1. Gelation could adsorb water molecules as it is hydrophilic in nature. Upon incorporation of glutaraldehyde, the swelling property of the gelatin film was found to decrease possibly due to the increase in hydrophobicity of the matrix [42]. Another reason that could be attributed to this phenomenon was the increase in the crosslinking density between the glutaraldehyde and gelatin [43]. When glutaraldehyde was added to gelatin, the reaction between the amine (NH 2 ) group of gelatin and the carbonyl (C=O) groups of glutaraldehyde would occur leading to the formation of a gelatin hydrogel network [27]. EDC/NHS crosslinking of gelatin film along with glutaraldehyde further reduced the swelling behavior of the gelatin film which could be possibly due to high crosslinking at longer duration (48 h). This is also possible from the production of short-range molecular crosslinks since reaction of EDC/NHS with gelatin matrices brought gelatin films more low-swelling structure [44]. In general, the degree of swelling was reduced for the polymer with high crosslinking and hence among all, the dual-crosslinked gelation film possessed a small rate of swelling indicating the low water adsorption capacity and increased hardness of the material [45].
Mechanical Properties of Gelatin Films
It was reported that glutaraldehyde crosslinking affects the stiffness of gelatin films [31]. Figure 3 shows the typical stress-strain curves recorded from gelatin films crosslinked with glutaraldehyde (C-1) and EDC/NHS/glutaraldehyde (C-2). A decrease in the extensibility and increase in the stress at break were observed for the C-2 gelatin film. The calculated Young's modulus for the C-1 and C-2 gelatin films was 8.55 ± 0.57 and 23.72 ± 2.04 kPa, respectively. From the results, it was noted that an increase in the Young's modulus would result in lower elasticity and higher size stability. This was possibly due to the compact space between the films contributed by higher crosslinking density. Thus, the structure of film was retained without any stretching. Cao et al. [32] reported a similar trend for polycarbonate film. This result indicates the improved mechanical strength of C-2 gelatin film and hence dual crosslinking makes gelatin film highly durable to physical pressure and is suitable for screen-printing. The mechanical strength test cannot be performed in the un-crosslinked gelatin film due to its poor mechanical properties. C-2 gelatin film was used as a substrate for printing electrode arrays.
Mechanical Properties of Gelatin Films
It was reported that glutaraldehyde crosslinking affects the stiffness of gelatin films [31]. Figure 3 shows the typical stress-strain curves recorded from gelatin films crosslinked with glutaraldehyde (C-1) and EDC/NHS/glutaraldehyde (C-2). A decrease in the extensibility and increase in the stress at break were observed for the C-2 gelatin film. The calculated Young's modulus for the C-1 and C-2 gelatin films was 8.55 ± 0.57 and 23.72 ± 2.04 kPa, respectively. From the results, it was noted that an increase in the Young's modulus would result in lower elasticity and higher size stability. This was possibly due to the compact space between the films contributed by higher crosslinking density. Thus, the structure of film was retained without any stretching. Cao et al. [32] reported a similar trend for polycarbonate film. This result indicates the improved mechanical strength of C-2 gelatin film and hence dual crosslinking makes gelatin film highly durable to physical pressure and is suitable for screen-printing. The mechanical strength test cannot be performed in the un-crosslinked gelatin film due to its poor mechanical properties. C-2 gelatin film was used as a substrate for printing electrode arrays.
Mechanical Properties of Gelatin Films
It was reported that glutaraldehyde crosslinking affects the stiffness of gelatin films [31]. Figure 3 shows the typical stress-strain curves recorded from gelatin films crosslinked with glutaraldehyde (C-1) and EDC/NHS/glutaraldehyde (C-2). A decrease in the extensibility and increase in the stress at break were observed for the C-2 gelatin film. The calculated Young's modulus for the C-1 and C-2 gelatin films was 8.55 ± 0.57 and 23.72 ± 2.04 kPa, respectively. From the results, it was noted that an increase in the Young's modulus would result in lower elasticity and higher size stability. This was possibly due to the compact space between the films contributed by higher crosslinking density. Thus, the structure of film was retained without any stretching. Cao et al. [32] reported a similar trend for polycarbonate film. This result indicates the improved mechanical strength of C-2 gelatin film and hence dual crosslinking makes gelatin film highly durable to physical pressure and is suitable for screen-printing. The mechanical strength test cannot be performed in the un-crosslinked gelatin film due to its poor mechanical properties. C-2 gelatin film was used as a substrate for printing electrode arrays.
Gelatin Film as Screen-Printing Electrode Substrate
Screen printing has evolved as a potential fabrication tool because it enables simple, rapid, and inexpensive electrode array preparation on a large scale [33]. In this work, we use C-2 gelatin film as a substrate on which carbon and silver electrode arrays were realized by employing screen-printing technique.
Adhesion Test of SPE
Adhesion strength is a significant factor for the reliability and functionality of metal electrode arrays onto various substrates. Both carbon and silver inks were screen-printed onto C-2 gelatin and PC substrates. In order to determine the adhesion capacity of the crosslinked C-2 gelatin film, the percentage of the adhesion was determined according to the procedure explained by ASTM D-3359-95 standard test methods and compared with PC substrate. From the test results (Figure 4), the screen-printed carbon ink and silver ink onto the PC film were rated as 4B and 5B, respectively. Carbon ink on C-2 gelatin film revealed poor adhesion (Grade-1B). However, silver ink on C-2 gelatin film exhibited strong adhesion (Grade-5B). The adhesion test confirmed that the silver electrode has a strong adhesion strength to the C-2 gelatin film substrate. Hence, silver screen-printed electrode was chosen for subsequent experimental analysis.
Gelatin Film as Screen-Printing Electrode Substrate
Screen printing has evolved as a potential fabrication tool because it enables simple, rapid, and inexpensive electrode array preparation on a large scale [33]. In this work, we use C-2 gelatin film as a substrate on which carbon and silver electrode arrays were realized by employing screen-printing technique.
Adhesion Test of SPE
Adhesion strength is a significant factor for the reliability and functionality of metal electrode arrays onto various substrates. Both carbon and silver inks were screen-printed onto C-2 gelatin and PC substrates. In order to determine the adhesion capacity of the crosslinked C-2 gelatin film, the percentage of the adhesion was determined according to the procedure explained by ASTM D-3359-95 standard test methods and compared with PC substrate. From the test results (Figure 4), the screenprinted carbon ink and silver ink onto the PC film were rated as 4B and 5B, respectively. Carbon ink on C-2 gelatin film revealed poor adhesion (Grade-1B). However, silver ink on C-2 gelatin film exhibited strong adhesion (Grade-5B). The adhesion test confirmed that the silver electrode has a strong adhesion strength to the C-2 gelatin film substrate. Hence, silver screen-printed electrode was chosen for subsequent experimental analysis.
Electrochemical Characterization of SPE
The fabricated silver SPEs on C-2 gelatin film were characterized by cyclic voltammetry (CV) in potassium ferricyanide solutions and their performances compared with silver SPEs on PC substrate. Analytical data obtained from CV studies are shown in Figure 5. The results showed that the cyclic voltammograms for Ag electrode on PC and C-2 gelatin film almost exhibited the same common features. There were two redox peaks in each curve, which could be attributed to the redox of ferric ions. The upward peak is an anodic peak, reflecting the oxidation process from ferrous ion to ferric
Electrochemical Characterization of SPE
The fabricated silver SPEs on C-2 gelatin film were characterized by cyclic voltammetry (CV) in potassium ferricyanide solutions and their performances compared with silver SPEs on PC substrate. Analytical data obtained from CV studies are shown in Figure 5. The results showed that the cyclic voltammograms for Ag electrode on PC and C-2 gelatin film almost exhibited the same common features. There were two redox peaks in each curve, which could be attributed to the redox of ferric ions. The upward peak is an anodic peak, reflecting the oxidation process from ferrous ion to ferric ion. Correspondingly, the downward peak is a cathodic peak, reflecting the reduction process from ferric ion to ferrous ion [34]. The sigmoidal response and its degree of symmetry indicated the irreversible nature (between silver ink and potassium ferricyanide) of the electroactive substances. This CV response suggests that the SPEs on soft gelatin substrate are very suitable for obtaining electrical signals from biological cells.
Polymers 2020, 12, x FOR PEER REVIEW 8 of 11 ion. Correspondingly, the downward peak is a cathodic peak, reflecting the reduction process from ferric ion to ferrous ion [34]. The sigmoidal response and its degree of symmetry indicated the irreversible nature (between silver ink and potassium ferricyanide) of the electroactive substances. This CV response suggests that the SPEs on soft gelatin substrate are very suitable for obtaining electrical signals from biological cells.
Cell Viability Assay
MTT assay was executed to test the cell viability on C-2 gelatin substrate. L929 fibroblast cells were cultured on PC film and C-2 gelatin film for three days both in the presence and absence of Ag-SPE and the biocompatibility test with MTT assay results are shown in Figure 6. The MTT assay results exhibited that the proliferation of L929 fibroblasts is insignificant on C-2 gelatin substrate and C-2 gelatin substrate with Ag-SPE. On the first day, the cells proliferated, and then the growth became stagnant for all groups, although PC film and Ag-SPE gelatin film showed significant difference (p < 0.05). This result clearly indicates that C-2 gelatin substrate and Ag-SPE are not cytotoxic toward cell proliferation. Thus, the C-2 gelatin film could provide a biocompatible surface with exposed ligands that promotes cell attachment and proliferation by integrin-mediated interactions [23].
Cell Viability Assay
MTT assay was executed to test the cell viability on C-2 gelatin substrate. L929 fibroblast cells were cultured on PC film and C-2 gelatin film for three days both in the presence and absence of Ag-SPE and the biocompatibility test with MTT assay results are shown in Figure 6. The MTT assay results exhibited that the proliferation of L929 fibroblasts is insignificant on C-2 gelatin substrate and C-2 gelatin substrate with Ag-SPE. On the first day, the cells proliferated, and then the growth became stagnant for all groups, although PC film and Ag-SPE gelatin film showed significant difference (p < 0.05). This result clearly indicates that C-2 gelatin substrate and Ag-SPE are not cytotoxic toward cell proliferation. Thus, the C-2 gelatin film could provide a biocompatible surface with exposed ligands that promotes cell attachment and proliferation by integrin-mediated interactions [23].
Polymers 2020, 12, x FOR PEER REVIEW 8 of 11 ion. Correspondingly, the downward peak is a cathodic peak, reflecting the reduction process from ferric ion to ferrous ion [34]. The sigmoidal response and its degree of symmetry indicated the irreversible nature (between silver ink and potassium ferricyanide) of the electroactive substances. This CV response suggests that the SPEs on soft gelatin substrate are very suitable for obtaining electrical signals from biological cells.
Cell Viability Assay
MTT assay was executed to test the cell viability on C-2 gelatin substrate. L929 fibroblast cells were cultured on PC film and C-2 gelatin film for three days both in the presence and absence of Ag-SPE and the biocompatibility test with MTT assay results are shown in Figure 6. The MTT assay results exhibited that the proliferation of L929 fibroblasts is insignificant on C-2 gelatin substrate and C-2 gelatin substrate with Ag-SPE. On the first day, the cells proliferated, and then the growth became stagnant for all groups, although PC film and Ag-SPE gelatin film showed significant difference (p < 0.05). This result clearly indicates that C-2 gelatin substrate and Ag-SPE are not cytotoxic toward cell proliferation. Thus, the C-2 gelatin film could provide a biocompatible surface with exposed ligands that promotes cell attachment and proliferation by integrin-mediated interactions [23]. Figure 6. The biocompatibility test of gelatin film by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay using L929 fibroblast cells. Data were expressed as means with standard deviations (mean ± SD). Statistical significance was set at a level of * p < 0.05 when compared with the control group. | v3-fos-license |
2020-11-22T14:09:25.754Z | 2020-11-17T00:00:00.000 | 227102656 | {
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} | pes2o/s2orc | Structural Basis of CYRI-B Direct Competition with Scar/WAVE Complex for Rac1
Summary Rac1 is a major regulator of actin dynamics, with GTP-bound Rac1 promoting actin assembly via the Scar/WAVE complex. CYRI competes with Scar/WAVE for interaction with Rac1 in a feedback loop regulating actin dynamics. Here, we reveal the nature of the CYRI-Rac1 interaction, through crystal structures of CYRI-B lacking the N-terminal helix (CYRI-BΔN) and the CYRI-BΔN:Rac1Q61L complex, providing the molecular basis for CYRI-B regulation of the Scar/WAVE complex. We reveal CYRI-B as having two subdomains - an N-terminal Rac1 binding subdomain with a unique Rac1-effector interface and a C-terminal Ratchet subdomain that undergoes conformational changes induced by Rac1 binding. Finally, we show that the CYRI protein family, CYRI-A and CYRI-B can produce an autoinhibited hetero- or homodimers, adding an additional layer of regulation to Rac1 signaling.
INTRODUCTION
Regulation of the cytoskeleton has a direct impact on cellular shape, polarity, migration, and homeostasis. Actin is the key effector protein shaping cytoskeleton dynamics at membrane interfaces (Krause and Gautreau 2014). Recently, a novel effector of Rac1, Fam49B also known as CYRI-B (CYFIP-related Rac interactor), has been reported to bind to active Rac1 and oppose the activation of the Scar/WAVE complex (Fort et al., 2018). It was described as a local inhibitor of branched actin assembly, and is thought to be recruited by the activating signal-active Rac1-and suppresses Scar/WAVE activity (Fort et al., 2018). This local inhibition buffers the actin polymerization process at the leading edge (Fort et al., 2018), increases membrane dynamics, suppresses T cell activation (Shang et al., 2018) and can promote resistance to Salmonella infection (Yuki et al., 2019).
CYRI-B, its isoform CYRI-A and the Scar/WAVE complex all possess an evolutionarily conserved Rac1 binding domain (RBD) termed the DUF1394 domain. Based on this homology, CYRI's suppression of Rac1-mediated actin polymerization was proposed to be through direct competition with the Scar/ WAVE complex (Fort et al., 2018). Nevertheless, the structural basis of this interaction and the interplay with Scar/WAVE binding are not known.
The Scar/WAVE complex is composed of five subunits; CY-FIP, NCKAP1, Scar/WAVE, ABI, and HSPC300. It has been proposed that the Scar/WAVE complex undergoes an activating conformational change (Chen et al., 2010) induced by its recruitment to the plasma membrane through interactions with negatively charged phospholipids and binding to ''active'' GTPbound Rac1 (Chen et al., 2010;Hoeller et al., 2016;Veltman et al., 2012). Activation of the Scar/WAVE complex releases the C-terminal VCA region of Scar/WAVE, promoting binding to and activation of Arp2/3, leading to branched actin polymerization (Davidson and Insall, 2011). While Rac1 binding to Scar/ WAVE and CYRI has been biochemically detected, little is known about the binding interface. Based on the crystal structure of Scar/WAVE, a Rac1 binding site on CYFIP termed the ''A'' site was proposed and validated using pulldown experiments (Chen et al., 2010). A recent cryo-EM structure of the Scar/ WAVE complex fused to Rac1 also revealed a secondary Rac1 interaction site, which has been named the ''D'' site (Chen et al., 2017). Nevertheless, mutagenesis studies revealed that the ''A'' site was the most potent regulator of lamellipodia activity in cells (Chen et al., 2017;Schaks et al., 2018). Importantly, the atomic level interaction and structural basis of Rac1-mediated activation and release of the VCA region remains unresolved.
Here, we present the crystal structures of mouse CYRI-B (residues 26-324 hereafter termed CYRI-BDN) and human CYRI-BDN in complex with the constitutively active mutant Rac1Q61L.GppNHp. This is the first structure of a DUF1394 domain in complex with Rac1 and provides the structural details dictating Rac1 specificity for this module. Based on these structures, we define a novel Rac1 interacting domain and provide clues to how CYRI can directly and locally compete with the Scar/WAVE complex. Furthermore, the complex structure leads to a model for how Rac1 activates the Scar/WAVE complex. Finally, we show that CYRI proteins can homo-or heterodimerize. Dimerization uses the same interface as Rac1 binding, leading us to propose an autoinhibition mechanism for the regulation of CYRI.
RESULTS
Crystal Structure of CYRI-BDN CYRI proteins are evolutionarily conserved and show low sequence identity (18% in humans) to the Scar/WAVE complex subunit CYFIP, which shares a DUF1394 domain. As there is no structural data for CYRI, we set out to solve its crystal structure.
Despite mouse CYRI-A and CYRI-B sharing 80% sequence identity, significant differences in solubility were observed, with the latter showing better solubility. We, therefore, focused our crystallographic efforts on CYRI-B. Initial crystal screens were set up for the full-length CYRI-B and although crystals were obtained, they diffracted poorly and resisted optimization. To obtain crystals capable of diffracting to high resolution, the N-terminal residues (1-25) involved in membrane binding were truncated, but the entire DUF1394 domain (residues 26-324) was retained. We solved the crystal structure to 2.37Å resolution (Table 1), using experimental phasing, as molecular replacement using CYFIP was not successful.
The structure of each CYRI-BDN monomer consists of 12 alpha helices packed into two bundles running perpendicular to each other to produce an L-shape fold ( Figure 1A). The asymmetric unit contains two molecules of CYRI-BDN with each monomer ''slotting'' against the other to form a compact dimer ( Figure 1B). The dimer interface produces a large contact area of 1524Å 2 , yet there are relatively few bonding contacts (Figures 1C,1D) between the CYRI monomers. Briefly, R161 form a hydrogen bond with A192, R165 and M166 binds to Q324, I168 with R320, N169 with S321 and Q324. With such a low density of bonding interactions between each monomer, suggests that despite a large binding interface the potential dimerization may be weak.
Using the DALI server (http://ekhidna2.biocenter.helsinki.fi/ dali/), we compared our structure to all PDB entries. This showed that the only protein having reasonable structural homology (RMSD <5Å for the entire protein) with CYRI-B is CYFIP1 (Figure 1E). Remarkably, given the divergence in sequence, the two proteins overlay with an RMSD of 2.7Å for all Ca atoms, and 1.9Å for residues 26-214 of CYRI-B. The region of greatest divergence corresponds to the C-terminal helical bundle where the alignment of residues 215-324 of CYRI-B to CYFIP gives an RMSD of 5.1Å for Ca atoms due to an insertion of an antiparallel b-hairpin in CYFIP ( Figure 1E). Based on the structural alignment, we define CYRI-B DUF1394 domain as a module comprising two helical bundles in an L-shape fold ( Figure 1F). CYRI-B runs as a monomer on size exclusion chromatography ( Figure S3A). Thus to test if the CYRI-B dimer observed in the asymmetric unit can occur in solution and if it can heterodimerize with CYRI-A, we used pulldown assays, which revealed a weak but reproducible interaction between MBP-CYRI-A or CYRI-B with HA-CYRI-B (Figures S1A and Figure 4B). Since the structure of the CYRI-B:CYRI-A heterodimer is not available to validate the heterodimer interface, we mutated residues on CYRI-A based on the CYRI-B homodimer structure. Both single and double mutation of the two conserved arginine residues in CYRI-A reduce the dimerization by up to 50% (Figures S1A and S1B). To investigate if the heterodimerization could occur in a cellular context, we performed proximity ligation assay (PLA). A positive signal was strongly observed with wild-type CYRI-A but is significantly reduced in the RRDD mutant, confirming the possibility of heterodimerization in cells (Figures S2B and S2C).To determine whether the CYRI dimer interface could be conserved with the Scar/WAVE complex, we overlaid the crystal structure of the Scar/WAVE complex (PDB: 3P8C) with each monomer of the CYRI-B dimer ( Figure S3B). The resulting structure shows minimal steric clashes between each Scar/WAVE complex BDN's C-terminus was identified that successfully produced crystals of the complex that diffracted to 3.1Å resolution ( Figure 2A, Table 1). The crystal structure contains one copy of the CYRI-BDN in complex with one Rac1Q61L molecule in the asymmetric unit where Rac1Q61L binds in trans with CYRI-BDN from a symmetry mate (Figures S3E and S3F). Examination of the CRYI-BDN in complex with Rac1Q61L shows that binding is primarily mediated through extensive contacts between the N-terminal helical bundle of CYRI-BDN and the switch-I loop (residues 25-39) of Rac1Q61L along with residues on the adjacent b-strand but no contribution from the switch-II loop of Rac1 ( Figure 2B), resulting in a large interface of 1097Å 2 . As the DUF1394 domain shows no sequence homology to other Rac1 effectors, we compared the CYRI-BDN:Rac1Q61L complex with other Rac1 effector complexes; Rac1: Arfaptin (PDB: 1I4T), Rac1: pPhox67 (PDB: 1E96) and Rac1: PRK (PDB: 2RMK) ( Figures 2C, 2D, and 2E). Although binding of pPhox67 to Rac1 utilizes the switch-I of Rac1, additional contacts are also made through Q162, A159 L160, and Q162 of Rac1 (Lapouge et al., 2000), which are not observed in CYRI-B binding. The interface with PRK and Arfaptin is mediated through fewer switch-I contacts than CYRI-BDN but both make additional interactions through the switch-II loop of Rac1 (Modha et al., 2008;Tarricone et al., 2001) that are also not observed in CYRI-BDN. This results in a different switch-I conformation in Rac1 when bound to CYRI-BDN compared with Arfaptin and Rac1 from p67Phox complex Rac1 from Arfaptin complex Relative fold change WT S157A S157R Q153R WT MBP-CYRI-B
GST-Rac1Q61L
GST-Rac1Q61L S41A (legend continued on next page) ll OPEN ACCESS Article PRK ( Figure 2F). Thus, CYRI-B binds to Rac1 with a unique interface. While the interface between CYRI-B and Rac1 is unique, its contact with the switch-I loop of Rac1 explains the specificity for active GTP-bound Rac1. Binding is mediated through a combination of polar and hydrophobic interactions. Despite the size of the interface, there are relatively few interactions ( Figures 3A and 3B). The R160 residue of CYRI-B forms a salt bridge with the side chain of Rac1 D38 and an additional hydrogen bond with the main-chain carbonyl group of Rac1 F37. Q153 and R64 of CYRI-B form a hydrogen bond with S41 and N26 of Rac1, respectively. The main-chain carbonyl of M147 forms a hydrogen bond with N52 of Rac1 and S157 of CYRI-B peptide carbonyl forms a hydrogen bond with the side chain of N39 of Rac1. This relatively large interface, has low interaction density explaining the modest affinity between Rac1 and CYRI-B. Rac1 P29S is a clinically important mutation (Hodis et al., 2012;Krauthammer et al., 2012), as it allows Rac1 to undergo spontaneous nucleotide exchange in the absence of a GEF, producing a shift to active Rac1 (Davis et al., 2012). It has been reported that the dual mutation P29S Q61L increases the affinity of Rac1 to CYRI-B and not other effectors such as the CRIB domain (Fort et al., 2018;Whitelaw et al., 2019). As P29 is located at the binding interface between Rac1 and CYRI-B ( Figure 3A), one could speculate that mutation to a serine could result in additional hydrogen bonds, perhaps with R161 to increase the affinity of the complex.
Previously we have shown that CYRI-B R160 and R161 are important in binding to Rac1 (Fort et al., 2018) and another report indicates that P150 is also involved (Yuki et al., 2019). Examination of the complex shows that R160 forms hydrogen bonds with D38 and mutating R160 to an aspartic acid would introduce a repulsive charge-charge interaction and thus disrupt complex formation ( Figure S4A). The R161 residue is adjacent to Rac1's switch-I loop and although the distance to the nearest Rac1 residue is 4.4Å , a reversal of the charge will also disrupt the interface ( Figures S4A and S4B). We also showed this to be the case for CYRI-A ( Figures S4C and S4D). P150 is in close proximity to bound Rac1 and although not directly involved in making contacts, a mutation to arginine would introduce steric clashes, inhibiting complex formation (Yuki et al., 2019) ( Figure 3C). To further validate the complex, we rationally introduced mutations at the interface of the complex, S157 on CYRI-B was mutated to both alanine and arginine, Q153 was mutated to arginine and S41 on Rac1 was mutated to alanine ( Figure 3D). Following pulldown experiments, all mutations resulted in reduced binding ( Figures 3E and 3F). We conclude that the DUF1394 domain of CYRI-B binds Rac1 through a unique effector-binding interface and we identify residues involved in this interaction, suggesting a model for how the DUF1394 domain specifically interacts with active Rac1.
Rac1 Sequestration by CYRI Is Regulated through Autoinhibited CYRI Dimers
Overlaying CYRI-BDN:Rac1Q61L complex structure with that of the CYRI dimer, we can see that the two directly compete for the same binding interface ( Figure 4A). This raises the possibility that CYRI dimers autoinhibit Rac1 binding and regulate CYRI functionality. To test if the dimer interface competes with Rac1 binding, the pulldown assays were repeated in the presence of increasing levels of active Rac1. As seen in Figures 4B, 4C, and 4D, both homo and heterodimers of CYRI are disrupted by the addition of active Rac1.
Interestingly, following the overlay of the CYRI-BDN structure with that of the CYRI-BDN:Rac1Q61L complex, we can see that the C-terminus of CYRI-BDN undergoes a conformational change such that it undergoes a domain swap with a symmetry mate ( Figure 5A, Video S1). The conformational change is induced through a steric clash between residue Q2 of Rac1 and Y305 of CYRI-B ( Figure 5A). The domain-swapped C-terminal bundles occupy the same site as in the CYRI-BDN structure but is shifted by 5Å , allowing Rac1 to bind without steric clashes and might act as an alternative mechanism for CYRI dimerization ( Figure 5C). When overlaying CYRI-BDN structure with that of the Scar/WAVE complex (PDB: 3P8C) ( Figure 5D), no steric clash with Rac1 is observed. This implies that the steric clash with Rac1 is either specific to CYRI-B or that the crystal structure of the Scar/WAVE complex has captured a conformation where no Rac1 clashes would occur. However, as Rac1 failed to induce dimerization of CYRI-B in solution, we conclude that the dimerization mechanism via domain swapping is most likely a crystallization artifact. Nonetheless, the C-terminus of CYRI does undergo a conformational change induced through Rac1 binding. Therefore, assuming that CYRI evolved as a fragment of CYFIP, which retained Rac1 binding activity, it has acquired new and defining features, distinguishing it from a pure Rac1 binding module. The destabilization of intramolecular contacts through GTPase binding is a common feature in Rho family effector complexes and is seen in other proteins, such as the formins (K€ uhn et al., 2015), and IRSp53 (Kast et al., 2014). As the C-terminal alpha-helical bundle (residues 215-324) is not involved in binding to Rac1, and Rac1 binding interactions are mediated through the N-terminal alpha-helical bundle (residues 26-215), we conclude that the CYRI-BDN DUF1394 domain contains two subdomains. We define these subdomains as the N-terminal Rac1 binding subdomain (RBD) and the C-terminal Ratchet subdomain ( Figure 5A), as it undergoes a conformational change upon Rac1 binding. Binding of active Rac1 to CYRI-BDN destabilizes the contacts between the Ratchet subdomain and RBD through steric clashes. The precise biological role of the Rac1-induced conformational change to the Ratchet subdomain in CYRI remains elusive. Molecular Basis for Rac1 Specificity of DUF1394 over Other GTPases It remains a major question in the field how CYRI and by analogy CYFIP proteins interact specifically with Rac1, while WASP proteins can bind either Rac1 or Cdc42. We, therefore, performed a sequence alignment for Rac1 against Cdc42 and other less related GTPases RhoA and RND1 ( Figure 6A). Together with a structural comparison of Cdc42 (PDB: 4JS0) with our complex, the basis for selectivity becomes apparent. Residues A27, G30 in Rac1 are lysine and serine in Cdc42; both residues result in steric clashes with CYRI-B ( Figures 6B-6E). S41 in Rac1 is an alanine in Cdc42 resulted in breaking a hydrogen bond with Q153 of CYRI-B whereas W56 is a phenylalanine residue in Cdc42, which reduces the extent of hydrophobic interactions. Mutation of Rac1 S41A used to validate the interface also serves to validate Rac1 selectivity, as this mutant mimics Cdc42. This mutation disrupts binding by 30% ( Figure 3D, E). Sequence comparison to more distantly related members of the Rho GTPase family RhoA and RND1 ( Figure 6A) demonstrates even greater levels of sequence variation and together provides the basis for Rac1 selectivity to CYRI. As CYRI has structural similarity to CYFIP, it is reasonable to extrapolate that Rac1 would bind to CYFIP with a similar binding mode. Therefore, it is tempting to reason that the sequence variations explaining Rac1 selectivity to CYRI may also explain the selective activation of the Scar/WAVE complex by Rac1. For example, a Rac1 mutation A27K (a Cdc42 sequence variation) could induce steric clashes with CYRI-B ( Figure 6B), mimicking cdc42, and this could prevent it from binding to the ''A'' site of CYFIP and activating the Scar/WAVE complex. In summary, the DUF1394 domain is a Rac1 selective binding module, and sequence alignments with other Rho family GTPases provide us with evidence to explain this binding specificity.
CYRI and CYFIP Have a Conserved, but Distinct Rac1
Interface Both CYRI-A and CYRI-B can bind to active Rac1 ( Figure S4). Examination of the residues involved in this interaction ( Figure 6A) shows that all of them are conserved between CYRI isoforms with the exception of M147N; however, this amino acid's side chain is not involved in Rac1 contacts, as binding is mediated by a main-chain hydrogen bond ( Figure 3B). Furthermore, mutation of residues at the interface between CYRI and Rac1 (R159D, R160D in CYRI-A and R160D, R161D in CYRI-B) showed comparable reductions in Rac1 binding between the two CYRI isoforms ( Figure S4). Thus, Rac1 binding is conserved between CYRI isoforms and is likely to involve the same interface.
As there is limited structural data for Rac1 binding to the ''A'' site of the Scar/WAVE complex, we sought to use our complex as a ll OPEN ACCESS Article template to model this interaction. Overlaying of the CYRI-BDN:Rac1Q61L structure with the Scar/WAVE complex (PDB: 3P8C) shows that Rac1 contacts the ''A'' site (residues on CYFIP adjacent to a4-a6 on the meander region of WAVE1) as previously proposed (Chen et al., 2010) (Figures 7A and 7B). All CYFIP residues identified as involved in Rac1 binding are at the docked interface. Of the residues reported (R190, C179, E434, F626, and M632), only R161 (R190 in CYFIP) is conserved with CYRI. We, therefore, set out to identify a minimal conserved DUF1394 domain Rac1 binding interface, using a sequence alignment of CYFIP1/2 and CYRI-A/B coupled with our CYRI-BDN:Rac1Q61L complex ( Figures 6A, S5A, and S5B). Despite the low sequence identity, nine residues (Q68, N154, S157, R160, R161, N185, S188, L189 and A192) at the interface are either conserved or have similar chemical properties. This supports that the binding mechanism is conserved between CYRI and CYFIP allowing for a more informed model of how Rac1 interacts with the Scar/ WAVE complex. A previous model proposed that Rac1 would either interact directly with the meander region of WAVE1 or Rac1 binding would serve to alter the meander regions stability -allowing for the VCA region to become Arp2/3 binding competent (Chen et al., 2010). Using our model, we observe no direct interaction between Rac1 and WAVE1. Instead, a steric clash is observed between the switch-I loop of Rac1 and a loop on CYFIP, which is adjacent to the a4 helix of WAVE (Figure 7C). We propose that binding of Rac1 would induce a conformational change in CYFIP, mediated through steric clashes with Rac1's switch-I, which would destabilize the meander region of WAVE and a structural rearrangement in the adjacent VCA helices to facilitate the binding and activation of the Arp2/3 complex.
Phosphorylation of Y151 in WAVE has also been implicated in Scar/WAVE activity (Stuart et al., 2006). Y151 is buried and therefore unlikely to be phosphorylated without a necessary conformational change. In our model of Rac1 binding, Rac1 would induce conformational changes in CYFIP residues that bind to Y151 of WAVE. It is therefore feasible that these conformational changes would allow Y151 to be exposed for its phosphorylation, to enhance Scar/WAVE activity.
In summary, the Rac1 binding interface is evolutionarily conserved among DUF1394-containing proteins. This conservation allows for the docking of Rac1 onto the ''A'' site of the Scar/ WAVE complex (Chen et al., 2017). Our model provides a starting point for further examination of functionally important residues to further elucidate the activation mechanisms of the Scar/WAVE complex.
DISCUSSION
Actin polymerization provides a driving force for membrane protrusion during lamellipodia extension and migration. It is increasingly clear that positive and negative feedback loops control actin dynamics and allow cells to respond quickly to environmental cues such as chemoattractants, adhesion molecules, or changes in rigidity. The Scar/WAVE complex is the major controller of actin in lamellipodia downstream of Rac1 and was recently shown to be negatively regulated by CYRI proteins (Fort et al., 2018). Understanding how positive and negative signals influence actin dynamics is crucial to our understanding of cell migration, developmental processes, and pathological conditions such as cancer. While the interaction between Rac1 and the Scar/ WAVE complex has been modeled based on their individual 3D structures (Chen et al., 2017), we here elucidate the structural interface between Rac1 and CYRI proteins, which serve as a structural analogue of the CYFIP:Rac1 binding ''A'' site.
Here, we report the novel crystal structures of the CYRI-BDN alone and in complex with Rac1. CYRI-B binds Rac1 through a unique binding interface using extensive contacts with the switch-I loop and is distinct from other Rac1 effector complexes. Using this structure and sequence alignments, we provide a molecular basis for DUF1394 domain selectivity toward Rac1 over other members of the Rho GTPase family. Our study reveals how Rac1 interacts with the DUF1394 and the conservation between CYFIP and CYRI proteins, albeit with some important differences that impact on the likely mechanism of their competition for active Rac1.
Article
The crystal structure of CYRI-BDN reveals the presence of an autoinhibited dimer present in solution and in cells as revealed through pulldowns and PLA. The dimerization of CYRI proteins could provide a basis for cooperative inhibition of lamellipodia protrusions in keeping with the known ability of CYRI to sharpen and increase lamellipodia dynamics (Fort et al., 2018;Linsay et al., 2019). Removal of autoinhibition will increase the affinity of CYRI to Rac1. Whether specific post-translational modifications or a membrane environment is required to regulate this autoinhibition is an intriguing possibility. Alternatively, CYRI association with the Scar/WAVE complex could allow specific regulation beyond sequestration of Rac1 proteins. Indeed, if autoinhibitory dimers are a conserved feature of DUF1394 domains, the possibility that Scar/WAVE could also form autoinhibited dimers and that CYRI could disrupt such interactions cannot be excluded. In fact, there have been several reports suggesting that the Scar/WAVE complex could form oligomeric structures (Pipathsouk et al., 2019), making this an attractive question for further investigation.
CYRI-B, unlike CYFIP or the Scar/WAVE complex, is myristoylated (Fort et al., 2018) and is thought to be transiently membrane-associated (Model, Figure S6). CYFIP and the Scar/ WAVE complex depend on their interactions with the membrane-bound active Rac1 and acidic phospholipids to promote its own membrane association in ''permissive zones'' where these favorable conditions exist (Model, Figure S6). It would be interesting to study further the biological function of the Rachet subdomain, and whether its dynamics affect CYRI-B activity. It is conceivable that ensembles of CYRI are present in solution, where an open or a closed conformation can be stabilized by Rac1 binding or dimerization. This could allow CYRI-B to be regulated and switched on/off in response to Rac1 binding. Nevertheless, the existence of such a Ratchet subdomain and the implications of the conformational change suggest further tests of biological function to reveal how the Rac1 -CYRI-B -Scar/WAVE complex feedback loop is controlled are necessary. Since our crystal structure revealed a similar fold between CYRI and CYFIP DUF1394, we modeled Rac1 binding to the CYFIP ''A'' site. Due to the sequence conservation, overlaying of our complex structure with that of the Scar/WAVE complex (Chen et al., 2010) reveals the likely interface between Rac1 and CYFIP at the ''A'' site. Strikingly, the structure reveals that there is no basis for the previously suggested direct competition between Rac1 and the meander region of CYFIP (Chen et al., 2010). Instead, we observe a steric clash between switch-I of Rac1 and CYFIP and we propose this as the driving force behind the release of the VCA region of WAVE. The caveat remains that the crystal structure of Scar/WAVE does not contain the proline-rich (residues 187-484) region of WAVE, and direct competition with these residues cannot be ruled out. Nevertheless, our model provides the basis for further experimentation and brings us closer to understanding Rac1 driven activation of the Scar/WAVE complex.
In conclusion, our structures of CYRI-BDN and CYRI-BDN:Rac1Q61L reveal the nature of the inhibition of Rac1mediated activation of the Scar/WAVE complex by CYRI proteins and affirm the DUF1394 domain as an evolutionarily conserved Rac1 binding module. We reveal how Rac1 binding can drive a dramatic conformational change in CYRI, provide a basis for autoinhibited dimerization and the ability of CYRI to compete with the Scar/WAVE complex for Rac1 binding. Docking of our complex onto the Scar/WAVE complex additionally provides structural evidence that current models for how Rac1 activates Scar/WAVE complex will need to be revised.
Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Shehab Ismail, email: [email protected].
Materials Availability
All the materials generated in this study are available from the Lead Contact upon request.
Data and Code Availability
Protein structures were deposited in the PDB accession codes: PDB: 7AJL for CYRI-BDN and PDB: 7AJK for CYRI-BDN:Rac1Q61L.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Unlabelled protein was expressed and purified from Escherichia coli BL21 (DE3) cells grown in LB media at 37 C until OD 600 reached 0.6. Cells were cooled to 20 C and induced with 0.2mM IPTG and left to express for 20 hours. Selenomethionine labelled protein was expressed using B834 (DE3). Cells were grown in minimal media at 37 C until OD 600 reached 0.6. Cells were cooled to 20 C washed in PBS and added to selenomethionine labelled minimal media (Molecular Dimensions) and left to express overnight.COS7 cells (Cercopithecus aethiops-ATCC) are cultured using standard culturing method with DMEM (Gibco) supplemented with 10% serum, 1X glutamine and 1X Penicillin/Streptomycin. Cells are maintained at 37 C at 5% CO2 and are passaged every Monday and Friday. Cells are used for experiments no longer than P20.
Construct Design
All GST-tagged Rac1Q61L and its mutants were cloned into pGEX2T backbone, while CYRI-B and its mutants used for pulldowns were cloned into pMAL-C5X vector (NEB). MBP CYRI-BDN and MBP CYRI-BDN:Rac1Q61L fusion were cloned into pRSF_Duet cloned to have a His 10 tagged MBP that was TEV cleavable at the N-terminus. Mutagenesis was done using the Q5 Site-directed Mutagenesis kit (NEB, #E0554) according to the manufacturer protocol. Primers were designed using NEB BaseChanger website. The human CYRI-BDN: Rac1 fusion was synthesised as a codon optimised gBlock (Integrated DNA Technologies) for expression in E. coli. The construct contained human CYRI-B residues 26-324, a ten-residue linker GSAGSAGSAG and human Rac1Q61L residues 2-177.
Protein Expression and Purification
All GST fusion constructs were grown in the presence of 100ugml -1 ampicillin, all cleavable-MBP fusion constructs used for crystallography were grown in the presence of 50ugml -1 kanamycin, whilst all MBP fusion constructs for pulldown assays were grown in the presence of 50ugml -1 ampicillin. All constructs were expressed in BL21 pLysS (Promega). Cells were grown at 37 C until OD 600 reached 0.4-0.6 and induced with 0.4mM IPTG and left to express at 20 C for 16 hours.
Purification of GST-Rac1
Cells were lysed using a microfluidizer at 20,000psi in a buffer containing 20mM Tris pH 8.0, 300mM NaCl, 5mM MgCl 2 and 2mM betamercaptoethanol (BME). Cell lysates were clarified by centrifugation followed by filtration through a 0.45mm filter. Clarified lysate was loaded onto a GSTrap column (GE Healthcare). Once loaded, the column was washed with 50ml of lysis buffer before adding thrombin (Sigma) and cleaving on the column overnight at 16 C. The next day the protein was pooled, concentrated and passed over an S75 size exclusion column equilibrated in 20mM Tris, 150mM NaCl, 5mM MgCl 2 and 2mM DTT.
Expression and Purification of MBP-CYRI-BDN for Seleno-Methionine
To ensure efficient incorporation of seleno-methionine into the expressed protein, B834 (DE3) cells (Novagen) were used. An overnight culture was grown at 37 C in LB. Unlabelled media (Molecular Dimesions) was inoculated with 10ml of overnight culture and grown until OD 600 reached 0.5. Cells were harvested and washed three times in PBS before resuspending the pellets in seleno-methionine labelled media (Molecular Dimension). After a 40 minute incubation at 20 C, cells were induced with 0.4mM IPTG and left to express for 16 hours.
Harvested cells were lysed using a microfluidizer at 20,000 psi in a buffer containing 50mM Tris pH 8.0, 300mM NaCl, and 5mM BME. Cell lysates were clarified by centrifugation at 20,000xg and filtered through a 0.45mm filter. Lysates were then loaded onto a HisTrap column (GE Healthcare) before washing with 20mM imidazole. The protein was eluted with a linear gradient of imidazole from 0-300mM. Fractions containing protein were pooled and dialysed overnight at 4 C in the presence of TEV in a buffer containing 20mM Tris pH 8.0, 150mM NaCl, 5mM imidazole, and 2mM BME. The following day, proteins were passed over a HisTrap column and the flowthrough collected which was concentrated and passed over a Superdex S200 (GE Healthcare) column equilibrated in 10mM Tris pH7.5, 50mM NaCl and 2mM DTT. Purification of MBP-CYRI-BDN-Rac1Q61L Fusion and HA-CYRI-B Constructs Harvested cells were lysed using a microfluidizer at 20,000 psi in a buffer containing 50mM Tris pH 8.0, 300mM NaCl, 5mM MgCl 2 and 5mM BME. Cell lysates were clarified by centrifugation at 20,000xg and filtered through a 0.45mm filter. Lysates were then loaded onto a HisTrap column (GE Healthcare) before washing with 20mM imidazole. The protein was eluted with a linear gradient of imidazole from 0-300mM. Fractions containing protein were pooled and dialysed overnight at 4 C in the presence of TEV in a buffer containing 20mM Tris pH 8.0, 150mM NaCl, 5mM imidazole, 5mM MgCl 2 and 2mM BME. The following day, proteins were passed over a HisTrap column and the flowthrough collected and concentrated to 1ml. Nucleotide exchange was performed for 2 hours by adding EDTA to a final concentration of 50mM and GppNHp at a 10:1 molar ratio. Finally, the protein was passed over an S200 size exclusion column (GE Healthcare) equilibrated in a buffer containing 10mM Tris pH 8.0, 50mM NaCl, 4mM MgCl 2 and 5mM DTT. Small-Scale Purification of GST-tagged and MBP-tagged Proteins for Pulldown Assay Day 1, 200ml of transformed BL21 competent bacteria is grown overnight at 37 C in LB with the appropriate antibiotics. Day 2, 10ml of the preculture is transferred to 1L of LB and grow until the OD reaches 0.4. The bacteria are then induced with 0.2mM of IPTG overnight at room temperature. Day 3, the bacteria are then centrifuged at 3000rpm for 10min, then lysed in Buffer A (50mM Tris 7.5, 50mM NaCl, 5mM MgCl 2 ) containing 0.25mM DTT using sonication. The lysate is cleared by spinning at 20,000rpm for 20min and then incubated with pre-equilibrate GST beads (Glutathione Sepharose 4B #GE 17-0756-01) for 1h at 4 C. The GST-tagged protein is then eluted by incubating with Buffer A supplemented with 0.1% Tx100 and 10mM Glutathione. For MBP-tagged proteins, instead of using Buffer A as above, we use MBP Buffer (100mM NaCl, 10mM Tris pH7.5). Once the protein is conjugated to MBP beads, the beads can be kept at 4 C in the MBP Buffer until use.
The proteins are checked with Coomassie for purity and correct molecular weight.
Crystallisation of CYRI-BDN and CYRI-BDN-Rac1Q61L.GppNHp Following a large sparse matrix crystallisation screen, initial rectangular crystals of CYRI-BDN were obtained in Morpheus G8 at 291K at 8mgml -1 . Crystals were optimised using reagents purchased from Molecular Dimension and contained 0.1M carboxylic acid, 8% MPD_P1K_P33 and 0.1M MOPS/HEPES-Na pH 8.0. Crystals were cryoprotected in the same condition with 20% MPD_P1K_P33 and flash-frozen in liquid nitrogen. | v3-fos-license |
2017-05-30T19:39:33.027Z | 2013-01-01T00:00:00.000 | 680601 | {
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} | pes2o/s2orc | In Vivo Evaluation of Curcumin-loaded Nanoparticles in a A549 Xenograft Mice Model
Lung cancer is a disease with poor overall survival. 5-year survival rate of patients with lung cancer of all stages is only 16% (Jemal et al., 2009; Rocks et al., 2012). Therefore, it is important to identify potential regimens and explore more efficient therapeutic strategies for the treatment of lung cancer. Curcumin (Cum), the principal polyphenolic curcuminoid, obtained from the turmeric rhizome Curcuma longa, has been reported for its potential chemopreventive and chemotherapeutic activity by influencing different stages of carcinogenesis, including cell cycle arrest, differentiation, and apoptosis in a series of cancers (Pan et al., 2000; Surh et al., 2001; Aggarwal et al., 2003; Duvoix et al., 2005; Aggarwal et al., 2009; EI-Azab et al., 2011). For example, In vitro and in vivo experiments show the ability of Cum in inhibiting skin squamous cell carcinoma growth and blocking tumor progression (Phillips et al., 2011). In our previous research, we prepared Curcuminloaded methoxy poly (ethylene glycol)-polycaprolactone (mPEG–PCL) nanoparticles (Cum-NPs) (Yin et al., in press). The drug loaded content of Cum-NPs is more than 15% and the encapsulation efficiency is around 85%. The
Introduction
Lung cancer is a disease with poor overall survival. 5-year survival rate of patients with lung cancer of all stages is only 16% (Jemal et al., 2009;Rocks et al., 2012). Therefore, it is important to identify potential regimens and explore more efficient therapeutic strategies for the treatment of lung cancer.
Curcumin (Cum), the principal polyphenolic curcuminoid, obtained from the turmeric rhizome Curcuma longa, has been reported for its potential chemopreventive and chemotherapeutic activity by influencing different stages of carcinogenesis, including cell cycle arrest, differentiation, and apoptosis in a series of cancers (Pan et al., 2000;Surh et al., 2001;Aggarwal et al., 2003;Duvoix et al., 2005;Aggarwal et al., 2009;EI-Azab et al., 2011). For example, In vitro and in vivo experiments show the ability of Cum in inhibiting skin squamous cell carcinoma growth and blocking tumor progression (Phillips et al., 2011).
In our previous research, we prepared Curcuminloaded methoxy poly (ethylene glycol)-polycaprolactone (mPEG-PCL) nanoparticles (Cum-NPs) (Yin et al., in press). The drug loaded content of Cum-NPs is more than 15% and the encapsulation efficiency is around 85%. The
In Vivo Evaluation of Curcumin-loaded Nanoparticles in a A549 Xenograft Mice Model
Hai-Tao Yin 1& , De-geng Zhang 2& , Xiao-li Wu 3& , Xin-En Huang 4 *, Gang Chen 5 * in vitro release test demonstrated the sustained release pattern of Cum-NPs at room temperature. Moreover, in vitro cytotoxicity test showed that Cum-NPs inhibited the growth of A549 cells in a time and dose dependent manner. Apoptotic staining demonstrated the superior pro-apoptotic effect of Cum-NPs over the free drug.
In the current study, we hypothesize that Cum-NPs could have in vivo anticancer efficiency in a xenograft model of A549 cells. The growth curve of tumor volume and bodyweight of the mice will be measured every other day. At the end of in vivo experiment, mice will be sacrificed to detect the influence of Cum-NPs on the peripheral blood parameters and liver and kidney functions.
Materials
Curcumin, was purchased from Sigma Chem. Co., (St. Louis, USA). PEG samples were dehydrated by azeotropic distillation with toluene, and then vacuum dried at 50 ℃ for 12 h before use. ε-CL was purified by drying over CaH2 at room temperature and distillation under reduced pressure. Stannous octoate (Sigma) were used as received. All other chemicals were of analytical grade and used without further purification. Human lung cancer cell line A549 was obtained from Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China).
Male and female nude mice (nu/nu; 6-8 weeks old and weighing 18-22 g) were purchased from Model Animal Research Center of Nanjing University (Nanjing, China). The mice were housed and maintained in the animal facility of the Animal Center of Nanjing Medical University. The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
Preparation and characterization of Cum-loaded mPEG-PCL nanoparticles
Cum-NPs were prepared by a nano-precipitation method as described previously with minor modification (Yin et al., in press). Briefly, 10 mg of each copolymer and 2 mg Cum were dissolved in 0.3ml hot acetone. The obtained organic solution was added dropwise into 10 times volumes of distilled water under gentle stirring at room temperature. The solution was dialyzed in a dialysis bag (molecular weight cut-off 4kd, Sigma) to remove acetone thoroughly. The resulted bluish aqueous solution was filtered through a 0.22 μm filter membrane to remove non-incorporated drugs and copolymer aggregates. The prepared nanoparticles were lyophilized for further use. In vivo antitumor efficacy Nude mice implanted with A549 cell line were used to qualify the antitumor efficacy of Cum and Doc, alone or in combination, through intravenous administration. The mice were raised under specific pathogen-free (SPF) circumstances and all of the animal experiments were performed in full compliance with guidelines approved by the Animal Care Committee of Nanjing Medical University. The mice were subcutaneously injected at the left axillary space with 0.1 ml of cell suspension containing 4-6*10 6 A549 cells. Treatments were started after 7-8 days of implantation. The mice whose tumor reached a tumor volume of 100 mm 3 were selected and this day was designated as "Day 0".
On Day 0, the mice were randomly divided into four groups, with each group being composed of 6 mice. The mice were treated intravenously with saline, blank NPs, free Cum and Cum-NPs, respectively. Cum was administered at a equivalent dose of 15 mg/kg. All mice were tagged, and tumors were measured every other day with calipers during the period of study. The tumor volume was calculated by the formula (W 2 *L)/2, where W is the tumor measurement at the widest point, and L is the tumor dimension at the longest point.
Each animal was weighed at the time of treatment so that dosages could be adjusted to achieve the mg/ kg amounts reported. Animals also were weighed every other day throughout the experiments. After 15 days of injections, the mice were sacrificed for the detection of peripheral blood parameters as well as liver and kidney functions.
Statistical analysis and research experience
Results were presented as Mean±SD. Statistical comparisons were made by t test or ANOVA analysis. The accepted level of significance was P value < 0.05. We have enough experience in conducting medical researches, and have published some results elsewhere (Huang et al., 2004;Zhou et al., 2009;Jiang et al., 2010;Yan et al., 2010;Gao et al., 2011;Huang et al., 2011;Yan et al., 2011;Zhang et al., 2011;Gong et al., 2012;Yu et al.,2012).
Results
In vivo antitumor evaluation of Cum-NPs against A549 xenograft Antitumor efficacy of Cum-NPs was investigated in A549 human lung cancer xenografts in nude mice. It is observed from Figure 1A that blank NPs showed no tumor growth inhibition effect compared to control group, while both Cum and Cum-NPs significantly inhibited the growth of lung cancer since Day 4 (P <0.05 vs control). Moreover, delivery of Cum in nanoparticles inhibited the growth of tumor more efficiently than free Cum (P <0.05). Among the four groups, the group that received Cum-NPs was observed to maintain the greatest amount of antitumor activity ( Figure 1A&C). At the end of treatment, The RTV of the group received free Cum is 10.42±1.23. The RTV of the group received Cum-NPs is 6.24±0.59, which is the lowest among all the groups indicating the strongest tumor inhibition. Statistical analysis reveals the significant differences between the group receiving Cum-NPs and the group receiving free Cum. Figure 1C showed the shrinkage of tumors during the treatment groups. It could be observed clearly that the tumors taken from the mice receiving Cum-NPs were obviously smaller than those of other groups. An analysis of body weight variations generally defined the adverse effects of the different therapy regiments ( Figure 1B). No significance was observed among the four groups. However, the mice receiving free Cum were in a weak state in the aspects of movement and spirit, whereas no obvious alteration was observed in the Cum-NPs treated animals. Table 1 indicates the influence of different agents on the peripheral blood parameters of the mice. No adverse effect was observed in mice treated by Blank NPs. As expected, Cum-NPs and Cum showed no severe influence on WBC and Hb. Neither did each of the agents damaged the liver and kidney function in the experimental time (Table 2).
Discussion
Here we reported that a spherical coreshell structure Cum-NPs formed by amphilic mPEG-PCL block copolymers demonstrated superior antitumor efficacy against lung cancer in vivo. In the current research, a xenograft model of human lung cancer was established in nude mice to evaluate the efficiency and toxicity of Cum-NPs. Previous study from the author's lab proved the dose dependent growth inhibition effect of Cum-NPs against A549 cells (Yin et al., in press). The present study further evaluated the in vivo antitumor efficacy of Cum-NPs in A549 xenografts. Cum-NPs significantly delayed tumor growth when compared with free Cum. In addition, Cum-NPs showed no adverse influence on peripheral blood parameters and organ functions.
The targeted release of Cum-NPs together with chemotherapy drugs will be paid more attention to further expand the application of this current research. For example, co-delivery of Cum and Paclitaxel would be of great significance in future in that the synergistic antitumor effect of Cum and Paclitaxel has been already demonstrated in previous studies (Ganta et al., 2009;Hossain et al., 2012). Moreover, increasing the targeting ability of Cum-NPs by receptor-targeting peptides remains to be the most attractive research focus (Rothdiener et al., 2010;Franzen et al., 2011;Guo et al., 2011). It is hypothesized that a combination of passive targeting, with receptor targeting peptides, may significantly amplify the antitumor activity of the drug delivery system (Yu et al., 2010;Cutler et al., 2013;Liu et al., 2013). Due to the molecular recognition of peptides by tumor cell surface receptors, site-specific drug uptake by tumor cells may be raised, which may then lead to enhanced cytotoxicity (Hosta-Rigan et al., 2010;Almansour et al., 2012).
The current research reported the satisfied antitumor efficiency of a spherical core-shell structure Cum-NPs formed by amphilic mPEG-PCL block copolymers. In vivo evaluation further demonstrated the superior anticancer efficacy of Cum-NPs compared to free Cum in an established A549 transplanted mice model. Moreover, Cum-NPs showed little toxicity to normal tissues including bone marrow, liver and kidney at its therapeutic dose. It is concluded that nano formulation of Cum delivery is a most promising way in countering the spread of lung cancer, and continuing research will definitely advance the current study. | v3-fos-license |
2016-05-12T22:15:10.714Z | 2014-11-12T00:00:00.000 | 16597213 | {
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} | pes2o/s2orc | A Polymeric Prodrug of 5-Fluorouracil-1-Acetic Acid Using a Multi-Hydroxyl Polyethylene Glycol Derivative as the Drug Carrier
Purpose Macromolecular prodrugs obtained by covalently conjugating small molecular drugs with polymeric carriers were proven to accomplish controlled and sustained release of the therapeutic agents in vitro and in vivo. Polyethylene glycol (PEG) has been extensively used due to its low toxicity, low immunogenicity and high biocompatibility. However, for linear PEG macromolecules, the number of available hydroxyl groups for drug coupling does not change with the length of polymeric chain, which limits the application of PEG for drug conjugation purposes. To increase the drug loading and prolong the retention time of 5-fluorouracil (5-Fu), a macromolecular prodrug of 5-Fu, 5-fluorouracil-1 acid-PAE derivative (5-FA-PAE) was synthesized and tested for the antitumor activity in vivo. Methods PEG with a molecular weight of 38 kDa was selected to synthesize the multi-hydroxyl polyethylene glycol derivative (PAE) through an addition reaction. 5-fluorouracil-1 acetic acid (5-FA), a 5-Fu derivative was coupled with PEG derivatives via ester bond to form a macromolecular prodrug, 5-FA-PAE. The in vitro drug release, pharmacokinetics, in vivo distribution and antitumor effect of the prodrug were investigated, respectively. Results The PEG-based prodrug obtained in this study possessed an exceedingly high 5-FA loading efficiency of 10.58%, much higher than the maximum drug loading efficiency of unmodified PEG with the same molecular weight, which was 0.98% theoretically. Furthermore, 5-FA-PAE exhibited suitable sustained release in tumors. Conclusion This study provides a new approach for the development of the delivery to tumors of anticancer agents with PEG derivatives.
Introduction
Cancer is one of the most life-threatening diseases worldwide, which seriously endangers human health and survival [1,2]. Surgery, radiotherapy, chemical medication, biological immunization therapies are the major treatment strategies, among which chemotherapy plays an important role in the treatment of cancer [3][4][5][6][7][8][9]. Regarding chemotherapies, 5-fluorouracil (5-Fu) is one of the most widely used antimetabolites in clinic [10], which shows significant inhibitory effect against a broad spectrum of solid tumors [11][12][13]. Traditional chemotherapies such as 5-Fu are cytotoxic agents that inhibit rapidly proliferating cancer cells. Due to its low specificity, side effects such as myelosuppression, mucositis, dermatitis and diarrhea are commonly observed during the clinical application of 5-Fu [14][15][16]. Additionally, 5-Fu has a very short half life of about 20 minutes and is rapidly eliminated after administration. The irregular oral absorption and the low bioavailability often results in poor clinical therapeutic outcome [17][18][19].
To address the aforementioned problems, researchers have tried various methods to improve the efficacy and to reduce the toxicity of 5-Fu, including modification of the chemical structure, formulation strategies and novel delivery systems. Several small molecular prodrugs of 5-Fu were developed, such as 5-fluoro-29deoxyuridine, 1-(2-tetrahydrofuryl)-5-fluorouracil and 3, 5dioctanoyl 5-fluoro-2-deoxyuridine [20][21][22]. Various delivery systems have been developed for the targeted delivery of 5-Fu [23]. Menei et al developed biodegradable microspheres to obtain sustained delivery of 5-Fu for the treatment of glioblastoma [24]. Liposomes have been used as a sustained delivery system for 5-Fu [25]. In recent years, macromolecular carrier/delivery systems have been studied extensively. Macromolecular prodrugs obtained by combining small molecular drugs with polymeric carriers could slowly release the therapeutic agents in vivo with an improved half-life [26][27][28][29][30][31]. Moreover, the enhanced permeability and retention (EPR) effect may contribute to the accumulation of macromolecular prodrugs within the solid tumor, which would lead to a tumor-targeted drug delivery and reduced toxicity to normal tissues [32][33][34]. Moreover, the EPR effect has been regarded as the ''golden rule'' in the design of antitumor drugs. Based on the EPR effect, numerous tumor-targeted drug delivery systems were developed using macromolecules such as albumin (65 kDa), transferrin (90 kDa), IgG (immunoglobulin, 150 kDa), a2-macroglobulin (240 kDa) and ovomucoid of chicken eggwhite (29 kDa, highly glycosylated protein), and some have entered clinical trials [35].
In addition to the aforementioned macromolecular materials, polyethylene glycol (PEG) has become a material of great interests due to its low toxicity, low immunogenicity and high biocompatibility [36][37][38]. The molecular weight of PEG used in forming macromolecular prodrugs would impact the in vivo behaviors of the conjugates because the retention time of the prodrugs increased with the molecular weight of the carriers [30]. Prolonged retention of the prodrug is critical to the tumor accumulation of the therapeutic agents loaded. However, for linear PEG macromolecules, the number of available hydroxyl groups for drug coupling does not change with the length of the polymeric chain, which limits the application of PEG for drug conjugation purposes. Therefore,the development of new PEG derivatives to improve its drug loading efficiency has become a hot topic in material science and is of great significance to the tumor-targeted delivery of small molecular agents and 4-arm PEG derivatives were thus developed [39], and the 4-arm PEG based prodrugs have entered clinical trials with promising results [40][41][42][43][44]. For small molecular drugs such as 5-Fu, treatment requires a high therapeutic concentration, while the macromolecular based prodrugs have a relatively low drug loading efficiency. Thus, the modification of linear PEG creates derivatives with high drug loading efficiency which will have great significance for anticancer drug development [45].
In this study, a macromolecular prodrug, 5-fluorouracil-1 acid-PAE derivative (5-FA-PAE), was designed and synthesized to increase the drug loading efficiency, achieve delivery to the tumor and prolong the retention time. PEG with a molecular weight of 38 kDa was selected as the starting material to obtain the multihydroxyl PEG derivative, which was then coupled with 5fluorouracil-1 acetic acid (5-FA), to afford the prodrug. The in vitro drug release, pharmacokinetics, in vivo distribution and antitumor effect of the prodrug were investigated, respectively.
Synthesis of multi-hydroxyl polyethylene glycol derivative (polyethylene glycol-allyl glycidyl ether-mercaptoethanol, PAE) Polyethylene glycol-allyl glycidyl ether (PA) was synthesized as described before [46,47] with some modifications. Briefly, 10.0 g of PEG was melted in an oil bath at 120uC with stirring under vacuum for about 3 h to remove the adsorbed moisture before adding 120 mg of NaH. The mixture was stirred for 4 h at 120uC, and 2.0 ml of AGE was added. The product was recrystallized with isopropanol to remove the micromolecular materials.
Synthesis of 5-FA-PAE prodrug 5-FA was synthesized as previously described [48,49] with some modification. Briefly, 6.5 g of 5-Fu was dissolved in 25 ml of aqueous solution of potassium hydroxide (4 M), then 15 ml of aqueous solution of chloroacetic acid (5 M) was added dropwise with stirring. The pH value of the reaction mixture was monitored and kept at 10 by adding an aqueous solution of potassium hydroxide (10 M) during the addition of chloroacetic acid and throughout the whole course of the reaction. The mixture was heated to 50uC in an oil bath with stirring for 8 h, and then acidified by HCl to obtain 5-FA.
A solution of 5-FA (0.496 g) in 1 ml of dimethylformamide was added dropwise to a solution of 0.5 g of PAE in 20 ml of dimethylformamide, then 0.196 g (1.7 mmol) of NHS and 0.4 g (2.09 mmol) of EDC?HCl were added sequentially. After a further 16 h of incubation at room temperature away from light, the mixture was precipitated with 150 ml of isopropanol. The obtained residue was recrystallized by isopropanol several times until the reagents and uncoupled 5-FA were totally removed (monitored by TLC and HPLC), then dried in vacuum at 40uC overnight.
HPLC analysis
HPLC assay was established for the determination of 5-FA in PBS, plasma or tissues homogenates, which was performed using Shimadzu instruments (Chiyoda-Ku, Japan) consisting of a CTO-10A column thermostat, two LC-10AT pumps and a SPD-10A UV detector. A Scienhome ODS column (5 mm, 15064.6 mm, Tianjin, China) was used to separate samples. Phosphate buffer (0.05 M, pH 2.5) was used as the mobile phase at a flow rate of 1 ml/min. The temperature of the column was kept at 35uC and the effluent was detected at 270 nm. Studies showed that the precision, accuracy, and recovery of this HPLC method all met the measurement requirements.
Safety evaluation
All animal experiments were approved by the Institutional Animal Care and Ethic Committee of Sichuan University (Approved No. SYXK2013-185). All animals were fed on a light and dark cycle and allowed free access to standard chow and water. Temperature and relative humidity were kept at 25uC and 50%, respectively. After experiment, mice were sacrificed by neck dislocation, and all efforts were made to minimize suffering. Myelosuppression is one of the major side effects of 5-Fu [14]. To assess the suppression level, 60 male Kunming mice (20-25 g, purchased from Laboratory Animal Center of Sichuan University) were randomly divided into 5 groups (n = 12) and were intravenously administered with 5-Fu (27.66 mg/kg), 5-FA (40 mg/kg), PAE (338 mg/kg) or 5-FA-PAE (378 mg/kg) (equivalent to 0.213 mmol/kg 5-FA). The control group was given physiological saline (0.009 g/ml). Zero point one mL blood samples were collected at prearranged time intervals (one day before injection and 1, 4, 7, and 10 days post injection). The white blood cells (WBC) and the blood platelets number were counted by MEK-6318K Automated Hematology Analyzer (Nihonkohden, Shinjuku-ku, Japan) as an index of myelosuppression.
In vitro drug release
The in vitro drug release of 5-FA-PAE was investigated in physiological saline (0.009 g/ml), PBS with various pH values, 50% mouse plasma (diluted with PBS, pH 7.4, v/v) and 50% mouse tumor homogenate which was obtained from the H22 tumor loaded mice (homogenized and diluted with physiological saline). An aqueous solution of 5-FA-PAE (100 ml) was added to 4 ml of preheated release medium (physiological saline or PBS with pH = 3.04, 4.51, 6.02, 7.41, 8.99). The mixture was maintained in a water bath at 37uC under continuously stirring, and 100 ml of each sample was collected at fixed time intervals (i.e. 0.25, 1, 3, 6, 10, 24, 48, 72, 96 h). The samples from physiological saline and PBS was acidified by 100 ml hydrochloric acid (1 M), diluted with 300 ml mobile phase and analyzed by HPLC. The samples from mouse plasma and tumor homogenate were obtained in duplicate at each time point (100 ml each). For hydrolysis, samples were mixed with 50 ml of aqueous solution of 5-bromouracil (96 mg/ml, 50 ml) as the internal standard, and then supplemented with 100 ml sodium hydroxide (1 M) and acidified by 100 ml hydrochloric acid (1 M), and extracted by 3.3 ml of ethyl ester for 15 min. After centrifugation at 10,000 rpm for 5 min, 2.7 ml of the ethyl ester portion was collected, concentrated in a nitrogen gas flow, redissolved in 100 ml of the mobile phase and centrifuged at 10,000 rpm for 10 min before HPLC analysis. The other group was not subjected to hydrolysis by substituting sodium hydroxide solution with saline and acidifying with 50 ml hydrochloric acid. The differences of 5-FA in the two groups at the same time point was the unreleased 5-FA in each sample. The decrement method was used to calculate the release rate. All experiments were conducted in triplicate.
Pharmacokinetics study
Male Wistar rats were purchased from The laboratory Animal Center of Sichuan University. 12 Wistar rats (body weight: 200 g620 g) were divided into two groups randomly (n = 6). The control group and the test group were administered intravenously with 20 mg/kg of 5-FA and 189 mg/kg 5-FA-PAE (equivalent to 20 mg/kg of 5-FA) dissolved in physiological saline, respectively. The blood samples were collected into heparinized centrifuge tubes at predetermined intervals (see Table S2 in file SI) by retroorbital puncture, and the plasma was separated by centrifugation. Each plasma sample of the test groups was divided into two portions. They were treated as hydrolyzed and unhydrolyzed as described in the ''In vitro drug release'' section. The two portions of the samples were analyzed by HPLC to determine the plasma concentrations of released 5-FA and total 5-FA of the conjugate whereas the plasma samples of the control group were treated as unhydrolyzed samples.
In vivo distribution
Murine H22 hepatocarcinoma cells (purchased from Type Culture Collection of Chinese Academy of Sciences) were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% fetal bovine serum (FBS) at 37uC with 5% CO 2 , and were passaged every 2 or 3 days. The tumor-bearing animal model was established by subcutaneous injection of H22 cells (1610 7 cells/ml, in 0.2 ml saline) into the right axillary region of Kunming mice. The sizes of tumors were monitored 7 days after inoculation and the tumor volumes were calculated as described in the ''Antitumor activity in tumor-bearing mice'' section. The mice with tumor volumes between 0.35 cm 3 and 0.65 cm 3 were randomized into two groups (n = 30). The control group and the test groups were administered intravenously with 20 mg/kg of 5-FA or 189 mg/kg 5-FA-PAE (equivalent to 20 mg/kg of 5-FA) dissolved in physiological saline (0.009 g/ml), respectively. The mice were exsanguinated and sacrificed by neck dislocation at predetermined time points. Tissues including heart, liver, spleen, lung, kidney, brain and tumor were collected, washed with physiological saline, weighed and homogenized with two fold concentrated physiological saline. The samples of the test group were treated as hydrolyzed samples as described in the section ''In vitro drug release'', whereas those of the control group were treated as unhydrolyzed samples. All data are presented as the concentration of 5-FA.
Antitumor activity in tumor-bearing mice
The tumor-bearing mice model was established as previously described in the ''In vivo distribution'' section. 72 h after inoculation, mice with no signs of tumor growth were exclude from this experiment. 48 tumor-bearing mice were randomly divided into 4 groups (n = 12). The control group was administered intravenously with 20 ml/kg of physiological saline. The other groups were administered intravenously with 30 mg/kg (0.160 mmol/kg) of 5-FA or 284 mg/kg 5-FA-PAE (equivalent to 0.160 mmol/kg 5-FA) dissolved in physiological saline. 5-Fu (20.47 mg/kg, 0.160 mmol/kg) was administered as a control. All animals were administered once on day 3,5,7,9,11,13,15 after the inoculation of H22 cells and sacrificed on day 20. Tumors and organs (heart, liver, spleen, lung, kidney, brain and thymus) were removed and weighed. The tumor volume and tumor control rate were evaluated. The tumor volume, organ/body weight index and tumor control rate were calculated as follows:
Data analysis
The data of pharmacokinetics and in vivo distribution study were processed using the Drug and Statistics Software 2.0 (DAS 2.0, Shanghai, China). The statistical analysis of the samples was performed by using one-way ANOVA and Student's t-test. p-values ,0.05 were considered as statistically different.
Results and Discussion
Synthesis and characterization of 5-FA-PAE prodrug As a polyether macromolecule, PEG is widely used for its suitable solubility and bioavailability in developing drug delivery systems [50][51][52][53]. However, as a drug carrier, the loading efficiency of prodrugs based on PEG is significantly constrained due to the limited positions for drug conjugation, i.e., two hydroxy groups in the linear PEG molecule [45]. Thus, the modification of PEG to create derivatives with higher drug loading efficiency is greatly needed. PEG with a molecular weight of 38 kDa was selected as the starting material to synthesize the derivative (Fig. 1A). Allyl glycidyl ether was coupled to both ends of PEG under the catalysis of sodium hydride to form an intermediate, namely PA, with multi-double bonds on the side chains. PA was further reacted with small molecules through the addition reaction of the double bonds and the thiol group to afford various PEG derivatives with multi-hydroxyl groups. 1 H-NMR showed that the double bonds disappeared completely in PAE ( Figure S1 in file SI). The GPC analysis demonstrated that PA and PAE had similar molecular weight distribution as the starting material PEG ( Table 1). As a common drug carrier, the molecular weight of PEG greatly influenced the in vivo behaviors of prodrugs [54]. As the molecular weight increases, the in vivo clearance rate decreases. Thus, PEG with a higher Mw is likely to prolong the retention time of prodrugs and increase the drug accumulation in a tumor. It is suggested that the Mw of PEG should be no less than 30 kD to prevent the prodrug from quick elimination from kidney [55]. Accordingly, PEG of 38 kD was used as the starting material.
However, 5-Fu could not be directly coupled with the carriers due to the lack of available hydroxyl groups in the structure. The derivative of 5-Fu, 5-fluorouracil-1-acetic acid (5-FA), was synthesized first. The macromolecular prodrug multi-hydroxyl polyethylene glycol-5-fluorouracil-1-acetic acid (5-FA-PAE) was obtained by covalently conjugating 5-FA with the PEG derivative under the catalysis of carbodiimide condensing agents ( Figure 1B). The successful synthesis of the 5-fluorouracil derivative and the prodrug was confirmed by 1 H-NMR ( Figure S1 in file SI). A higher molecular weight (Mw) and polydispersity (PDI) of 5-FA-PAE were observed compared with those of PAE (Table 1). HPLC analysis indicated that after the double bond-thiol addition reaction, multiple hydroxyl groups were introduced on the PEG backbone thus making it capable of loading more drugs. The drug loading efficiency of 5-FA-PAE was determined as 10.58%, much higher than the maximum drug loading efficiency of PEG with the same molecular weight, which was calculated as 0.98% theoretically. The drug loading efficiency of 5-FA-PAE was improved by
Safety evaluation
To investigate the myelosuppression levels after 5-FA or 5-FA-PAE treatment, hematological parameters (i.e. the number of white blood cells and blood platelets) were measured at different time points after drug administration. Changes in these parameters presumably reflect the occurrence of myelosuppression and abnormality in the immune system. As shown in Table 2, the WBC count decreased after intravenous injection of 5-Fu, 5-FA and 5-FA-PAE. Only the group of 5-Fu exhibited significant reduction of WBC (5.3962.17610 9 /L one day after injection, p, 0.05). Then the WBC level increased gradually. Notably, the increase of WBC in the 5-Fu group was slower, leading to a lower WBC level at day 10 (8.5962.39610 9 /L) compared with the level before administration (9.5561.28610 9 /L), while the WBC level of other groups recovered within 4 days. Another major indicator of myelosuppression is the change in blood platelets number. Table S1 in file SI shows that though the platelets number of 5-Fu decreased 1 day after injection, the blood platelets of all groups didn't exhibit any significant changes, indicating that both 5-FA and 5-FA-PAE hardly affect the platelets level at such doses. Taken together, these results indicated that the prodrug of 5-Fu, 5-FA-PAE, showed a lower toxicity than 5-Fu.
In vitro drug release
The in vitro drug release behavior of 5-FA-PAE was investigated using phosphate buffered saline (PBS) of various pH values, physiological saline, mouse plasma and tumor homogenate as the release media. The release rate of 5-FA-PAE was pH-dependent. As the pH increased, the release rate increased significantly, reaching 94.1%65.88% at 96 h when the pH was 8.99 (Figure 2A). This is mostly likely due to the hydrolysis of ester bonds under basic conditions. However, if the conjugation with the PEG derivative increased the retention time in plasma, this would possibly enhance the drug accumulation in a tumor ( Figure 2B). The release rate of prodrug 5-FA-PAE in plasma was 89.46%66.36% at 10 h, and reached 98.15%61.96% at 24 h, while the rate was 55.9%60.61% in tumor homogenate at 24 h, suggesting that the ester bond can be easily degraded by easterases in plasma.
Pharmacokinetics study
A major drawback of 5-Fu is the relatively short half-life, which results in poor patient compliance and side effects. 5-FA, the derivative of 5-Fu, shows the same metabolism and clearance rate as 5-Fu. Moreover, after conjugation with PEG, the retention time in plasma was greatly prolonged, which might enhance tumor accumulation. After administration intravenously, 5-FA was rapidly eliminated from the blood circulation, which led to a complete removal at 6,8 h after administration, whereas the elimination rate of 5-FA-PAE was much lower than that of 5-FA, and the blood retention time of this macromolecular prodrug reached more than 96 h (Figure 3). The detailed plasma concentration of 5-FA and 5-FA-PAE at different time points are shown in Table S2 in file SI. Some pharmacokinetic parameters, such as the area under the curve (AUC), the mean retention time (MRT) and the elimination half-life (t 1/2 ) of 5-FA-PAE were much higher than those of 5-FA, i.e., 25.6 times for AUC (546.6636.7 mg/ml ?h vs 21.3764.36 mg/ml ?h), 11.7 times for MRT (7.96260.400 h vs 0.67960.142 h) and 14.4 times for t 1/2 (22.1065.92 h vs 1.53860.419 h), indicating a much longer blood circulation times and a remarkably enhanced bioavailability of the macromolecular prodrug (Table 3). Meanwhile, the amount of 5-FA released from 5-FA-PAE in rat plasma was determined. Although the total amount of 5-FA-PAE in rat plasma was significantly higher than that of 5-FA, the concentration of released 5-FA from 5-FA-PAE was not as much as 5-FA. It was lower than the 5-FA group within 30 min after administration and then increased slightly afterwards (Table S2 in file SI). This may concentration at 5 min), which is consistent with the previous in vitro release study. Comparing the concentration of the free 5-FA of unhydrolyzed and hydrolyzed 5-FA-PAE group, it can be concluded that the unreleased 5-FA-PAE was intact in the blood circulation, which could accumulate in tumor tissue in the form of prodrug and then slowly release 5-FA at the tumor site to achieve antitumor effect. 5-FA was shown to be rapidly eliminated from blood, while after conjugation with PAE, the retention time was significantly prolonged, which may be attributed to a protective role of PEG. Thus, the prolonged retention time of 5-FA not only extended the duration time and enhanced the bioavailability, but also improved the delivery of macromolecular drugs to a tumor.
In vivo distribution
Small molecular drugs eliminate quickly after intravenous administration, which could distribute them to normal tissues through capillaries nonspecifically. To analyze the in vivo biodistribution of 5-FA-PAE, the murine hepatic cancer cell line (H22) was used to establish the tumor-bearing animal model. The hepatoma H22 model has been widely use as a tumor model in the study of antitumor drugs and its mechanism. Generally, it is believed that the orthotopic tumor can better simulate the pathologic process of tumor development. However, due to the high mortality rate of animals and the complexity of operation, we Table 3. Pharmacokinetic parameters of 5-FA and 5-FA-PAE after i.v. injection in rats. Fig. 5B. 45 min after administration, the concentration of 5-FA in tumor was lower than that in plasma, and then increased rapidly afterwards. 4 h after injection, the ratio of tumor/plasma concentration reached 1.99, suggesting that 5-FA could distribute from plasma to tumor within a short time, and that the clearance rate of 5-FA in plasma was higher than that in the tumor. 6 h after administration, the 5-FA concentration was almost undetectable in both plasma and tumor. The ratio of tumor/plasma concentration of 5-FA-PAE increased steadily within 48 h after injection and was lower than 0.5. Between 48 h to 72 h, the ratio increased quickly and reached 1.56 at 72 h, indicating that the clearance of 5-FA-PAE from tumor was much lower than that from the plasma. These results indicated that the amount of 5-FA-PAE in tumor lasted longer compared with that of 5-FA, exhibiting a sustained-release profile. Though the initial concentration and tumor/blood concentration ratio of 5-FA were higher than 5-FA-PAE, a high elimination rate of 5-FA severely limited its therapeutic effect in clinic. In comparison, the concentration of 5-FA-PAE in the tumor could be maintained at a relatively high level, which lasted for more than 70 h, despite the large variation (probably due to inter-individual difference). Similarly, the tumor/blood concentration ratio of 5-FA-PAE showed a gradually increasing trend.
Antitumor effect in tumor-bearing mice 5-Fu is the first-choice antimetabolite in the treatment of colon cancer and colorectal cancer. 5-FA, a derivative of 5-Fu, has been reported to be effective and safe [56][57][58][59]. To address the antitumor activity of the prodrug, 5-FA and 5-Fu were both used as controls. In the pharmacokinetics studies of anticancer drugs, two dosing regimens are commonly used. One is the preventive administration strategy in which drugs are administered at the beginning of the tumor growth. The other one is the therapeutic administration with drugs administered when the tumor growth reached a certain size. Since the relatively high mortality rate of the H22 tumor model in the later period of this experiment, we adopted a prophylactic administration scheme, i.e. 72 h after inoculation, mice with no signs of tumor growth were excluded from this experiment. Based on the pharmacokinetics and biodistribution results, we administered the drugs every other day (from day 3 to day 15 after inoculation). The antitumor effect of 5-FA-PAE was assessed by analyzing tumor volume, tumor control rate and the organ/body weight index of tumor-bearing mice. From the beginning of administration, the tumor volume of 5-FA-PAE group was smaller than that of the saline group, showing the highest antitumor activity ( Figure 6A and 6B). The 5-FA and 5-Fu groups also displayed some antitumor effect. However, after the last administration on day 15, the tumor volume of these two groups increased obviously, while the tumor size of the 5-FA-PAE group did not, which suggested that the antitumor activity of 5-FA-PAE could last for a longer time. This is compatible with the pharmacokinetics results in which 5-FA-PAE showed a much longer retention time than that of 5-FA. 20 days after inoculation, the average tumor volume of 5-FA-PAE group was significantly smaller than that of the 5-Fu and saline groups (p,0.01). However, no significant differences were observed between the 5-FA and 5-FA-PAE groups. This may be due to the large variation of the 5-FA group.
Though the tumor control rates of the 5-FA-PAE and 5-FA groups were not significantly different (p.0.05), the tumor control rates of the 5-FA-PAE group (51.9611.2%) and the 5-FA group (35.0617.6%) were significantly higher than that of the 5-Fu and saline groups (Table 4). Since the tumor growth can affect the weight of normal organs, the organ/body weight index was used to assess the impact. The tumor/body index of the 5-FA-PAE (3.38161.224) group was much lower than those of the 5-Fu (8.81663.578) and saline groups (7.08861.961, p,0.05, Table 5). No significant differences were observed in other organ/body indices.
Owing to the conjugation with PEG, 5-FA-PAE exhibited a longer retention time, which led to a long-lasting antitumor effect. Notably, during the administration period, the death rate in the tumor-bearing mice of the 5-FA-PAE group is relatively high. This is probably due to the tumor growth and the toxicity of 5-FA-PAE, which is also a drawback of our present regimen and needs further refinement. However, after administration of all doses, no more deaths were observed in the 5-FA-PAE group, indicating that the toxicity caused by repeated administration of 5-FA-PAE was reversible. While in the 5-FA and 5-Fu groups, large number of animal deaths were observed after all administrations, suggesting a shorter duration of their antitumor effect.
Conclusion
To solve the paradox of drug loading and the molecular weight of PEG, we synthesized a PEG multi-hydroxyl derivative (PAE). PAE was coupled with 5-FA via ester bonds to afford 5-FA-PAE, and the drug loading efficiency was shown to be 10.8-fold higher than using unmodified PEG. Besides, the retention time and bioavailability of 5-FA-PAE were greatly improved compared to 5-FA, showing a prolonged half-life and improved antitumor efficacy in vivo. Owing to the improved drug loading efficiency and prolonged half-life, the multi-hydroxyl PEG derivative PAE proves to be an efficient carrier for 5-Fu. Future study should focus on further improving the tumor-targeting efficiency and the antitumor effect of 5-FA-PAE while reducing its toxicity. This paper provides some insights for the future development of antitumor drugs using PEG as a drug carrier. | v3-fos-license |
2018-12-27T10:21:15.584Z | 2013-10-12T00:00:00.000 | 86380370 | {
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} | pes2o/s2orc | The Analysis on Fat Characteristics of Walnut Varieties in Different Production Areas of Shanxi Province
To gain knowledge of the fat characteristics of walnut varieties in different production areas of Shanxi Province, kernel oil content and its fatty acid composition of 6 walnut varieties were analyzed by using soxhlet extraction method and gas chromatography. The experiment sites were Yicheng and Tunliu. Yicheng has 800 m of altitude and 11 oC of annual mean temperature while Tunliu has 1100 m of altitude and 9.3 oC of annual mean temperature. The results indicated between Yicheng and Tunliu County, the most variety’s oil content was increased with increasing of altitude, but had no significant difference (64.9% > 64.3%, P > 0.05). Both the content of oleic acid and linoleic acid showed a significant difference between Tunliu and Yicheng, and there is a negative relationship between the contents of oleic and linoleic acid. Annual mean temperature has an obvious influence on α-linolenic acid (ALA) content of walnut. The content of ALA from Yicheng with higher annual mean temperature is higher than that from Tunliu with lower annual mean temperature. The nutrition analysis showed that Tunliu’ walnut kernel has a lower saturated fatty acids (SFA) (7.6% < 8.2%, P < 0.05) and a higher unsaturated fatty acids (UFA) (92.5% > 91.8%, P < 0.05) compared to Yicheng, respectively, but the polyunsaturated fatty acids (PUFA) content of Yicheng was higher than that of Tunliu (76.3% > 68.5%, P < 0.05), and its ratio of N-6/N-3 was also better compared to Tunliu (5.4:1 < 5.9:1). These results showed that with the different altitude and annual mean temperature, the walnut nutrition of fat is also different. Higher altitude and lower annual mean temperature can help to produce oleic-rich walnut while lower altitude and higher annual mean temperature is helpful for the higher content of ALA in walnut kernel.
Introduction
Walnut kernel contains around 60-70% of oil, and can be praised as "the oil store on the tree" (Li et al., 2009).Walnut is a rich source of energy for life.People prefer plant oils to animal fat due to concerning about animal cholesterol (Pyorala, 1987).Plant oils don't contain any animal cholesterol; but it contains a large amount of unsaturated fatty acids (UFA) that benefit human health.The UFA content of walnut oil is more than 90%.
To obtain the perfect nutrition characteristics of fatty acid composition of walnut, a lot of investigations have been done about variety (Wang et al., 2004;Chen et al., 2007;Wu et al., 2007).However, the results are not quite consistent even the same cultivars.It is reported that the external site conditions including altitude, air temperature, average annual relative humidity, longitude, may affect the single fruit kernel weight, accumulation of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) during the plant growing season (Zhang et al., 2011;Li Guo et al., 2007;Xu et al., 2009).In order to gain knowledge of the fat characteristics of walnut in different production areas, the effect of production area on walnut oil quality is necessary to be investigated.Shanxi belongs to mountainous region which develops different climate conditions.Moreover, its total plant area of walnut is also large (Cheng, 2011), so we selected Shanxi as experimental site to determine the effect of producing region of walnut on oil quality.
Materials
Walnut samples were collected at Yicheng County and Tunliu County, in Shanxi province of China during 8-9, 2012.The condition data of the two sites were shown in According to the method 3-5 g of walnut kernel powder was weighted, wrapped with filter paper and laid in Soxhlet extractor.A 50-60 ml solvent of petroleum ether (60-90 ºC) was added and refluxed for 8h.The oil content of walnut kernel was determined gravimetrically after complete evaporation of solvent to the dryness at 45 ºC using a rotary evaporator.
Analysis of Fatty Acids by Gas Chromatography
20-30mg of walnut oil was weighted and esterified through sodium methoxide.Fatty acid methyl esters were analyzed on a Lufen company gas chromatograph GC6500 with flame-ionization detector.The glass column packed with 20% DEGS on Chromsorb (4 mm i.d.× 1.1 m length) was employed; N2 was used as a carrier gas; the column temperature was 190 ºC and the injector and detector temperatures were 250 ºC.The content of fatty acid was obtained by calculating the peak area percentage method.Figure 1 is the typical fatty acid chromatography of Liaoning No.1 walnut oil from Tunliu County.
Analysis of Oil Content and Fatty Acid Content of Walnut in Different Production Areas
6 varieties of walnut were selected to inspect the effects of two different production areas (Tunliu and Yicheng) on walnut oil content and fatty acid content.Figure 2 showed that the oil content of the same varieties from Tunliu was more than that of Yicheng except Zhonglin series (No.1 and No.3), but the average value of Tunliu was a little higher than that of Yicheng (64.9% > 64.3%).The factors (Li et al., 2010) influencing oil content includes genetic quality, seed maturity, and fruiting number, etc.In our study these factors were controlled to be as similar as possible.However, there are still some of differences of climatic conditions such as altitude and yearly precipitation.Altitude has a positive effect on walnut single fruit kernel weight and kernel percent (Zhang et al., 2011), but it can be considered with water factor as far as the oil content.Altitude of Tunliu is about 1100m more than 800 m of Yicheng, and the yearly precipitation is also higher than that of Yicheng.During fruit development, the moisture of soil was higher in Tunliu of high altitude than that in Yicheng of lower altitude, and water help to the nutrition absorption for the synthesis of oil, which was seen in most of variety except for Zhonglin No.1 and Zhonglin No.3.
Figure 2. The oil content of walnut with increasing of altitude Frost-free period means plant growth period, but even the frost-free period in Yicheng was approximate to one month longer than that in Tunliu, there was not obvious increment in oil content.This indicated that the synthesis of oil can be related with altitude and water rather than plant growth time.The pH value of soil can have different influences on oil content due to walnut variety.Zhonglin series including Zhonglin No.1 and Zhonglin No.3 were suitable in milder alkaline soil of Yicheng, while higher pH value of soil of Tunliu was more suitable for the most of varieties.Tree-oil crops preferred calcareous soils (Li et al., 2010) where higher pH value of soil was usually seen.However, the content of oil of the most varieties from Tunliu was more than that from Yicheng.It was found that the climate played a more important effect on walnut compared to soil (Cao, 1994).From the view of climate, the oil content of higher altitude was more than that of lower altitude, but the difference was insignificant (P > 0.05).This might be due to the little sample number.
From Tables 2-3, there was no difference in main composition kinds of fatty acid.But there was significant difference in oleic content, the major monounsaturated fatty acid (MUFA) in walnut; the average content from Tunliu was more than that from Yicheng as far as MUFA (23.8% > 15.0%, P < 0.05).On the contrary, linoleic acid content of Tunliu was less than that of Yicheng, (58.4% < 64.2%, P < 0.05).Higher altitude can produce the higher oleic acid content, especially with lower annual mean temperature.An extremely negative correlation is observed between the contents of oleic and linoleic acid in cameillia oleifera fruit trees (Wang et al., 2008) and this same relationship was also proved existed in walnut trees in our results.This phenomenon probably rises from the saturate conversion of the two fatty acids of same carbon chain length.Although the samples are from two different production areas, the content relationship between oleic acid and linoleic acid appears to be same.Therefore, the differences of oleic and linoleic acid from two production areas can be considered as one index of fatty acid to compare.Our results showed that Tunliu is suitable for MUFA-rich or oleic-rich walnut, in other words, Yicheng is suitable area for linoleic acid-rich walnut.
Comparison of Nutrition Characteristics of Walnut From Two Production Areas
The nutrition characteristics of walnut fatty acids was compared on Tables 4-5.Tunliu has a lower of SFA (7.6% < 8.2%, P < 0.05) and a higher UFA (92.5% > 91.8%, P < 0.05) compared to Yicheng, respectively, but the polyunsaturated fatty acids (PUFA) content of Yicheng was higher than that of Tunliu (76.3% > 68.5%, P < 0.05), and its ratio of N-6/N-3 was also better compared to Tunliu (5.4:1 < 5.9:1).Air temperature has a positive effect on PUFA amount (Li et al., 2007).Annual mean temperature of Yicheng (11.0 ºC) was higher than that of Tunliu (9.3 ºC).However, there was no difference in PUFA/SFA.This indicated that the unsaturated conversion of the two fatty acids of same carbon chain length are confined to definite unsaturated acids rather than saturate acids, despite of the difference of annual mean temperature in two production areas.There was a significant difference of α-linolenic acid content (ALA) between Yicheng and Tunliu.The content of ALA of every variety from Yicheng was higher than that of Tunliu correspondingly (Figure 3).And then the average of α-Linolenic acid was also more than that of Tunliu (12.1% > 10.2%, average value).Annual mean temperature can affect the synthesis of ALA.With the annual mean temperature the content of ALA increases (Li et al., 2007).In our study, higher annual mean temperature of Yicheng (11.0 ºC) had an increased amount of ALA compared to Tunliu (9.3 ºC).This result can be explained for the differences of annual mean temperature in production areas.Higher annual mean temperature was always accompanied with longer frost-free period and illumination time, and these climate conditions can be also helpful to ALA biosynthesis.
Figure 3. ALA content of walnut between Tunliu and Yicheng ALA is the precursor of synthesize EPA (eicosapentaenoic acid) and DHA (Docosahexaenoic acid), and have also physiologically active like decreasing blood lipid, decreasing blood pressure, antithrombosis and preventing and curing athrosclerosis, etc.For the appropriate ratio of the N-6 to N-3 fatty acids (Simopoulos, 2002) also needs an important increase in ALA concentration.So it is strongly advised that the higher annual mean temperature should be considered to produce the ALA-rich walnut.
Conclusion
The content of oil of the most varieties from Tunliu with higher altitude was more than that of Yicheng of lower altitude, but the difference was insignificant.
The content of oleic acid and linoleic acid showed a significant difference between Tunliu and Yicheng.The differences of oleic and linoleic acid from two production areas can be considered as one index of fatty acid to compare.There is a negative relationship between the contents of oleic and linoleic acid despite of different production areas of walnut.
Annual mean temperature from different production areas has an obvious influence on α-linolenic acid (ALA) content of walnut.The content of ALA of Yicheng with higher annual mean temperature is higher than that of Tunliu with lower annual mean temperature.
Higher altitude and lower annual mean temperature can help to produce oleic-rich walnut while lower altitude and higher annual mean temperature is helpful for the higher content of ALA in walnut kernel.
Table 1 .
Some climate and soil characteristic in walnut producing regions
Table 2 .
The fatty acid composition of walnut kernel in Yicheng County (%)
Table 4 .
Fat nutrition characteristic of 6 walnut varieties yielded from Yicheng County ΣSFA, ΣUFA, ΣMUFA and ΣPUFA are abbreviated for the total amount of SFA, UFA, MUFA, and PUFA, respectively.
Table 5 .
Fat nutrition characteristic of 6 walnut varieties yielded from Tunliu County | v3-fos-license |
2019-04-10T13:12:24.757Z | 2018-11-06T00:00:00.000 | 196670816 | {
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"pdf_src": "BioRxiv",
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} | pes2o/s2orc | A chemogenetic approach for optical monitoring of voltage in neurons
Optical monitoring of neuronal voltage using fluorescent indicators is a powerful approach for interrogation of the cellular and molecular logic of the nervous system. Here we describe a Semisynthetic Tethered Voltage Indicator (STeVI1) based upon Nile Red that displays voltage sensitivity when genetically targeted to neuronal membranes. This environmentally sensitive probe allows for wash-free imaging and faithfully detects supra- and subthreshold activity in neurons.
Main
Complementation and substitution of electrophysiology methods with non-invasive optical imaging of neuronal activity is a major technological challenge in neuroscience 1,2 . Calcium imaging with genetically encoded indicators is widely used to interrogate the connectivity and function of neural circuits at different spatial and temporal resolutions 2,3 . However, as a surrogate for underlying electrical activity, calcium imaging has a number of shortcomings. For example, calcium indicators lack the sensitivity to register subthreshold activity, and their slow kinetics and the nature of the calcium transient itself often preclude recording of high-frequency firing 2 . Direct readout of neuronal membrane voltage is therefore necessary for imaging subthreshold and inhibitory activity, and for investigating fast-coordinated phenomena 4,5 . As such, significant efforts are being invested in developing probes and improving microscopy for optical monitoring of voltage [6][7][8][9] .
Fluorescent indicators for membrane potential can be divided into two groups; synthetic voltage sensitive dyes (VSD) 6 , and genetically encoded voltage indicators (GEVI) based upon voltage sensitive proteins such as opsins, channels and phosphatases 10 . Organic VSDs possess excellent photophysical properties and fast kinetics for live imaging 6,11 . However, their application in vivo is limited by unspecific staining of tissue, compromising signal-tonoise ratio (SNR) and cell identity. GEVIs provide a valuable alternative as they can be genetically targeted to subsets of cells 7,8,12 . However, GEVIs often suffer from low brightness, poor photostability and slow kinetics 7,8 .
They may also localize poorly to the plasma membrane and exhibit cellular toxicity. To circumvent these problems, hybrid voltage indicators have been proposed which combine the superior optical properties of smallmolecule fluorophores with genetically encoded voltage sensors [13][14][15] . Examples include a precursor VSD that is converted to an active membrane-bound dye by a genetically encoded enzyme 16 , and click chemistry-and enzyme-mediated ligation of organic fluorophores to rhodopsin to function as FRET donors 17,18 .
Here we considered an alternative approach for hybrid voltage sensor design; localization of a synthetic voltage indicator to cells of interest using genetically encoded protein tags 19,20 . We focused on enzyme-based small protein tags such as the self-modifying enzyme SNAP 21,22 -tag, and transferase-mediated labeling of the acyl carrier protein (ACP) 23,24 -tag, as these technologies allow for rapid, irreversible labeling, and are compatible with in vivo imaging 25,26 . For the VSD component we found that derivatives of Nile Red, an environment-sensitive ('fluorogenic') dye that shows fluorescence enhancement upon transition from aqueous to hydrophobic solvent 27,28 , register membrane potential with high fidelity. We named the resulting voltage sensor Semisynthetic Tethered Voltage Indicator 1 (STeVI1).
We initially investigated the voltage sensitivity of the Nile Red-derivative NR12S (Fig.1a), a fluorogenic probe which contains a zwitterionic group and hydrocarbon chain, and has been used to monitor lipid order 29,30 . NR12S readily labeled the membranes of HEK293T cells with a signal-to-noise ratio (SNR) of 10.45±1.3 under wash-free conditions (SI Fig. 1 and SI Fig.4a). To quantify its voltage sensitivity, we used whole cell voltage clamp to control membrane potential, and simultaneously recorded the fluorescence signal from the membranes at an illumination power of 12 mW/mm 2 . Upon application of rectangular depolarizing voltage steps of various magnitudes from a -60 mV holding potential, fluorescence signal decreased linearly in the physiological range of membrane potential (R 2 =0.97, Fig.1b). Voltage sensitivity expressed as fractional fluorescence change F/F% (normalized to fluorescence at a holding potential of -60 mV) achieved -5.1 ± 0.4 % per 100 mV (n= 10 cells). NR12S fluorescence responded to applied voltage steps with a mean rise time on of 1.9 ± 0.4 ms and a weighted decay time off of 1.9 ± 0.2 ms (the fast component represented >85% of response).
We further enquired whether NR12S could report action potentials in dissociated dorsal root ganglion (DRG) sensory neurons. Imaging at 333 fps allowed for detection of current injection-triggered action potentials in single trials, with a F/F% per action potential of -1.9 ± 0.3% (n=4 neurons) corresponding to a peak SNR of 9.5 ± 1.6 ( Fig. 1c-d). Because of the large dynamic range of the probe and its fast kinetics, NR12S fluorescence signals closely followed the action potential shape, permitting the optical monitoring of sub-and suprathreshold neuronal events. Moreover, the orange emission of membrane-bound NR12S (max = 581 ± 1 nm in live cells, SI Fig. 2), favors this fluorophore over green-emitting VSDs and GEVIs for use in vivo. Fluorescence responses in a typical NR12S-labeled HEK293T cell subjected to 500 ms-long square voltage steps of various magnitudes. Responses were normalized to fluorescence at the −60-mV holding potential. Line colors match the colors of data points at the main graph. c) Typical NR12S fluorescence response to single trial recordings of action potentials DRG neurons triggered by current injections of incrementing amplitude (80 ms, 80 -560 pA, top trace). d) NR12S response to single trial recordings of action potentials in DRG neurons triggered by current injection (80 ms, 600 pA). Full width at half-maximum of the action potential of the voltage trace (left) was 4.5 ms and of its fluorescence readout was 6.3 ms (right). Fluorescence was recorded every 5 ms for b) and 3 ms for c) -d). Light power density was 12 mW/mm 2 . Cells were labeled at 500 nM of NR12S for 7 mins at room temperature.
We next asked whether Nile Red derivatives could be genetically localized to cells of interest through binding to a protein tag (Fig.2a). We first synthesized Nile Red derivatives that bind to SNAP-tag, and expressed the corresponding tag on the extracellular surface of HEK293T cells via a glycosylphosphatidylinositol (GPI) anchor signal sequence (SI Fig. 3a). Polyethylene glycol (PEG) linkers of n=11 repeats (4.8 nm) and charged groups were introduced in the molecules to improve water solubility and reduce nonspecific interactions 31,32 (SI Scheme 1). Although compounds specifically labeled membranes of cells expressing SNAP-tag, negligible voltage sensitivity was observed as tested by simultaneous patch clamp and imaging (SI Fig. 3c-d). Measurements of emission spectra of the compounds from live cells revealed red-shifted fluorescence in comparison to NR12S (SI Table 1), suggesting that compounds were probing hydrophobic surfaces 33 of the protein tag rather than the membrane environment.
To resolve this issue, we reasoned that a smaller tag may give the probe better access to the membrane electric field. We selected the ACP-tag (8-kDa) and synthesized Nile Red-Coenzyme A (CoA) conjugates with variable PEG repeats (n=5, 2.5 nm; n=11, 4.8 nm; Fig. 2b, SI Scheme 2). The ACP-tag was expressed on the surface of HEK293T cells via a GPI anchor, and functionality was verified by labeling with CoA-ATTO532 in the presence of phosphopantetheinyl transferase (SFP-synthase, SI Fig. 4d). Nile Red compounds also specifically labeled ACP-GPI expressing HEK293T cells with minimal background (Fig. 2c, SI Fig. 4), SNR of the membrane fluorescence over background under wash-free conditions were 10.7±1.5 for CoA-PEG11-NR and 12.4 ±1.3 for CoA-PEG5-NR. Typical fluorescence responses of CoA-PEG11-NR in a HEK293T cell subjected to a 500 ms-long square voltage steps of various magnitudes. Responses were normalized to fluorescence at the −60-mV holding potential. Line colors correspond to different membrane voltages. d) Fluorescence response of CoA-PEG 11 -NR bound to ACP-GPI to rectangular voltage steps of 160 mV, applied at 10 Hz (10 ms-duration). Fluorescence recorded every 5 ms for c) and 2 ms for d).
ACP-targeted compounds displayed a significant voltage sensitivity in HEK293T cells which, similar to NR12S, was almost linear (R 2 =0.97 and R 2 =0.96 for PEG11 and PEG5; Fig. 2c and SI Movie 1). Fractional fluorescence change per 100 mV was -5.5 ± 0.4 % and -4.9 ± 0.3 % for the CoA-PEG5-NR and CoA-PEG11-NR compounds (n=9 and n=8 cells). This is comparable to the voltage sensitivity of the non-targetable hemicyanine dye di-4-ANEPPS 34 . We tested the ability of ACP-tag targeted probes to detect trains of action potential-like voltage steps in HEK293T cells (Fig.2d). The kinetics of the CoA-PEG11-NR fluorescence response to applied voltage steps was fast, with a mean rise time on=1.8 ± 0.2 ms and a weighted decay time off = 2.6 ± 0.1 ms (the fast component represented >85% of the response).
We next investigated voltage sensitivity of CoA-PEGn-NR probes in isolated DRG neurons. ACP-GPI was expressed via recombinant adeno-associated virus (AAV)-mediated gene delivery, and functionality was verified by labeling with CoA-TMR (SI Fig. 5). STeVI1 compounds selectively labeled the neuronal membranes of cell body and axons with negligible intracellular signal (Fig.3a) and no toxicity. Neurons were patch clamped, and action potentials were evoked via current injection. CoA-PEGn-NR compounds tracked single and trains of action potentials in the cell body (Fig.3b,c, SI Fig. 6, SI Movie 2). F/F% amplitudes per action potential reached -2.0 ± 0.3 % for PEG11 and -2.2 ± 0.3 % for PEG5 respectively (n=5 neurons). Corresponding action potential peak SNR were 13.8 ± 1.5 for PEG11 and 16.2 ± 3.0 for PEG5. ACP-GPI expression and probe insertion in the membrane did not cause significant differences in action potential amplitude and duration, or membrane capacitance between control and labeled neurons (One-Way ANOVA, p=0.11, p=0.72 and p=0.58). (80 ms, 80 pA). Full width at half-maximum of the action potential of the voltage trace (top) was 2 ms and of its fluorescence readout was 5 ms (bottom). c) Representative single-trial recordings of current-triggered injection (80 ms, 80 pA) action potentials in DRG neurons with 2 M CoA-PEG11-NR probe bound to ACP-GPI. Fluorescence recorded at 500 fps. d) Fluorescence change vs. membrane potential (mean ± standard deviation) displays the linearity of the voltage sensitivity in neurons (data from the trace in c). Fluorescence changes corresponding to membrane voltage binned to 10 mV intervals were averaged and then fitted with a linear function.
Importantly, in neurons Nile Red fluorescence reported voltage changes linearly (R 2 =0.94, slope = -0.027; Fig. 2d). Fluorescent traces from cell membranes closely mimicked the shape of electrophysiological recorded action potentials, as evidenced by the close match of full width at half-maximum between the fluorescent and electrical traces ( Fig. 1c and 3b). Indeed, from the voltage imaging, it was possible to discern the inflection on the falling phase of action potentials that is indicative of nociceptive neurons 35 (see for example SI Fig. 6). We further investigated whether the Nile Red probes would allow for the detection of spontaneous activity in neuronal processes. Wide-field fluorescence imaging of neurons labeled with CoA-PEG11-NR faithfully reported spontaneous spikes at the level of cell body and axons in single-trial optical recordings (Fig. 4, SI Movie 4). F/F amplitude per spontaneous spike at 10 Hz was -2.3 ± 0.1 % at the cell body membrane corresponding to a peak SNR of 7.6 ± 0.1. In conclusion, we demonstrate that Nile Red derivatives display an intrinsic voltage sensitivity that can be exploited to monitor membrane potential in genetically tagged cells. STeVI1 probes were able to detect the shape of subthreshold depolarizations and fast neuronal activity with sensitivity comparable to GEVIs, but required 2-fold less light power (comparison data and references in SI Table 2). Key to the success of the approach was the small size of the ACP-tag which allowed for positioning of the Nile Red in the membrane environment. In future applications, this small size may also enable insertion of the ACP-tag in exposed loops of channels or other membrane proteins, to direct expression to defined subcellular compartments. | v3-fos-license |
2017-08-03T01:47:01.011Z | 2016-11-02T00:00:00.000 | 2238423 | {
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} | pes2o/s2orc | Ryanodine-induced vasoconstriction of the gerbil spiral modiolar artery depends on the Ca2+ sensitivity but not on Ca2+ sparks or BK channels
Background In many vascular smooth muscle cells (SMCs), ryanodine receptor-mediated Ca2+ sparks activate large-conductance Ca2+-activated K+ (BK) channels leading to lowered SMC [Ca2+]i and vasodilation. Here we investigated whether Ca2+ sparks regulate SMC global [Ca2+]i and diameter in the spiral modiolar artery (SMA) by activating BK channels. Methods SMAs were isolated from adult female gerbils, loaded with the Ca2+-sensitive flourescent dye fluo-4 and pressurized using a concentric double-pipette system. Ca2+ signals and vascular diameter changes were recorded using a laser-scanning confocal imaging system. Effects of various pharmacological agents on Ca2+ signals and vascular diameter were analyzed. Results Ca2+ sparks and waves were observed in pressurized SMAs. Inhibition of Ca2+ sparks with ryanodine increased global Ca2+ and constricted SMA at 40 cmH2O but inhibition of Ca2+ sparks with tetracaine or inhibition of BK channels with iberiotoxin at 40 cmH2O did not produce a similar effect. The ryanodine-induced vasoconstriction observed at 40 cmH2O was abolished at 60 cmH2O, consistent with a greater Ca2+-sensitivity of constriction at 40 cmH2O than at 60 cmH2O. When the Ca2+-sensitivity of the SMA was increased by prior application of 1 nM endothelin-1, ryanodine induced a robust vasoconstriction at 60 cmH2O. Conclusions The results suggest that Ca2+ sparks, while present, do not regulate vascular diameter in the SMA by activating BK channels and that the regulation of vascular diameter in the SMA is determined by the Ca2+-sensitivity of constriction.
Background
Cochlear function is sensitive to dynamic changes in cochlear blood flow that is responsible for the delivery of oxygen and glucose and the removal of CO 2 [1,2]. Regulation of cochlear blood flow is essential for hearing and is important as a treatment strategy for the restoration of hearing loss in humans [3][4][5][6][7][8]. Homeostatic regulation of blood flow in the cochlear capillary beds is achieved by the dynamic adjustment of the vascular diameter or "tone" of pre-capillary arteries and arterioles against systemic changes of pressure, nerve and metabolic activity [9][10][11][12][13][14]. The mechanisms involved in such regulation of the spiral modiolar artery, the principal artery of the cochlear blood supply, remain to be elucidated.
Smooth muscle cells of most arteries exhibit "Ca 2+ sparks", which are transient local elevations of Ca 2+ caused by the opening of ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) [15,16]. In most smooth muscle cells, Ca 2+ sparks activate BK channels, leading to membrane hyperpolarization, reduced activity of L-type voltage-dependent Ca 2+ channels (VDCCs), decrease in [Ca 2+ ] i and smooth muscle relaxation [15,[17][18][19]. Thus, the triad of Ca 2+ sparks, VDCCs and BK channels effectively regulates intracellular Ca 2+ to oppose vasoconstriction and maintain blood flow to the underlying tissue. Activation of BK channels by Ca 2+ sparks is a potent vasodilatory mechanism to regulate SMC global [Ca 2+ ] i and vascular diameter and a prominent feature in blood vessels of the cerebral, kidney, mesenteric and cardiac microcirculation [17][18][19][20][21].
We have recently demonstrated Ca 2+ sparks in smooth muscle cells of the intact SMA [22]. In this study, we investigate whether Ca 2+ sparks regulate the global Ca 2+ and vascular diameter in the SMA. Our results demonstrate that Ca 2+ sparks are also present in the pressurized SMA but do not regulate vasodilation of the SMA by activating BK channels. Instead, the effects produced by ryanodine, which eliminates Ca 2+ sparks, are dictated by the pressuredependent changes in the Ca 2+ sensitivity of contraction.
Ethics statement
All procedures involving animals were approved by the Institutional Animal Care and Use Committee at Kansas State University (IACUC#: 2961 and 3245).
Isolation of the spiral modiolar artery (SMA)
Female gerbils between the ages of 4 to 12 weeks (Charles River, Wilmington, MA) were anesthetized with tri-bromo-ethanol (560 mg/kg i.p.) and sacrificed by decapitation. Auditory bullae were harvested and the spiral modiolar arteries (SMAs) separated from the cochlea by microdissection in HEPES-buffered physiological saline solution (PSS) at 4 C°.
Pressurization and superfusion
Segments of the SMA were pressurized and perfused in a custom-built bath chamber using a variable hydrostatic pressure column connected to a motorized set of concentric glass pipettes (Wangemann Instruments, Kansas State University, KS) mounted on an inverted microscope (Axiovert 200, Carl Zeiss, Göttingen, Germany) [12]. Briefly, arteries were held by a holding pipette and luminally perfused with a perfusion pipette at one end while the other end was occluded using a blunt glass pipette. All pipettes were prepared using a custom-built micro-forge. The pressurized vessel was superfused in the bath with either HEPES-buffered PSS at a rate of 1.6 ml/min, permitting one complete exchange of the bath volume (~70 μl) within~3 s. Experiments were conducted at 37°C. Bath temperature was maintained by a triple heating system consisting of regulating the temperatures of the superfusate (8-line heater, CL-100, Warner Instruments, Hamden, CT, USA), the bath chamber (TC 324B, Warner Instruments) and the microscope objective (TC 324B, Warner Instruments).
Measurements of cytosolic Ca 2+ signals
Cytosolic Ca 2+ signals in SMCs were monitored as spatial and temporal changes in the fluorescence intensity of the indicator dye fluo4. For loading the dye, pressurized vessel segments were incubated in 2.5 μM fluo4-AM (Invitrogen, Carlsbad, CA, USA) for 15 min at 37°C , followed by wash and superfusion with HEPESbuffered PSS. The dye loaded virtually exclusively into SMCs. Fluo4 was excited by a 488 nm argon laser. Fluorescence emissions were filtered by a 488 notch and two long-pass filters (490 nm and 505 nm) and recorded by a photomultiplier through an open pinhole (LSM 510 Meta, Carl Zeiss).
Ca 2+ sparks
Ca 2+ sparks in SMCs of pressurized SMA were detected in frame scans and line scans. For frame scans, tangential images of the vascular wall (32.14 μm × 10.04 μm) were recorded using an oil-immersion objective (Plan-Neofluar 40× 1.3 N.A., Carl Zeiss) at a temporal resolution of~61 images/s (16.3 ms/frame) and a spatial resolution of 0.25 μm × 0.25 μm per pixel. Spark sites were identified using custom-designed software, SparkAn, developed by Dr. Adrian D. Bonev (University of Vermont, VT, USA) in IDL 5.0.2 (Research Systems, Boulder, CO) and kindly provided for use by Dr. Adrian D. Bonev and Dr. Mark T. Nelson (University of Vermont). Ca 2+ sparks were detected by dividing an area spanning 2.01 μm (8 pixels) × 2.01 μm (8 pixels) in each frame by a baseline (F 0 ) that was obtained by averaging 10 frames without Ca 2+ spark activity. Spark traces and 2-dimensional pseudo-color images were obtained as F/F 0 . 3-dimensional ratio images were generated by SparkAn.
Line-scan recordings of 5 s duration each were performed at a Ca 2+ spark site to determine the temporal parameters of Ca 2+ sparks in SMCs. Lines (0.15 μm × 12.4 μm) were recorded using an oil-immersion objective (Plan-Neofluar 40× 1.3 N.A.) at a temporal resolution of~521 lines per second (0.82 ms per line). For spark measurements at different pressures, three 5 s line-scans were performed first at 60 cmH 2 O, followed by three line-scans at 40 cmH 2 O, followed by three line scans at 60 cmH 2 O. Time intervals between consecutive linescans were 15 s to allow for recovery. Time intervals between pressure-changes were 45 s. For experiments in ryanodine and tetracaine, 10 μM ryanodine or 100 μM tetracaine was introduced after the third line-scan in PSS and scans were resumed after 2 min. Recordings were analyzed as described earlier [11]. For presentation, a single 5 s line-scan image was contrast-enhanced to highlight the occurrence of Ca 2+ spark events.
Determination of length and height of smooth muscle cells
To calculate Ca 2+ spark density, cell length and cell height of single SMCs were estimated from scanned images of pressurized SMA loaded with BCECF (Sigma-Aldrich). SMC length and height were determined to be 132 ± 17 μm and 3.2 ± 0.1 μm based on images of 9 vessels that each covered 20 -30 cells. These values correspond to a cell volume of~1 pl, which is consistent with SMCs from other vessels [15].
Simultaneous measurements of vascular diameter and global cytosolic Ca 2+
Pressurized vessels (40 or 60 cmH 2 O), loaded with the indicator dye fluo4 as described above, were superfused with HEPES-buffered PSS. To record the inner diameter of pressurized vessels simultaneously with changes in the global cytosolic Ca 2+ in SMCs, images (225 μm × 225 μm) were recorded using an oil-immersion objective (Plan-Neofluar 40× 1.3 N.A.) with a temporal resolution of 1 image/s (983 ms/frame) and a spatial resolution of 0.44 μm × 0.44 μm per pixel. In addition to filtering and recording fluorescence emissions as described above, the transmitted light from the argon laser was detected by a second photomultiplier. Inner diameter was measured by automatic edge detection by a method developed and described in Reimann et al. 2011 [12]. Inner diameter (ID) was detected from acquired real time transmitted light images, using a custom written data acquisition program (Dr. W. Gil Wier, University of Maryland). Edge detection data were analyzed using a custom written analysis program in Origin 6.0 (Dr. P. Wangemann, Kansas State University). Inner diameter changes were normalized against the average of 30 data points obtained in PSS at the beginning of the experiment (basal vascular tone). Fluorescence intensity values from 5-10 SMCs per pressurized vessel were averaged and normalized between the fluorescence values in Ca 2+ free solution and the fluorescence value in PSS before the addition of drugs according to the formula Norm 2+ ] PSS is marked in Figs. 2b, 3b, 3e, 4b, 5b, and 7b. a
Ca 2+ sensitivity
Simultaneous measurements of diameter and global cytosolic Ca 2+ were performed to determine the Ca 2 + -sensitivity of constrictions. In these experiments, following equilibration in HEPES-buffered PSS for 15 min, arteries were superfused with saline solutions containing 0, 1, 3 and 10 mM Ca 2+ in 2 min steps, first at 60 cmH 2 O followed by the same protocol at 40 cmH 2 O. Data points for concentration curves were obtained by averaging diameter and fluorescence intensity measurements over the last 30s of each Ca 2+ step and normalizing against the average value obtained in PSS at 60 cmH 2 O. Data points from individual vessels were fitted to a modified Hill equation:
Results
Ca 2+ sparks in the pressurized spiral modiolar artery Ca 2+ sparks and waves were recently reported in the intact unpressurized gerbil SMA [22]. We now report Ca 2 + sparks and Ca 2+ waves in SMCs of the pressurized gerbil SMA (Fig. 1). Ca 2+ spark sites were consistently observed in frame scans (Fig. 1a), with super Ca 2+ sparks, of larger cross-sectional area and longer duration of elevated Ca 2+ observed occasionally, which may reflect the combined synchronized activity of two or more closely spaced Ca 2+ sparks sites. The average spatial area of spark sites was 14 ± 3 μm 2 , corresponding to a spatial width of~4 μm. Two out of 13 recorded Ca 2+ spark sites had larger spatial areas between 25 -50 μm 2 (Fig. 1b). An average of 1.3 ± 0.2 Ca 2+ spark sites were present in 200 ± 10 μm 2 of recorded area (n = 10), corresponding to~47 % of one cell, giving a spark density of 2.8 ± 0.3 spark sites/cell. The frequency of Ca 2+ spark occurrence per site and other temporal parameters were measured in pressurized SMA using line-scans (Fig. 1c). Spark frequency per site increased from 0.6 Hz to 0.9 Hz, as pressure increased from 40 cmH 2 O to 60 cmH 2 O (Table 1). However, increasing pressure from 40 to 60 cmH 2 O did not alter the spark amplitude, the rise-time or the half-time of decay (Table 1). Ca 2+ sparks were completely eliminated in the presence of 10 μM ryanodine (Fig. 1c) and significantly decreased in frequency in the presence of 100 μM tetracaine (Fig. 1d). Ca 2+ oscillations exhibiting wave-like phenomena were also observed in SMCs and were likewise abolished by application of 10 μM ryanodine (Fig. 2a).
Effects of inhibitors of Ca 2+ sparks and BK channels on global Ca 2+ and vascular diameter of the SMA In most arteries, inhibition of Ca 2+ sparks and/or BK channels has been shown to increase SMC global Ca 2+ to cause a robust vasoconstriction in a non-additive fashion, reflecting that Ca 2+ sparks and BK channels are part of the same mechanism to hyperpolarize the membrane and limit Ca 2+ influx, leading to vasorelaxation [17,18,20]. In the pressurized (40 cmH 2 O) SMA, application of 10 μM ryanodine inhibited Ca 2+ sparks and appeared to similarly increase the average global cytosolic Ca 2+ followed by a robust vasoconstriction (Fig. 2). However, application of 100 μM tetracaine, another known inhibitor of ryanodine receptors and Ca 2+ sparks [24], or application of 100 nM iberiotoxin, a potent BK channel inhibitor, did not cause any change in global Ca 2+ or vascular diameter similar to that produced by ryanodine (Fig. 3). Activation of BK channels is dependent on the local [Ca 2+ ] i as well as the membrane potential of the smooth muscle membrane [25]. It is possible that lack of an effect of iberiotoxin is a consequence of unopened BK channels caused by a hyperpolarized resting membrane potential in the smooth muscle cells of the SMA. To account for such a possibility, the pressurized SMA was superfused with PSS solution containing 30 mM K + . High K + induced a transient increase in the global [Ca 2+ ] i and vasoconstriction. Under these conditions, 100 nM iberiotoxin remained without effect (Fig. 4). Furthermore, contrary to the effect at 40 cmH 2 O, 10 μM ryanodine increased global Ca 2+ modestly and did not constrict the SMA pressurized at 60 cmH 2 O (Fig. 5), even though spark frequency is increased significantly from 40 to 60 cmH 2 O (Table 1).
These effects suggest that, unlike cerebral arteries, the ryanodine-induced increase in global Ca 2+ and constriction at 40 cmH 2 O in the SMA may not be attributed to a loss of the hyperpolarizing influence of the Ca 2+ spark-BK channel signaling mechanism. Under these conditions, ryanodine-sensitive Ca 2+ sparks do not appear to regulate global Ca 2+ and vascular tone via BK channels in the SMA. This raises the question as to the mechanism involved in the ryanodine-induced increase in the global Ca 2+ and vasoconstriction. It is to be expected that at least a portion of the global Ca 2+ is the result of Ca 2+ influx via voltage-dependent Ca 2+ channels (VDCCs), which are open at the physiological resting membrane potential in SMCs. Evidence for active VDCCs in the SMA comes from the observation of a decrease in global Ca 2+ upon application of a reversible VDCC inhibitor, 2 μM nifedipine, in the presence of 100 μM tetracaine ( Fig. 3d and e). It is possible that the remainder of the ryanodine-induced increase in the global Ca 2+ is the result of ryanodine receptor-mediated Ca 2+ release from the sarcoplasmic reticulum (SR). It is well-established that at a concentration of 10 μM, ryanodine binds to open ryanodine receptors and modifies the channel to lock them in an irreversible sub-conductance state of 234 pS [16,26] that inhibits Ca 2+ release and instead "leaks" SR Ca 2+ into the cytosol. This is reflected in the transient increase in global Ca 2+ immediately upon application of 10 μM ryanodine, followed by a slowly decaying plateau phase devoid of Ca 2+ oscillations, indicating the relatively slow emptying of the SR through the partially open ryanodine receptors ( Fig. 2a and b).
It is to be noted that the ryanodine-induced vasoconstriction continues to increase as the corresponding average global Ca 2+ plateaus and then decreases (Fig. 2b and c). Indeed, the maximum vasoconstriction corresponds to the least increase in global Ca 2+ induced by ryanodine, suggesting an increase in the Ca 2+ sensitivity Diameter changes were normalized to the average of values recorded between 30 -60 s (average value indicated by 'a' was set to 1). [Ca 2+ ] i and diameter data were simultaneously acquired at 1 s intervals, however, for clarity, error bars (sem) are plotted only every 10 s of constriction following the initial increase in the global Ca 2+ . In other words, the constriction induced by ryanodine at 40 cmH 2 O may be attributed to enhanced Ca 2+ sensitivity of the SMA that is able to respond to the ryanodine-induced increase in intracellular Ca 2+ with vasoconstriction. The observation that the ryanodineinduced constriction at 40 cmH 2 O is enhanced (Fig. 2c) compared to that at 60 cmH 2 O (Fig. 5c) concentration was manipulated by altering the Ca 2+ concentration in the superfusate ( Fig. 6a and b). Normalized cytosolic Ca 2+ and corresponding vascular diameter measurements were plotted against each other and fitted to the Hill equation. A decrease in the pressure from 60 to 40 cmH 2 O shifted the Ca 2+ -diameter relationship to the left on the Ca 2+ axis, indicating a dramatic increase in the Ca 2+ sensitivity, with nearly 2-fold decrease in the Ca 2+ required for a half-maximal constriction at 40 cmH 2 O compared to that at 60 cmH 2 O (Fig. 6c), whereas a time control repeated at 60 cmH 2 O did not (Fig. 6d). Thus, the modest increase in global Ca 2+ caused by ryanodine at 60 cmH 2 O was insufficient to constrict the SMA at this pressure, whereas the enhanced Ca 2+ sensitivity at 40 cmH 2 O allowed for a robust constriction for an increase in global Ca 2+ .
Endothelin enhances the ryanodine-induced vasoconstriction
The result above implies that conditions that increase the Ca 2+ sensitivity at 60 cmH 2 O would increase the ryanodine-induced vasoconstriction. Consequently, the SMA pressurized at 60 cmH 2 O was first exposed to 1 nM endothelin-1 (ET-1) for 1 min. It has been previously shown that endothelin-1 acts via ET A receptors to increase the Ca 2+ sensitivity of the SMA in a rho-kinase dependent manner [27]. ET-1 caused a transient increase in the cytosolic Ca 2+ concentration and a persistent vasoconstriction, consistent with an increase in the Ca 2+ sensitivity (Fig. 7). Under these conditions, 10 μM ryanodine caused a vasoconstriction that was enhanced compared to that observed in the absence of ET-1 (Fig. 5). These results support the concept that ryanodine increases global Ca 2+ and constricts the SMA when the Ca 2+ sensitivity is high. Ca 2+ sensitivity of SMC contraction is hence a critical factor in the regulation of the vascular diameter of the SMA in response to changes in pressure and cytosolic global Ca 2+ .
Discussion
Salient findings of the present study are 1) Ca 2+ spark frequency in the pressurized SMA increases with pressure; 2) Inhibition of Ca 2+ sparks with ryanodine increases global Ca 2+ and causes a robust vasoconstriction, however, ryanodine-induced effects on global Ca 2+ and vascular diameter are not reproduced by other inhibitors of Ca 2+ sparks or by inhibitors of BK channels as would be expected if Ca 2+ sparks activated BK channels to regulate membrane potential, global Ca 2+ and vascular diameter.
3) The ryanodine-induced vasoconstrictions depends on the Ca 2+ sensitivity, which is higher at 40 cmH 2 O compared to that at 60 cmH 2 O and can be enhanced with endothelin-1.
Ca 2+ sparks
Ca 2+ sparks in the pressurized SMA occurred with a lower frequency than in unpressurized SMA [22], but with a similar frequency, spatial width and spark site density as observed in smooth muscle cells of cerebral pial arteries [15,19,28] and pressurized mesenteric arteries [21,29]. The increase in Ca 2+ spark frequency in response to a 20 cmH 2 O (~14 mmHg) increase in pressure, is also consistent with observations made in cerebral arteries [28]. However, as in unpressurized SMA, Fig. 7 Inhibition of Ca 2+ sparks with ryanodine increases global Ca 2+ and constricts SMA at 60 cmH 2 O following an increase in Ca 2+ sensitivity by endothelin-1. a Representative recordings of cytosolic Ca 2+ changes from single smooth muscle cells from a SMA loaded with fluorescent dye fluo4 and pressurized to 60 cmH2O in response to 10 μM ryanodine after treatment with 1 nM endothelin-1 (ET-1). b Average of normalized traces of cytosolic Ca 2+ changes at 60 cmH 2 O (64 cells from 7 arteries). Traces in a and b were normalized as described in Methods. c Average trace of corresponding changes in vascular diameter of SMA pressurized at 60 cmH 2 O (15 arteries). Diameter changes were normalized to the average of values recorded between 30-60 s (value indicated by 'a' was set to 1). Ca 2+ and diameter data were simultaneously acquired at 1 s intervals, however, for clarity, error bars (sem) are plotted only every 10 s the time of half decay of Ca 2+ sparks was far shorter (~17-19 ms) than that observed for Ca 2+ sparks in cerebral arteries, but closer to that found in rat heart [16,30]. Ca 2+ spark amplitudes and decay times are generally a reflection of the number as well as the isoform of ryanodine receptors (RyRs) present in a spark cluster. Typically, Ca 2+ spark sites are composed of 4 -6 RyRs, giving a punctate staining pattern in immunolocalization studies. In the SMA, the distribution pattern of RyRs in SMCs shows a uniform expression throughout the SR rather than a punctate expression expected of ryanodine receptors clustered in spark sites [22] and may underlie the observed differences in the temporal properties and functional role of Ca 2+ sparks in the SMA.
Absence of the Ca 2+ spark/BK channel hyperpolarizing mechanism in the SMA The Ca 2+ spark/BK channel signaling complex provides an important vasodilatory mechanism in preventing or mitigating pressure-or agonist-induced vasoconstrictions in arteries. This negative feedback mechanism in regulating vascular tone is evident from observations that pharmacological inhibition of Ca 2+ sparks and/or BK channels or SMC-specific genetic manipulation of BK channel or RyR expression leads to the loss of this hyperpolarizing signal leading to membrane depolarization, increased VDCC activation, increase in Ca 2+ influx and global Ca 2+ and enhanced vasoconstriction [18,20,[31][32][33]. However, Ca 2+ sparks have not always been linked to a hyperpolarizing or vasodilatory mechanism. Other studies have observed excitatory roles for Ca 2+ sparks and RyR-mediated Ca 2+ release in small diameter arterioles. Kur et al. [24] reported that, contrary to the conventional hyperpolarizing mechanism, Ca 2+ sparks in retinal arterioles combined to form Ca 2+ waves and enhanced the myogenic tone. Westcott et al. [34] reported SMCs of murine cremaster muscle feed arterioles to express diffused staining of RyRs without manifesting Ca 2+ sparks and no coupling with BK channels, while SMCs of upstream feed arteries exhibited clustered staining of RyRs, robust Ca 2+ sparks and spatial and functional coupling to BK channels, indicating heterogeneity of RyR function within the same vascular tree. In the SMA, the effects of ryanodine on global Ca 2+ and vascular diameter at 40 cmH 2 O seemed to suggest, at first, a vasodilatory mechanism for Ca 2+ sparks acting via BK channels. However, the failure of tetracaine, an RyR inhibitor, which inhibits Ca 2+ sparks without depleting the SR, and iberiotoxin, a BK channel inhibitor, to produce similar effects on global Ca 2+ and diameter as ryanodine (Figs. 2 and 3) combined with the non-effect of BK channel inhibition following membrane depolarization by external application of high K + (Fig. 4) or increase in pressure (Fig. 5) disproves the regulation of vascular tone of the SMA by the Ca 2+ spark/BK channel mechanism.
Regulation of vascular tone by Ca 2+ sensitivity
Changes in SMC global Ca 2+ have generally been accepted as the central mechanism regulating SMC contractility in the development of pressure-dependent myogenic tone [35]. More recently, the contribution of Ca 2+ -independent processes that regulate the Ca 2+ sensitivity of the myofilament in the development of myogenic tone have been better described [36]. In cerebral and skeletal resistance arteries, increases in intravascular pressure are associated with increases in the Ca 2+ sensitivity achieved by balancing the relative activities of myosin light chain kinase and myosin light chain phosphatase in a PKC and rho-kinase-dependent manner. Such changes in Ca 2+ sensitivity further augment the Ca 2+ -dependent myogenic vasoconstrictions [36][37][38][39][40]. We have previously shown that myogenic tone in the male, but not female, gerbil SMA is regulated not by changes in the SMC global Ca 2+ but by rho-kinasemediated changes in the Ca 2+ sensitivity of contraction, which was revealed under inhibition of NO-mediated signaling [23]. The present study shows that the regulation of vascular tone in the female gerbil SMA is also determined by the Ca 2+ sensitivity of the myofilament, with the crucial difference that increase in intravascular pressure significantly lowered the Ca 2+ sensitivity (Fig. 7). This finding is consistent with the development of small myogenic tones with increasing intravascular pressures in the SMA [12]. Rho-kinase-dependent regulation of Ca 2+ sensitivity also plays a significant role in mediating the vascular effects of endogenous vasoconstrictors and agonists [41][42][43]. Further studies are required to elucidate the mechanisms underlying the relationship between pressure and Ca 2+ sensitivity in the SMA.
Conclusions
In conclusion, in this study, we have shown in the gerbil spiral modiolar artery that Ca 2+ sparks, while present, do not regulate vascular tone and global Ca 2+ by activating BK channels. Instead, ryanodine-receptor mediated increases in global Ca 2+ and vasoconstriction depend on the Ca 2+ sensitivity of SMC contraction, which is enhanced at lower pressures or by regulating rho-kinase activity.
It remains to be seen whether such mechanisms of vascular tone regulation as described in this study are applicable to spiral modiolar arteries in general or particularly unique to the gerbil spiral modiolar artery. Gerbils are commonly favored over other rodents such as mice and rats as hearing models for investigations into the causes for age-related hearing loss involving pathological changes in both peripheral and central auditory system components. Gerbils are uniquely suited for such investigations as they exhibit sensitive hearing in the low frequency ranges (below 4 kHz) that are relevant for human auditory perception, compared to the much higher thresholds in mice and rats for the same frequency range [44]. Thus, the differences observed in the regulation of the gerbil SMA by Ca 2+ sparks and BK channels compared to the observations made in arteries from other extensively studied rodent species become relevant in the choice of appropriate models for future interpretation of hearing studies and pharmacological interventions.
Funding
This study was supported by NIH-R01-DC04280 to PW and by Fortüne 2339-0-0 (University of Tuebingen, Tuebingen, Germany) to KR. The Confocal Microscopy Core facility was supported by the College of Veterinary Medicine at Kansas State University and by NIH-P20-RR017686.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article. | v3-fos-license |
2019-03-22T16:10:38.440Z | 2012-10-17T00:00:00.000 | 85002311 | {
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} | pes2o/s2orc | Effects of a UV-absorbing greenhouse covering film on tomato yield and quality
The effect of blocking the ultraviolet (UV) solar radiation using a UV-absorbing low density polyethylene (PE) film on tomato crop yield and fruit quality was evaluated in a two-year study in two arched roof greenhouses located in Central Greece. The UV-A and UV-B radiation transmission values of the greenhouse covered by the UVabsorbing PE film during the first year were 0.4% and 1.2%, respectively and increased to 0.8% and 1.3% in the 2nd year, while the respective values in the greenhouse covered by a traditional PE film were 20.7% and 12.5% during the 1st year and 28.7% and 26.7% during the 2nd year. Under the UV-absorbing film the number of insect injured fruit was reduced and the marketable yield was similar or higher than that under the common PE film, while fruit quality characteristics (size, shape), nutritional value (ascorbic acid and lycopene) and organoleptic quality (pH, titratable acidity and total soluble solids) were similar under both covering materials. Moreover, the reduction of incoming UV radiation had an appreciable effect on fruit skin color, indicating an effect on pigments other than lycopene. Additional key words: fruit quality; insects; polyethylene; transmission; ultraviolet radiation; UV-stabilizers.
Lycopene content and ascorbic acid are crucial quality parameters (Giuntini et al., 2005) but while ascorbic acid is present in all vegetables (Davey et al., 2006); lycopene is found only in red tomato and watermelon [Citrullus lanatus (Thunb.)]fruit (Bramley, 2000).Lycopene is considered one of the most important carotenoids in European and North American diet (Giuntini et al., 2005) and has attracted considerable interest as a health promoting phytochemical (Tadmor et al., 2005).
The aim of this work was to study the effects of UV-absorbing greenhouse covering films on fruit yield and quality characteristics of a common tomato cultivar (Solanum lycopersicum L., cv.Belladonna) in Greece.
Greenhouse facilities and plant material
The experiments were conducted during spring and summer 2003 and 2004 (transplanting at the end of February until July) in two similar arched roof greenhouses, N-S oriented, located at the University of Thessaly farm (39°22'N 22°44'E, altitude 85 m), on the continental area of Eastern Greece.The geometrical characteristics of each greenhouse were as follows: eaves height of 2.9 m; ridge height of 4.1 m; total width of 8 m; total length of 20 m; ground area of 160 m 2 , and volume of 524 m 3 .The greenhouses were equipped with two continuous side roll-up windows located at a height of 0.6 m above the ground with a maximum opening area of 27 m 2 (two vents of 15 m length × 0.9 m opening height) for both vents.A flap roof window was also located longitudinally on the whole greenhouse roof (20 m long) with 0.9 m maximum opening height (18 m 2 opening area).The vents were controlled automatically via a controller (Macqu, Geometions SA, Athens, Greece) and opened in steps; they began to open when greenhouse air temperature exceeded 22°C, and reached their maximum aperture when temperature reached 25°C.
Introduction
Greenhouses create an ideal environment not only for crop but also for pest and disease development.Chemicals pest and disease control are common practice for fresh vegetable protection.As an alternative, many growers use insect proof screen use, placing them in the ventilators.Thus, screening reduces ventilation (Katsoulas et al., 2006;Teitel, 2007) making the high internal temperature of Mediterranean greenhouses during summer even worse.
A complement to using insect screens is the use of a UV-absorbing film for greenhouse covering, which creates a light environment unfavourable to harmful insects.UV-absorbing films do not only block insect pests but also reduce spread of insect-borne viruses (Raviv & Antignus, 2004).Furthermore, UV-absorbing films can reduce crop diseases caused by a range of fungi that use UV as an environmental cue for sporulation (Nigel et al., 2005).
It is well documented that UV-absorbing films suppress several foliar diseases (Raviv & Antignus, 2004), but their impact on crop yield and quality still needs investigation.In Israel, no significant differences were found on growth, yield, maturation time and fresh and dry weight values of tomatoes (Solanum lycopersicum L.) grown under UV-absorbing films (Raviv & Antignus, 2004).In studies carried out in Spain, an increase in tomato yield was reported when UV-absorbing films were used (González et al., 2004;Monci et al., 2004).Kittas et al. (2006) compared the effect of UV-absorbing films on eggplant (Solanum melongena L.) crop behaviour and production.In the absence of UV radiation eggplants were taller (21%), with a larger leaf area (17%) and produced more marketable fruit yield.The adoption of UV-absorbing films by growers as an alternative technique to chemical pest and disease control seems promising as long as fruit yield and quality and concomitantly the anticipated economic return is not negatively affected.
In terms of nutritional quality, it is well established that light affects lycopene content (Cox et al., 2003) as well as ascorbic acid (Giuntini et al., 2005) and other compounds that contribute to fruit composition.Some authors report that UV radiation affects plant secondary metabolism by restricting the production of UV-absorbing compounds including flavonoids and other phenolics (Allen et al., 1998;Caldwell et al., 2003).These compounds significantly affect fruit composition and therefore fruit nutritional quality.Effects of UV-absorbing covers on tomato quality or 18°C during the day and interrupted it at 16°C during the night or at 20°C during the day.Greenhouse soil was covered by a white on black plastic sheet.
One greenhouse was covered by a common low density PE film (C-PE, Plastika Kritis S.A., Heraclion, Crete, Greece) and the second one by a UV-absorbing PE film (UV-PE, Plastika Kritis S.A., Heraclion, Crete, Greece).Both films had 180 μm thickness and equal amounts of infrared, diffusion and ethyl-vinyl-acetate additives but had different UV-absorbers content in the main PE master batch formation, which resulted in differences in UV radiation transmissivity.
The tomato crop (Lycopersicon esculentum, cv.Belladonna) was transplanted on 22/02/2003 and 25/02/2004, at the stage of 5-6 true leaves in bags filled with perlite at a density of 2.4 plants m -2 .Fertigation was automatically controlled by a computer with set points for electrical conductivity of 2.1 dS m -1 and pH of 5.6.Plants were pruned to one stem, topped after the 7 th truss and treated equally with fungicides [chlorothalonil (72-75% wettable powder, wettable granules, concentrating liquid suspension)] and miticides [clotentezine (50% concentrating liquid suspension) and propargite (30% wettable powder) for Tetranychus eggs and adults respectively] application when deemed necessary.A beehive of bumblebees Bombus terrestris (L.), was installed in each greenhouse to facilitate pollination.Plants in each greenhouse were divided into four blocks having 90 plants each.
Measurements
The following climatic data were recorded outside (on a meteorological mast) and inside (centre) of each greenhouse using sensors calibrated before the experimental period: air temperature, relative humidity, global solar radiation (model Middleton EP08-E, Brunswick Victoria, AUS), photosynthetically active radiation (model LI-190SA; Lincoln, NE, USA), UV-B radiation (290-315 nm, model SKU 430, Sky instruments LTD, UK) and UV-A radiation (315-380 nm, model SKU 420, Sky instruments LTD, UK).Each sensor was scanned every 30 s and averaged every 10 min using a Delta-T data logger system (model DL3000, Delta-T devices, Cambridge, UK).
PE film spectral transmittance measurements were made in the laboratory on three samples per PE film taken before their installation in the greenhouses using a LI-COR portable spectroradiometer (model LI-1800, LI-COR, Lincoln, NE, USA) equipped with a 10 W glass halogen lamp and an external integrating sphere (model LI-1800-12S, LI-COR, Lincoln, NE, USA) (Kittas & Baille, 1998).
Fruit harvesting took place twice a week, at the light red stage of maturity, according to the classification of Grierson & Kader (1986).Harvested fruit from 24 selected plants in each greenhouse were weighed, their dimensions were measured and the total production was calculated.Fruit were sorted into marketable and rejected production.The rejected production was due to (i) physiological problems, including small fruit (weight < 100 g), fruit with defects, breaks, scars, blossomend rot and other physiological disorders; and (ii) insects damage (was assessed in the scale 1 -5: 1 = none, 2 = slight, 3 = moderate, 4 = severe, 5 = extreme).The most prevalent insect causing fruit injuries was thrips (Frankliniella occidentalis).Thrips number was monitored every week by means of 12 blue sticky HORIVER ® traps (25 × 10 cm) and measurements on ten tomato plants (top, middle, bottom leaves) in each greenhouse (Vatsanidou et al., 2009).
Two measuring procedures were used for fruit skin colour measurements.The first one was applied both years (2003,2004) recording colour of all harvested mature fruit.The second one, applied only during 2004 experimental period, included measurement of fruit on the vein for colour evolution evaluation through the six maturity stages (green, breaker, turning, pink, light red and red).Forty two non-shaded, randomly selected fruit of the same truss with uniform shape and size were labelled in each greenhouse and their colour was measured every 1 to 3 days, according to severity of colour change, from green maturity stage until fully ripe fruit.Colour was measured by a Miniscan TM XE Plus (Hun-terLab, Hunter Associates Lab, Inc., Reston, VA, USA) tristimulus colour analyzer and colour around fruit equatorial region was recorded (6 measurements per fruit).Measurements were reported in the L*, a*, b* system [CIELAB, L* varies between light (L* = 100) and dark (L* = 0), a* varies between green (a* = -50) and red (a* = 50), and b* varies between yellow (b* = 50) and blue (b* = -50) colour].Chroma and hue (h°) values were also calculated (McGuire, 1992).
Fresh fruit were macerated in a blender for titratable acidity, total soluble solids, ascorbic acid and lycopene determinations.Total soluble solids (TSS) content was measured using a refractometer (model PR-1, Atago, Tokyo, Japan).Titratable acidity was measured by titra-tion with 0.1 N NaOH to pH 8.2 and results were expressed as % citric acid.Ascorbic acid was extracted in 1% oxalic acid and measured with reflectoquant ascorbic acid test strips (RQflex 10 Reflectoquant, Merck KGaA, Darmstadt, Germany) in a reflectometer (model 116970 Reflectometer Merck KGaA, Darmstadt, Germany).Lycopene was extracted by homogenizing 1 g sample with 25 mL of acetone in a centrifuge tube shaken in the dark.Absorbance at 503 nm was measured by means of a spectrophotometer and lycopene content was calculated using the molecular extinction coefficient of 17.2 • 10 4 L mol -1 cm -1 and expressed as mg per 100 g fruit weight (Mencarelli & Saltveit, 1988).
Data analysis
Comparison of means was performed by one-way analysis of variance at a significance level of 0.05 using the SPSS statistical package (SPSS-16, Chicago, IL, USA).
Greenhouse microclimate
The average transmission coefficients for PAR, UV-A and UV-B radiation, as calculated by the ratio of inside to outside measured value of each parameter during 2003 experimental period were 74.3%, 20.7% and 12.5% in the C-PE greenhouse and 75.5%, 0.4% and 1.2%, in the UV-PE covered greenhouse, respectively.The values changed during the replicate experimental period of 2004 to 73.0%, 28.7% and 26.7% in the C-PE greenhouse and to 72.7%, 0.8% and 1.3% in the UV-PE greenhouse.These results indicate that the UV radiation absorbing properties of the UV-PE film were maintained for two summer seasons after its placement in the greenhouse.In addition, measurements conducted in the laboratory concerning films' spectral transmittance revealed that both films had similar radiation properties in blue (400-500 nm) and green to red (500-700 nm) spectrum regions during both years (data not shown).Thus, any differences that may have emerged on crop yield and fruit quality characteristics could be ascribed to differences of the covering materials in UV-radiation transmittance.According to Krizek et al. (2005) plant productivity of greenhouse crops is greatly influenced by the amount of UV radiation, photosynthetically active radiation (PAR) and IR transmitted through the covering material of these structures which is not the case in our tested films since their transmittance in blue, green to red and PAR radiation regions was the same.
Air temperature and vapour pressure deficit (VPD) data during 2003 experimental period are presented in Table 1.Temperature and VPD values followed a similar pattern during the experimental period in both greenhouses.Similar values were also found during 2004 replicate experimental period (data not shown).
The UV-radiation energy flux corresponds to about 3.8% of the total solar radiation energy flux outside the greenhouse (Robaa, 2004).Considering that the films tested differ in UV transmission by about 28%, the difference in the incoming solar energy flux is expected to be about 1% (= 28% × 3.8%), which,
VPD i (kPa)
UV-A (kJ m -2 day -1 ) Effects of UV-absorbing covers on tomato quality for an average temperature of 20°C, may induce a 0.2°C temperature difference between the two greenhouses.
Consequently, the only difference between the environments of the two greenhouses could be considered the quality of light.
Fruit yield
No significant differences (p ≥ 0.05) were found between the two greenhouses in total yield, fruit number, marketable mean fruit weight and non-marketable fruit production ought to physiological problems (meaning zipper, blossom end rot, cutface, sunscald, small size).Under the UV-PE greenhouse, a significant decrease of fruit with insect injuries was observed during both years (Table 2).This could be explained by the significantly lower number of thrips observed in the UV-PE greenhouse, as indicated by the significantly lower number of thrips caught in the sticky traps in the UV-PE greenhouse (Table 3).It has also been observed that under UV-absorbing films some crops such as tomato (González et al., 2004;Monci et al., 2004), eggplant (Kittas et al., 2006) and lettuce (Nigel et al., 2005;Diaz et al., 2006) show better performance and yield.
The majority of the reports that concern the effect of UV-absorbing films on crop, focus on pest and disease control.They measure the efficiency of UVabsorbing films on insect population and its impact on marketable yield, although Antignus et al. (1996) mention that the effect of UV-absorbing films on crop yield due to a decrease of virus transmission may be higher than the effect on yield due to reduced insect population.Park et al. (2007) found that the number of marketable fruit decreased as thrips density increased and they also suggest that thrips fruit damage should not be evaluated in terms of yield loss, but in terms of the percentage of damaged fruit.They stated that thrips caused direct damage to fruit by feeding on and laying eggs in the developing fruit causing darkness and malformation.Similar impacts on crop yield were also observed in the present study.Rheinländer et al. (2006) stated that between thirteen species of insects and mites found on tamarillo leaves and fruit, most of these were not likely to cause any damage on fruit (e.g.spiders, lacewings ladybirds and beetles) and declared that thrips was the only insect which could cause such scarring.
The cosmetic scars that were observed in the harvested fruits of the present study were similar to those presented by Rheinländer et al. (2006), according to which thrips was the major cause for fruit damages.It was found that, compared to the C-PE greenhouse, thrips' population was 70% lower in the UV-PE than in the C-PE greenhouse, during the total experimental period and that the percentage of harvested fruit affected by thrips (injury score > 2), varied from 5% to 25% during both experimental periods.
Fruit quality
Tomato specific fruit weight (g mL -1 ) was similar in the two greenhouses ranging from 1.08 to 1.16 and from 0.93 to 0.95 for 2003 and 2004 experimental periods, respectively.Fruit shape parameters, which are components of fruit quality according to the European Commission (1983), were similar in both greenhouses (Table 4).
Table 2. Marketable yield, fruit number per greenhouse area, fruit weight and rejected production due to physiological problems such as zipper, blossom end rot, cutface, sunscald, small size, and insect damage in the tomato crop grown under the greenhouse covered by the UV-absorbing (UV-PE) and the common polyethylene (C-PE) film during the two experimental periods.Values [mean ± SE (standard error)] followed by different letters within year are significantly different (p ≤ 0.05)
Year Greenhouse
Total yield (kg m -2 ) In fruit shape formation, pollinators play a major role (Al-Attal et al., 2003).As fruit shape parameters did not differ among UV environments of the present work it could be assumed that bumblebees' activity was not affected by the UV-absorbing film.
Total soluble solids (TSS), ascorbic acid, lycopene content, pH and titratable acidity values were similar under both PE-films (Table 4).TSS were increased with time as truss number also increased under both greenhouses (Fig. 1).Similar results have been also reported by Bertin et al. (2000)The influence of low UV-B radiation levels on ascorbic acid, lycopene (Giuntini et al., 2005) and TSS content (Krizek et al., 2006) depends on tomato genotype and accordingly, the results of the present study may be valid only for the tomato cultivar studied.The non significant differences in fruit ascorbic acid and TSS content that was found in the present study may indicate a neutral reaction to UV radiation reduction of the specific cultivar tested.
Colour values (L*, hue, chroma) of harvested fruit were similar under both greenhouses and experimental periods (Fig. 2A, B and C, data for 2004 are not shown).The fruit colour is mainly attributed to the β-carotene and lycopene content (Grierson & Kader, 1986).Since lycopene content was similar under both greenhouses (Table 4) it could be assumed that the observed colour differences are attributed to the differences in β-carotene content.Hue colour parameter could be used as an objective colour index for vine ripened tomato fruit (Lopez-Camelo & Gomez, 2004).In the present work, hue values did not show any difference (Figs.2B, 2E) between the two greenhouses and accordingly, it could be concluded that tomatoes produced under the UV-PE greenhouse are not inferior of those produced under the C-PE covered greenhouse.Lastly, on the vein colour evolution evaluation through the six maturity stages (green, breaker, turning, pink, light red and red) showed that fruit under the UV-PE greenhouse had significantly higher chroma values than under the C-PE during light red and red maturity stages (Fig. 2F).These differences could be attributed to differences in synthesis rate of others than lycopene pigments.
It could be concluded that use of UV-absorbing greenhouse covering films that modify the UV radiation light environment leads to reduction of number of insect injured Effects of UV-absorbing covers on tomato quality fruit and to similar or higher marketable yield, while fruit quality characteristics (size, shape), nutritional value (ascorbic acid and lycopene) and organoleptic quality (pH, titratable acidity, total soluble solids) are not affected.Therefore, UV-absorbing films can be considered as an effective practice in tomato crop protection from insect, without negative effects on fruit quality attributes.
Table 1 .
Monthly average values of solar radiation, air temperature and vapour pressure deficit measured outside and under the greenhouse covered by the UV-absorbing (UV-PE) and the common polyethylene (C-PE) film during 2003, for the period of day between 08:00 to 20:00.Standard deviation of the values is given in parenthesis
Figure 1 .
Figure 1.Evolution, along the crop cycle, of tomato fruit soluble solids content measured in the two greenhouses.Triangles: UV-PE greenhouse; circles: C-PE greenhouse.The vertical bars represent the standard error of means.
Figure 2 .
Figure 2. Skin colour values of harvested fruit (A, B, C) (experimental period 2003) and skin colour change on fruit on the vein (D, E, F) (experimental period 2004).Triangles: UV-PE greenhouse; circles: C-PE greenhouse.The vertical bars represent the standard error of means.
Table 3 .
Mean number of thrips caught on sticky traps per week under the greenhouse covered by the UV-absorbing (UV-PE) and the common polyethylene (C-PE) film during the two experimental periods.Values [mean ± SE (standard error)] followed by different letters within year are significantly different (p ≤ 0.05) | v3-fos-license |
2020-12-03T09:05:16.502Z | 2020-12-01T00:00:00.000 | 227296453 | {
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} | pes2o/s2orc | Development of an In Vitro Membrane Model to Study the Function of EsxAB Heterodimer and Establish the Role of EsxB in Membrane Permeabilizing Activity of Mycobacterium tuberculosis
EsxA and EsxB are secreted as a heterodimer and have been shown to play critical roles in phagosome rupture and translocation of Mycobacterium tuberculosis into the cytosol. Recent in vitro studies have suggested that the EsxAB heterodimer is dissociated upon acidification, which might allow EsxA insertion into lipid membranes. While the membrane permeabilizing activity (MPA) of EsxA has been well characterized in liposomes composed of di-oleoyl-phosphatidylcholine (DOPC), the MPA of EsxAB heterodimer has not been detected through in vitro assays due to its negligible activity with DOPC liposomes. In this study, we established a new in vitro membrane assay to test the MPA activity of N-terminal acetylated EsxA (N-EsxA). We established that a dose-dependent increase in anionic charged lipids enhances the MPA of N-EsxA. The MPA of both N-EsxA and EsxAB were significantly increased with this new liposome system and made it possible to characterize the MPA of EsxAB in more physiologically-relevant conditions. We tested, for the first time, the effect of temperature on the MPA of N-EsxA and EsxAB in this new system. Interestingly, the MPA of N-EsxA was lower at 37 °C than at RT, and on the contrary, the MPA of EsxAB was higher at 37 °C than at RT. Surprisingly, after incubation at 37 °C, the MPA of N-EsxA continuously decreased over time, while MPA of EsxAB remained stable, suggesting EsxB plays a key role in stabilizing N-EsxA to preserve its MPA at 37 °C. In summary, this study established a new in vitro model system that characterizes the MPA of EsxAB and the role of EsxB at physiological-relevant conditions.
Introduction
Tuberculosis (TB) is a leading infectious disease in the world that accounts for tens of millions of new cases and more than one million deaths per year [1]. Mycobacterium tuberculosis (Mtb), once inhaled, reaches alveoli where it comes into contact with dendritic cells [2] and macrophages [3]. The macrophages engulf Mtb into phagosomes. Under normal circumstances, the phagosome matures into a phago-lysosome by fusing with a lysosome, which causes a drop in pH and the release of reactive oxygen intermediates that digest mycobacteria. However, Mtb either prevents or avoids phagosome maturation via different mechanisms such as phagosome rupture, pH regulation [4], and phagosome-lysosome fusion arrest [5][6][7]. As a result, Mtb is translocated from the phagosome [8,9] into the cytosol, thereby avoiding digestion in the phago-lysosome. Mtb translocation activity is attributed to the RD1 locus that codes for a type VII secretion system and its substrates EsxA (ESAT- 6) and EsxB (CFP-10). These proteins have been described as essential virulence factors, as their absence attenuates mycobacterial virulence [8,[10][11][12][13][14][15][16].
EsxA and EsxB form a heterodimer (EsxAB) that is co-secreted when Mtb is phagocytized by macrophages [12,15,17]. In previous in vitro studies, EsxA protein has been shown to undergo significant conformational changes and exhibits strong membrane permeabilization activity (MPA) upon acidification [18]. By contrast, EsxB does not have MPA and does not undergo any significant conformational change [9,[18][19][20][21]. Though EsxB may not play any role in membrane permeabilization, it is required for EsxA secretion and may act as a chaperone [17,19,[22][23][24]. The EsxAB membranepermeabilizing mechanism has not been described yet and its role in mycobacterial phagosome escape and cytosolic translocation is still under debate. Current studies have suggested a model that the EsxAB heterodimer is dissociated at low pH to allow EsxA to interact and permeabilize the membrane [9,18,19,24]. However, macrophage infection assays with M. marinum indicated that phagosomal pH decrease did not trigger EsxAB-mediated membrane disruption [21], and there may be other cellular or bacterial factors involved. The absence of an efficient in vitro model to study EsxAB MPA makes the task more challenging. While the MPA of EsxA has been well characterized in liposomes composed of DOPC, a neutral fluidic lipid, the MPA of EsxAB has been poorly studied due to its extremely low activity with the DOPC liposomes. The role of EsxB in MPA has been speculative, and there is no in vitro experimental evidence to convincingly establish its role in the permeabilizing activity.
Our recent study has found that the MPA of EsxA can be significantly affected by the physical characteristics of lipid membranes, such as fluidity and charge [25]. Fluidity (the presence of unsaturated acyl chain) is required for EsxA to permeabilize the membrane, and the presence of negatively charged lipids enhances the MPA of EsxA [25]. The EsxA used in that study was purified from E. coli and lacked post-translational modifications. Our recent studies showed that EsxA derived from M. smegmatis is N-terminally acetylated [24]. Inspired by these findings, in this study, we used M. smegmatis-derived N-terminally acetylated EsxA for our studies. In our previous study using E. coli-derived EsxA, we showed that MPA activity was highly efficient with a physiologically-relevant liposome model (POPC:POPG) compared to DOPC neutral charged membrane lipids. In this study, we optimized our membrane model by titrating the anionic charged lipids (e.g., POPG) to determine their dose-dependent effects on the MPA of N-EsxA.
Next, we found that the optimized liposome system significantly enhanced the MPA of EsxAB to a level that has never been reached before. It is important to note, so far, that there are very few studies that establish the role of EsxB in MPA, and there are no in vitro assays that test the MPA of EsxAB. This study establishes an effective membrane model for the characterization of the MPA of EsxAB.
Thus far, most membrane permeabilization in vitro assays using lipid vesicles with EsxA and EsxAB were done between 20 • C and 33 • C [9,[18][19][20][21]24,26]. Because the melting temperature of EsxA is 35 • C, EsxA retains more than 50% of its native structure at 33 • C, while at 37 • C EsxA loses more than 50% of its native structure and become unstable [25]. Hence, it is imperative to re-investigate the MPA of EsxA and the role of EsxB in membrane permeabilization at 37 • C. In comparison, EsxAB is more thermodynamically stable at 37 • C with a Tm at~53 • C [22]. This suggests that EsxB plays a larger role in membrane permeabilization at 37 • C. To date, there is no effective membrane permeabilization in vitro assay that captures the role of EsxB at physiological temperature.
EsxA and EsxAB Heterodimer Expression and Purification
The pMyNT(EsxA:EsxB) plasmid was a generous gift from Dr. Matthias Wilmanns. Expression of this plasmid in M. smegmatis produces EsxAB heterodimer (from M. tuberculosis) with a His 6 tag at the N-terminus of EsxB. The heterodimer expressed in M. smegmatis also contains mycobacterial native post-translational modification (N a -acetylation) that was described in a previous study [24]. The pMyNT(EsxA(S35C):EsxB) plasmid was generated by site-directed mutagenesis described in a previous publication [26]. Protein purification was performed as previously described [25].
EsxAB Heterodimer Separation
The EsxAB heterodimer was separated by using a 6 M guanidine solution. The EsxAB-containing solution was concentrated to 1 mL and injected into a 5-mL His trap column, which was pre-equilibrated with guanidine solubilization buffer (25 mM NaH 2 PO 4 , 150 mM NaCl, 10 mM imidazole, and 6 M guanidine, pH 6.8). The His 6 -tagged EsxB bound to the column, while EsxA was collected in the flow through. The collected flow-through fractions were concentrated and extensively dialyzed in a 3-kDa cutoff membrane in a dialysis buffer (25 mM NaH 2 PO 4 , 100 mM NaCl, and 1 mM EDTA pH 7.4), in which the denatured EsxA was refolded. The EsxB in the column was eluted with a linear gradient of (10-100%) of an elution buffer (25 mM NaH 2 PO 4 , 150 mM NaCl, 500 mM imidazole, and 6 M guanidine). The eluted fractions were collected and extensively dialyzed in a 3-kDa cutoff membrane in the dialysis buffer (25 mM NaH 2 PO 4 , 100 mM NaCl, and 1 mM EDTA pH 7.4).
Time Lapse Intensity Measurement of EsxA Membrane Permeabilization by ANTS/DPX Fluorescence Dequenching
EsxA MPA was measured in real-time by using the 8-aminonapthalene-1,3,6-trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide(DPX) fluorescence dequenching assay, as described in previous publications [19,20,26]. Furthermore, we evaluated the effects of different lipid compositions and temperatures on the MPA of EsxAB heterodimer. The lipids were solubilized in 1 mL of chloroform:methanol (3:2) solution and dried in nitrogen gas. ANTS and DPX were weighed and added to a final concentration of 10 mM. The mixture of lipids, ANTS, and DPX were resuspended in 1 mL of 5 mM HEPES, pH 7.4. The samples were frozen and thawed for 6 cycles. After this, the mixture was extruded through a 200 nm pore size membrane 23 times (back and forth) using an Avanti Polar Lipids, Inc. mini extruder set (cat no. 610000). The excess ANTS and DPX were removed by passing the sample through a 5 mL desalting column. The liposome fractions were collected and stored at 4 • C until use.
The ANTS/DPX fluorescence dequenching was measured in an ISS-K2 multiphase frequency and modulation fluorometer with excitation at 380 nm, and emissions were recorded at 520 nm. The liposomes (800 µM) containing ANTS/DPX were diluted into 1.35 mL of gel filtration buffer at pH 7.4, which had 4.76 µM of EsxA or EsxAB. The liposomes and proteins were incubated for 15 min at RT or 37 • C in the fluorometer. Following incubation, the fluorescence base line of the samples was monitored for 30 sec, and 1/10 volume (150 µL) of 1 M sodium acetate (pH 4.0) was injected into the cuvette to decrease the pH to 4.0. The samples were continuously stirred during the experiment and crossed polarized on excitation and emission beams, and a 515 nm long-pass filter was used to reduce the background scatter. The fold changes of ANTS fluorescence with the samples were calculated by using EsxA in DOPC as a control. The rates of ANTS fluorescence dequenching were calculated in GraphPad Prism by curve fitting plateau followed by a one phase association ). One-way ANOVA was used for statistical significance analysis.
Time-Lapse Intensity Measurement of NBD Fluorescence for EsxA Membrane Insertion
is a fluorophore that increases its fluorescence as it transitions from a hydrophilic environment to a hydrophobic environment. In our previous publications, the NBD-labeled EsxA(S35C) was successfully used to directly confirm the insertion of EsxA into the liposome membranes [20,25,26,29,42]. In brief, the purified EsxA(S35C) was reduced with dithiothreitol (DTT) on ice for 20 min with a DTT/protein ratio of 250:1. Then DTT was removed from the sample by passing the sample through a desalting column with a buffer (50 mM HEPES, 150 mM NaCl, 50 mM sodium acetate pH 8). Then, IANBD was added in 20x molar excess and incubated at RT for 2 h in the dark. Finally, the excess of NBD was removed by passing the sample through a desalting column. The labeling efficiency for EsxA(S35C) was~75%. The NBD fluorescence was measured in the ISS-K2 multiphase frequency and modulation fluorometer with excitation at 488 nm and emission at 544 nm. The NBD-labeled EsxA(S35C) or EsxA(S35C)-B heterodimer (1.36 µM) were incubated with 800 µM of the liposomes at 4 • C for 30 min in the buffer (20 mM TrisHCl and 100 mM NaCl, pH 7.4) as previously described [25]. The liposome/protein mixtures were incubated for 15 min at RT or 37 • C in the fluorometer with continuous stirring. Excitation and emission beams were cross polarized, and a 520 nm long-pass filter was used to reduce the back-ground scatter. After the addition of 1/10 volume of 1 M sodium acetate (pH 4.0), the NBD fluorescence emission was monitored. The fluorescence fold of change was calculated for each sample by using the sample EsxA with DOPC liposome as a control.
EsxB Stabilization Effect on EsxA at 37 • C
Time lapse intensity measurement of ANTS/DPX dequenching was used to evaluate the stabilization effect of EsxB on EsxA structure at 37 • C. EsxA or EsxAB were pre-incubated at 37 • C for the increasing time lapses (from 0 to 30 min) before fluorescence measurement. The EsxA membrane disruption was triggered by acidification as described above. The fluorescence fold of change was calculated as described above. The experiment was done in triplicate and replicated at least three times for the statistical analysis. Two-way ANOVA with a multiple comparison test was used to compare EsxA and EsxAB after incubation at 37 • C for different amounts of time. Then, a multiple comparison Tukey-Kramer test was used to find significant differences for each group.
Incorporation of Negatively Charged Lipids Increased N α -Acetylated EsxA (N-EsxA) Membrane Permeabilization
In our previous studies, N-EsxA MPA was characterized with the liposomes made of DOPC, an unsaturated lipid that confers membrane fluidity [19,20,26]. Recently, we have shown that while membrane fluidity is required for EsxA to permeabilize the membranes, incorporation of negatively charged lipids at the concentrations similar to phago-endosomal membranes increases EsxA membrane permeabilization [25,43]. Here, we systematically titrated the concentrations of PG lipids relative to PC lipids to determine the effects of the PG lipids on the MPA of N-EsxA.
We first used the ANTS/DPX fluorescence dequenching assay, an approach for measuring membrane permeabilization, to test the liposomes made of DOPC/DOPG at various molar ratios (%), namely 100/0, 95/5, 90/10, 80/20, and 60/40, respectively. Like in our previous studies with EsxA, the MPA of N-EsxA was significantly enhanced by DOPG in a concentration-dependent manner. Incorporation of DOPG at 10%, 20%, and 40% concentration yielded 3.32, 4.82, and 6.4 times the increase in membrane permeabilization, respectively ( Figure 1A,C). The curves of ANTS fluorescence intensity were fit into a nonlinear regression (plateau followed by a one-phase association), and the initial rate of membrane disruption was calculated. The rate of N-EsxA DOPC at RT was set as a control. SD is represented in the error bars. One-way ANOVA with multiple comparison test was performed for (C,D) (p < 0.05) *, (p < 0.01) **, (p < 0.005) ***, (p < 0.0001) ****. The experiment was done in triplicate and replicated at least three times for the statistical analysis.
The New Liposome System Was Validated by NBD Fluorescence
In order to validate the results obtained in the new liposome system, we used the NBD-labeled EsxA to test the physical insertion of EsxA into the liposomal membrane as an independent approach ( Figure 2). The membrane insertion of EsxA was significantly increased with the liposomes containing POPC/POPG (%), compared to the pure DOPC and POPC liposomes (Figure 2A,B). The results of membrane insertion measured by NBD fluorescence are consistent with the results obtained in the ANTS/DPX membrane permeabilization assays (Figure 1). In summary, we used two independent assays (ANTS/DPX and NBD) and tested the MPA of N-EsxA in the new liposome systems made of either DOPC/DOPG or POPC/POPG. We found that incorporation of the negatively charged PG lipids greatly enhanced the MPA of N-EsxA, and it particularly exhibited higher MPA with the POPC/POPG liposomes.
Characterization of the Heterodimer's MPA in the PC/PG Liposomes
N-EsxA and EsxB are co-expressed and co-secreted as a heterodimer after Mtb is phagocytized by the macrophage [15]. Current studies have suggested the heterodimer needs to be dissociated prior to N-EsxA membrane insertion [18,19]. We recently showed that N-terminal acetylation is required for the dissociation of these two proteins [19,24]. However, the characterization of the heterodimer MPA had been a challenge with the DOPC liposomes due to low activity detected by ANTS/DPX assay. Here, we used the POPC/POPG (%) 80/20 liposomes to test the heterodimer's MPA.
The membrane permeabilization activity (fold change of ANTS intensity) of N-EsxA with the DOPC liposomes was set as the control. EsxA:EsxB (1:1) had nearly undetectable membrane permeabilization ( Figure 3). Interestingly, with the POPC/POPG liposomes, EsxA:EsxB (1:1) had a significant increase in membrane permeabilization, which was about three-fold of the control. The displayed MPA in POPC/POPG liposomes was high enough to provide us with a "big window" to titrate the inhibitory effect of EsxB on EsxA MPA. As expected, EsxB inhibited the MPA of N-EsxA in a dose-dependent manner, indicating that heterodimer dissociation is required for N-EsxA to permeabilize the membranes (Figure 3), which is consistent with our previous findings [24]. It is important to note that the heterodimer EsxAB was comprised of N-EsxA purified from M. smegmatis. 1). SD is represented in the error bars. One-way ANOVA with multiple comparison test was performed for (B) (p < 0.0001) ****. The experiment was done in triplicate and replicated at least three times for the statistical analysis.
EsxAB is More Stable at 37 • C Than N-EsxA
Subsequently, we set out to confirm the results in membrane insertion of N-EsxA and EsxAB. We incubated N-EsxA and N-EsxA:EsxB (1:1, 1:2, and 1:4 ratio) at RT and 37 • C. At RT, N-EsxA MPA was significantly inhibited by EsxB in a dose-dependent manner. This was shown as a sharp drop even at a 1:1 ratio of N-EsxA:EsxB, where the MPA was inhibited by more than 50% (Figure 4A,B). At 37 • C, the inhibition of EsxB on N-EsxA MPA was less significant, evidenced by a slow dose-dependent decrease ( Figure 4C,D). The result suggests that EsxAB is more active at 37 • C than at RT. They were transferred to a cuvette in a fluorometer, and NBD fluorescence was monitored in real-time at 37 • C. EsxA(S35C) membrane insertion was triggered by the addition of a 1/10 volume of 1 M sodium acetate. (D) The relative fold of change of NBD fluorescence intensity of the samples was calculated using the NBD intensity of EsxA(S35C) on DOPC liposome as the reference. SD is represented in the error bars. One-way ANOVA with multiple comparison test was performed for (B,D) (p < 0.001) **, (p < 0.0001) ****. The experiment was done in triplicate and replicated at least three times for the statistical analysis.
In our previous publication, CD analysis showed that the T m of EsxA was 35 • C and the T m of EsxAB was 53 • C [22], meaning that at 37 • C, EsxA will have less than 50% of its native structure, in contrast to EsxAB, which will have a conserved native structure. It is not clear how the temperature will affect the MPA of N-EsxA and EsxAB. Thus, we tested the MPA of N-EsxA and EsxAB at RT (22 • C) and 37 • C. For both N-EsxA and EsxAB with the POPC/POPG liposomes, the rate of membrane permeabilization at 37 • C was much higher than that at RT ( Figure 5A,B). Interestingly, however, the fold change of ANTS fluorescence of N-EsxA at RT was lower than at 37 • C, while the fold change of fluorescence intensity of EsxAB at RT was lower than 37 • C ( Figure 5C,D). This suggests that while N-EsxA and EsxAB had similar initial rates of membrane permeabilization, EsxAB was more stable and hence remained active longer than N-EsxA in membrane permeabilization at 37 • C. To test this hypothesis, N-EsxA and EsxAB were pre-incubated at 37 • C at varying time frames (0, 5, 15, and 30 min) and were then tested for membrane permeabilization. The result showed that N-EsxA MPA was significantly decreased as the incubation time at 37 • C increased, while EsxAB MAP remained constant over time ( Figure 5E). The data suggests that at 37 • C, N-EsxA was progressively unfolded in solution and lost MPA over time, and EsxB played a role in stabilizing N-EsxA at 37 • C to preserve its MPA until acidification.
Discussion
The MPA of EsxA has been extensively characterized [8,19,20]. We have shown that N-EsxA from Mtb exhibits a unique MPA that is absent in its ortholog from M. smegmatis [19]. We obtained direct evidence that N-EsxA inserts into the liposomal membrane and forms a membrane-spanning structure [20]. Most recently, we have shown that the EsxA protein produced in M. smegmatis instead of E. coli contains mycobacterial-specific N-terminal acetylation, which is required for membrane permeabilization. Once Thr2 of EsxA is acetylated, it creates a dragging force that pulls away EsxB from EsxA and allows heterodimer dissociation in low pH [24], an event critical prior to membrane disruption [9,18,19,24]. Numerous studies have implicated that EsxAB ruptures the phagosome membrane and assists mycobacterial escape into the cytosol [8,9,12,18]. In the current DOPC liposome model, however, EsxAB exhibits very low MPA, which suggests DOPC liposome may not be suitable for the characterization of EsxAB MPA. The lack of a good membrane model has hindered our understanding of the molecular mechanism of EsxAB-mediated virulence. In this study, we tested a new liposome system made of PC/PG lipids that greatly enhanced membrane permeabilization by EsxAB. The PC/PG liposome is a capable tool to study EsxAB membrane permeabilization in a physiological-like condition. The use of PC and PG lipids instead of phosphatidylserine makes this model accessible to multiple study groups. However, the physiological characteristics of the phagosome membranes are conserved by using these lipids. We expect that the PC/PG liposome model will contribute to elucidate the mechanism of EsxAB mediated permeabilization into membranes.
Our previous studies have shown that the physical characteristics of membrane lipids can modulate N-EsxA interaction with the membranes [25]. We also identified new membrane models that effectively capture membrane permeabilization by N-EsxA at room temperature. We showed that N-EsxA has a much higher efficiency of membrane permeabilization in the presence of POPC/POPG at a 4:1 ratio, which closely mimics the inner leaflet of the plasma membrane in terms of fluidity and charge. In the present study, we systematically characterized the effects of the incorporation of negatively charged PG lipids into the PC liposome on the MPA of N-EsxA and EsxAB (Figures 1 and 2). Furthermore, the EsxB titration experiment showed that EsxB inhibited N-EsxA membrane permeabilization in a dose-dependent manner ( Figure 3). The result is consistent with the previous findings that EsxA needs to dissociate from EsxB prior to the membrane disruption [19,24].
After establishing the new liposome system, we tested the effects of temperature on membrane permeabilization. Previous studies have shown that EsxB has no specific membrane interaction, and EsxAB needs to be dissociated to allow EsxA insert into the membrane [9,18,19,24]. Here, we acquired an incremental understanding of N-EsxA and EsxB interaction at 37 • C. Previous membrane permeabilization assays were performed either using the pure DOPC liposome at RT [19,24], or sheep RBCs (red blood cells) that were incubated at 33 • C [9,18]. Thermal stability of EsxA (S35C) through CD analysis suggests that EsxA has a melting temperature around 35 • C; thus, at RT or 33 • C, EsxA retains more than half of its native structure. This allows the protein to interact efficiently with the membrane without requiring EsxB, but EsxA loses more than 50% of its native structure at 37 • C. Earlier studies have shown that EsxAB has a melting temperature around 53 • C; hence, it is more stable at 37 • C than EsxA [22]. Our experiments showed that N-EsxA by itself induced lower membrane permeabilization than the heterodimer at 37 • C (Figures 4 and 5). For the first time, this experiment captured the role of EsxB in stabilizing the structure of N-EsxA at 37 • C to achieve optimum efficiency to permeabilize the membrane. EsxB assists in the maintenance of N-EsxA structure as a chaperone that increases the efficiency of membrane disruption and is further evidenced in the time course of incubation at 37 • C, in which the MPA of N-EsxA dropped quickly over time, while the MPA of EsxAB remained stable (Figures 4 and 5).
In summary, this new experimental model enabled us to characterize the membrane permeabilization of EsxAB at physiologically-relevant conditions. We conclude that anionic charged lipids are critical for EsxAB membrane binding followed by MPA and EsxB functions as a chaperone to stabilize N-EsxA at 37 • C prior to the acidification-driven heterodimer dissociation. | v3-fos-license |
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} | pes2o/s2orc | Cooperative Roles of Various Membrane Phospholipids in the Activation of Calcium-activated, Phospholipidldep&dent Protein Kinase*
Although phosphatidylserine is the sole phospholipid effective for the activation of Ca2+-activated, phospholipid-dependent protein kinase in the presence of a small amount of unsaturated diacylglycerol and micromolar concentrations of Ca2+ (Takai, Y., Kishimoto, A., Kikkawa, U., Mori, T., and Nishizuka, Y. (1979) Biochem. Biophys. Res. Commun. 91, 1218-1224), other species of phospholipids modulate the activation of enzyme considerably. When phosphatidylserine is supplemented with phosphatidylethanolamine, further enhancement of the enzymatic activity is observed. Inversely, the addition of phosphatidylcholine or sphingomyelin markedly diminishes the enzyme activation by phosphatidylserine. Phosphatidylinositol, which serves as the source of unsaturated diacylglycerol, and phosphatidic acid do not show significant effects. Kinetic analysis has indicated that phosphatidylethanolamine enhances the enzyme activation by marked increase in the affinity of enzyme for Ca2+ and also by slight increase in the affinity for phosphatidylserine as well as for unsaturated diacylglycerol without affecting the maximum reaction velocity. Phosphatidylcholine and sphingomyelin diminish the enzyme activation in an uncompetitive manner with respect to Ca2+ and in a competitive manner with respect to both phosphatidylserine and unsaturated diacylglycerol. These results suggest that each species of the various membrane phospholipids plays a specific role with positive or negative cooperativity in the activation of this unique protein kinase.
turnover has been most extensively investigated (for reviews, see Refs. 1-4). This phospholipid turnover, f i s t recognized by Hokin and Hokin (5) in 1955 in pigeon pancreas and guinea pig brain stimulated by acetylcholine, was subsequently recognized by many investigators in virtually all types of tissues that are activated by a wide variety of extracellular messengers including a-adrenergic and muscarinic cholinergic neurotransmitters, peptide hormones, and many other biologically active substances (1-4). A series of recent studies in this laboratory has uncovered a new species of cyclic nucleotideindependent protein kinase in most mammalian tissues, which is normally present as an inactive form but is reversibly activated by the simultaneous presence of Ca2' and membranes (6, 7). The active component associated with membranes has been identified as phospholipid (6, 7). Among various phospholipids tested, only phosphatidylserine is effective, particularly at lower concentrations of Ca2+. A small amount of unsaturated diacylglycerol markedly increases the affinity of enzyme for ea2+ to less than the micromolar range and such a dramatic effect is observed only in the presence of phosphatidylserine and not other phospholipids (8). Thus, unsaturated diacylglycerol derived from the phosphatidylinositol turnover mentioned above may serve as a second messenger for the selective activation of this protein kinase (8,9). Recent reports from this laboratory (10,11) have presented evidence that such a protein kinase system indeed operates in vivo in human platelets during activation by thrombin. The present paper will describe that various other phospholipids are inactive by themselves but nevertheless show positive or negative cooperative roles in the activation of this protein kinase. The Ca2+-activated, phospholipid-dependent protein kinase will be referred to tentatively as protein kinase C.
EXPERIMENTAL PROCEDURES
Materials and Chemicals-Protein kinase C and Ca2+-dependent protease were prepared from rat brain as described previously (12). The catalytically active fragment of protein kinase C was prepared from the native enzyme by limited proteolysis with Ca2+-dependent protease as specified earlier (12). These protein kinase preparations were practically free of endogenous phosphate acceptor proteins. HI histone was prepared from calf thymus as described previously (13). Phosphatidylinositol (bovine brain) and phosphatidic acid (egg yolk), purchased from Serdary Research Laboratories and Sigma, respectively, were purified by two-dimensional silica plate thin layer chromatography under the conditions described earlier (6). Phosphatidylserine (bovine brain), phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin (human erythrocytes) were generous slfts from Drs. T. Fujii and A. Tamura, Kyoto College of Pharmacy. These phospholipid preparations were chromatographically pure. Synthetic diolein was purchased from Nakarai Chemicals. [y-3ZPJATP was prepared by the method of Glynn and Chappell (14). All materials Phospholipid and Protein Kinase Activation 7147 and reagents employed for the present studies were taken up in water which was prepared by a double distillation apparatus followed by passing through a Chelex 100 column to remove as much Ca2+ as possible as specified earlier (9). Other chemicals were purchased from commercial sources.
Protein Kinase Assay-Protein kinase was assayed by measuring the incorporation of 32P into HI histone from [y3'P]ATP. The standard reaction mixture (0.25 ml) contained 5 pmol of Tris/HCl at pH 7.5, 1.25 pmol of magnesium nitrate, 50 pg of HI histone, 2.5 nmol of [y3'P]ATP (5-15 X IO4 cpm/nmol), 0.5 pg of protein kinase C. The concentration of CaC12 and lipids employed are indicated in each experiment. Various lipids were first mixed with one another in a small volume of chloroform. After the chloroform was removed in vacuo, the residue was suspended in 20 mM Tris/HCl at pH 7.5 by sonication with a Kontes sonifier, Model K881440, for 5 min at 0 "C, and was employed for the assay. These procedures were performed under nitrogen to prevent oxidation of the lipids as much as possible.
All reactions were carried out in plastic tubes. The incubation was carried out for 3 min at 30 "C. The reaction was stopped by the addition of 25% trichloroacetic acid and acid-precipitable materials were collected on a Toyo-Roshi membrane filter (pore size, 0.45 pm).
Determinations-Radioactivity of 32P was determined using a Nuclear Chicago Geiger Muller gas flow counter, Model 4338. Protein was determined by the method of Lowry et al. (15) with bovine serum albumin as a standard protein.
RESULTS AND DISCUSSION
Protein kinase C was normally inactive but was activated by the simultaneous addition of Ca2+, phospholipid, and unsaturated diacylglycerol (6-9). Among various phospholipids tested, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid were active in support of enzyme activation to variable extents a t relatively higher concentrations of Ca2+ (10-4-10-3 M), but only phosphatidylserine was effective at the micromolar range of CazC concentrations as shown in Table I. In this experiment, synthetic diolein was employed as an unsaturated diacylglycerol. Phosphatidylcholine and sphingomyelin were practically inactive over a wide range of Ca2+ concentrations. However, some of these inactive phospholipids showed marked effects on this enzyme activity in the presence of phosphatidylserine, unsaturated diacylglycerol, and micromolar concentrations of Ca2+. Fig. 1 shows the effects of various phospholipids in the presence of a fxed amount of phosphatidylserine at two different concentrations of Ca2+. Further enhancement of the enzymatic activity was observed when phosphatidylethanolamine was a supplement to phosphatidylserine. Inversely, the enzymatic activity was markedly and progressively inhibited by the addition of increasing amounts of either phosphatidylcholine or sphingomyelin. Phosphatidylinositol and phosphatidic acid showed practically no effect. Unsaturated diacylglycerol was always indispensable a t micromolar concentrations of Ca2+. Neither phosphatidylethanolamine, phosphatidylcholine, nor sphingomyelin appeared to interact directly with the catalytic site of the enzyme, since none of these phospholipids activated or inhibited the catalytically active fragment of enzyme which was obtained by limited proteolysis with Ca2+-dependent protease, as shown in Table 11. This fragment was fully active in the absence of Ca2+, phosphatidylserine, and unsaturated diacylglycerol as described earlier (6, 12, 16). These results indicate that the various phospholipids modulate the activation of enzyme rather than the catalytic process itself.
T h e next set of experiments was performed to explore the mode of action of the three species of phospholipids mentioned above. Since the activation of protein kinase C required Ca2+, phosphatidylserine, and unsaturated diacylglycerol, one of these activators was varied in the presence and absence of either phosphatidylethanolamine, phosphatidylcholine, or sphingomyelin. As shown in Fig. 2 Each value is the mean of triplicate determinations.
TABLE I1
Effects of various phospholipids on reaction velocity of protein kinase C and its catalytically active fragment Protein kinase C was assayed at 1 X M CaC12 under conditions similar to those specified in the legend to Fig. 1 one-tenth of the concentration needed in the presence of phosphatidylserine alone. The Vmax value was increased slightly. However, the enzymatic activity in this experiment was assayed in the presence of limited quantities of phosphatidylserine and diolein. When these two activators were employed in sufficient and saturated quantities, phosphatidylethanolamine did not affect the V,,, value. In a marked contrast to phosphatidylethanolamine, both phosphatidylcholine and sphingomyelin appeared to inhibit enzyme activation over a wide range of Ca2' concentrations as shown in Fig. 2, B and C.
The double reciprocal analysis of these results indicated that both phospholipids increased slightly the K , value for Ca2+ and decreased the V,,, value, implying that these phospholipids inhibited the activation of protein kinase C in an uncompetitive manner with respect to Ca". Fig. 3 shows the experiments in which phosphatidylserine was varied in the presence and absence of one of the other three phospholipids. The double reciprocal plots obtained from these results indicated that phosphatidylethanolamine decreased the K , value for phosphatidylserine. An apparent slight increase in the V,,, value shown in Fig. 3A again appeared to be due to this assay condition; here, Ca2+ and diolein were limited, i.e. when these two ingredients were sufficiently added, phosphatidylethanolamine did not show any effect on the V,,, value. Fig. 3, B and C, indicates that both phosphatidylcholine and sphingomyelin were inhibitory. The double reciprocal plots indicated that both phosphatidylcholine and sphingomyelin increased the K, value for phosphatidylserine and inhibited the enzyme activation competitively with respect to this phospholipid. Finally, in the experiment given in Fig. 4, unsaturated diacylglycerol, diolein in this case, was varied, and the result indicated that phosphatidylethanolamine again enhanced the enzyme activation by decreasing the K , value for diolein without affecting the VmaX value. Both phosphatidylcholine and sphingomyelin increased the K, value for diolein and inhibited the enzyme activation competitively with respect to 1 B I c l this diacylglycerol. These kinetic results for the mode of activation and inhibition of protein kinase C are summarized in Table 111.
The experimental results presented above together with those reported earlier (8) activation of protein kinase C. Upon stimulation by extracellular messengers, phosphatidylinositol produces unsaturated diacylglycerol which serves as a second messengei-for the activation of protein kinase C. Phosphatidylserine plays an inevitable role during the enzyme activation. Phosphatidylethanolamine appears to facilitate this activation process, whereas phosphatidylcholine and sphingomyelin are inhibitory. However, it may be noted that interpretation of these results must be limited, since the various lipids added are a l l insoluble and, thus, the concentrations listed do not necessarily represent the actual physiological picture. Under these conditions, the phospholipids appear to form uni-or multilamellar systems and to be suspended in water to different degrees. Mixed lipid systems seem to form mixed lamellae, and phase separations may be anticipated. It would also be expected that Ca2+ may affect the lamellae structures and diacylglycerol may intercalate between the lamellae. Presumably, protein kinase C may bind variably to the lamellae formed. Much work remains to be done to resolve the complex role of Ca", diacylglycerol, and each phospholipid in the activation of this unique protein kinase. It has been well established that, in erythrocytes and platelets, most of the phosphatidylcholine and sphingomyelin is located in the outer monolayer, whereas phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine are largely located in the inner monolayer of membranes (for reviews, see Refs. 17-19). Although it is not known whether such asymmetric distribution of the various phospholipids is favorable for the activation of protein kinase C, it is possible to assume that each species of the various membrane phospholipids displays a specific role with positive or negative cooperativity in the activation of this unique protein kinase. On the other hand, Hirata et al. (20-23) have recently proposed that methylation of phosphatidylethanolamine to produce phosphatidylcholine may be stimulated by some extracellular messengers such as P-adrenergic agonists, concana-valin A, and chemotactic peptides. Since phosphatidylserine is well known to serve as precursor to phosphatidylethanolamine (24), it is possible that this decarboxylation reaction is stimulated by some extracellular messengers. In any case, it is conceivable that the activation of protein kinase C is intimately related to the metabolism of not only phosphatidylinositol but also of other phospholipids, since various phospholipids, particularly phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine modulate the activation of protein kinase C as described in this paper. | v3-fos-license |
2019-02-16T15:06:11.186Z | 2018-10-15T00:00:00.000 | 92752102 | {
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} | pes2o/s2orc | Synthesis and biological evaluation of phosphatidylcholines with cinnamic and 3-methoxycinnamic acids with potent antiproliferative activity
A series of eight novel phosphatidylcholines containing cinnamic or 3-methoxycinnamic acids (3a-b, 5a-b, 9a-b, 10a-b) at sn-1 and/or sn-2 positions were synthesized and tested for their antiproliferative activity in an in vitro model against representative six human cancer cell lines (MV4-11, A549, MCF-7, LoVo, LoVo/DX, HepG2) and a normal cell line BALB/3T3. The structures of the new compounds were confirmed by spectral analysis. Biological evaluation revealed that all the tested conjugates exhibited higher antitumor activity than the corresponding free aromatic acids. Compounds 3b and 9b turned out to be the most active, with IC50 values of 32.1 and 30.5 μM against the LoVo/DX and MV4-11 cell lines, respectively. Studies of the mechanism of the antitumor action were carried out for 1-palmitoyl-2-cinnamoyl-sn-glycero-3-phosphocholine (5a), and it was shown to be active toward almost all the tested types of cancer cells, showing that this compound could effectively arrest the cell cycle in G2/M and decrease the mitochondrial membrane potential of leukemia MV4-11 cells. The obtained results proved that the strategy of the incorporation of cinnamic and 3-methoxycinnamic acids into phospholipids could expand their potential application in industry, as well as could improve their antiproliferative activity and selectivity toward cancer cell lines.
Introduction
Cancer diseases are among the most signicant health problems in the world, being the main cause of death. Chemotherapy is the basic method for cancer treatment; however, due to the fact that available chemotherapeutics also act on healthy cells, it is necessary to look for new anticancer agents, particularly those with a selective mechanism of action.
In the search for molecules that are non-toxic for humans and that have the potential to suppress tumor development and counteract cancers already developed, we became interested in cinnamic acid (CA) (1a) and its methoxy derivatives. These compounds have been known for centuries in treatments for cancer. The rst mention of this subject came from 1905 and indicated that a 10% sodium cinnamate solution was a substance that could support anticancer therapy. 1 Liu and coworkers proved that cinnamic acid has a cytostatic effect on human glioblastoma cells (A175, U251), melanoma (MEL 1011, A375(M), SKMEL 28), prostate cancer (PC3(M), Du145, LNCaP), and lung cancer (A549) at doses that have no signicant effect on normal cells. 2 The anticancer activity of CA (1a) has been reported to be a result of its inhibitory effect on 17b-hydroxysteroid dehydrogenase type 5 (AKR1C3), which indicated a potential use of this compound in the treatment of hormonedependent forms of cancers. 3 CA (1a) causes the cell cycle arrest of human cervical cancer cell lines (HeLa), malignant melanoma (Fem-x), and breast cancer (MCF-7) and induces apoptosis of human melanoma cells . 4 In studies of the mechanism of CA action on human leukemia cells (K562) it has been conrmed that this compound promotes cell cycle arrest by prolonging the G1/G0 phase and inducing cell apoptosis. 5 Methoxy derivatives of cinnamic acid suppress benzo(a) pyrene-induced neoplasia of the forestomach and inhibit invasion and metastasis in the melanoma cell lines. 6,7 In other studies, they turned out to be effective chemopreventive agents against 1,2-dimethylhydrazine in an in vivo model, which indicated their possible application in the prevention of colon carcinogenesis. 8 For methoxy derivatives of cinnamic acid, induction of an intrinsic apoptosis pathway (dependent on mitochondria) in the human colon cancer cell line (HTC-116) has also been conrmed. 9 Cinnamic acid (1a) and its methoxy derivatives are also known to exert a number of other benecial effects. Among these, antimicrobial, 10,11 hepatoprotective, 12 and antidiabetic 13,14 activities, as well as a protective effect against glutamate-induce neurodegeneration in cortical neurons, which should be especially emphasized. 15 Despite extensive literature data indicating the biological activities of cinnamic acid and its methoxy derivatives, it is difficult to achieve in practice an anticancer and pro-health impact of these compounds on the body. The biological effects of aromatic acids delivery to the organisms from natural sources depend not only on their chemical form but also on the level of their release from the food matrix via gut microbes. It has been also conrmed that even when they are supplied in the free form to the organism, their bioavailability is still very low, because of their fast metabolism and elimination in both urine and bile aer ingestion. Therefore, their effects that have been proved in in vitro studies are difficult to achieve in in vivo experiments. For this reason, in the food and pharmaceutical industries, the products of the lipophilization of aromatic acids are used. One of the most effective strategies to enhance their bioavailability in biological systems is through covalent bounding with phospholipids (PLs). Only a few papers concerning aromatic acids attached to PLs have been published so far. Yang and co-workers incorporated ferulic acid into the structure of phosphatidylcholine using lipase Novozym 435. 16 In another study, phospholipids derivatives of syringic and vanillic acids obtained by chemical synthesis were introduced as new food-based ingredients with potential application in the food industry. 17 Recently, we described phospholipid conjugates of methoxy derivatives of benzoic acid as potential anticancer chemotherapeutics. 18 Herein, we report the synthesis of phosphatidylcholines containing in their structures cinnamic acid (CA) (1a) and 3-methoxycinnamic acid (3-OMe-CA) (1b), which are known to be even more active antitumor agents than the methoxy derivatives of benzoic acid, like anisic or veratric acids.
Results and discussion
Chemoenzymatic synthesis of phospholipid with cinnamic (1a) and 3-methoxycinnamic acid (1b) residues All the target conjugates (3a-b, 5a-b, 7a-b, 8a-b) were synthesized as outlined in Scheme 1-3. First, symmetrically substituted phosphatidylcholines with cinnamic or 3-methoxycinnamic acid residues (3a-b) were prepared (Scheme 1). Cinnamic acid (CA) (1a) and 3-methoxycinnamic acid (3-OMe-CA) (1b) were esteried with the cadmium complex of sn-glycero-3phosphocholine (GPC Â CCl 2 ) (2) in the presence of 4-(N,Ndimethylamino)pyridine (DMAP) as a catalyst and N,N 0 -dicyclohexylcarbodiimide (DCC) as a coupling agent. The products 3a-b were then puried by column chromatography. 1,2-Dicinnamoyl-sn-glycero-3-phosphocholine (3a) and 1,2-di(3-methoxycinnamoyl)-sn-glycero-3-phosphocholine (3b) were obtained in high yields, 93.5% and 94%, respectively. The structures of 3a and 3b were conrmed by 1 H, 13 C, and 31 P nuclear magnetic resonance (NMR) spectroscopy. Correlation spectroscopy (COSY), heteronuclear single-quantum correlation spectroscopy (HSQC), and mass spectra (ESI-MS) were also applied for this purpose. In the 1 H NMR spectra of 3a and 3b, not only were the signals characteristic for PC fragments visible, but also the signals from the aromatic acids incorporated into its structure. Signals of CH 2 -1 0 from the glycerol skeleton at d ¼ 4.17 and 4.31 for 3a and d ¼ 4.19 and 4.33 for 3b were observed as a doublet of doublets and as multiplets, respectively. The multiplets of H-2 0 were observed in the spectra of 3a and 3b at characteristic chemical shis of 5.18 and 5.21 ppm for sn-2 substituted phosphocholines, respectively. The signals at 2.96 and 2.99 ppm from three N-methyl groups from the choline were visible as singlets in the spectra of 3a and 3b. Characteristic signals of protons of aromatic moiety were found in the range 6.69-7.29 ppm. In the spectrum of 3a, the peaks at 6.22 and 7.45 ppm, as well as in the spectrum of 3b the peaks at 6.21 and 7.41 ppm, were from the olenic protons. In the 13 17 and 166.56 ppm and conrmed the presence of ester connections between aromatic acids and glycerol. The presence of an aromatic ring was also conrmed by the signals at 112.61-134.89 ppm observed in the 13 C NMR spectra of 3a and 3b. The 13 P NMR data conrmed that the phosphatidyl part in new molecules was retained, while the ESI-high-resolution mass spectra (HRMS) showed the expected mass values.
Two asymmetrically substituted phosphatidylcholines containing CA or 3-OMe-CA at the sn-2 position (5a-b) were obtained according to the reaction presented in Scheme 2. The starting lysophosphatidylcholine (PA-LPC) 4 was synthesized using phospholipase A 2 (PLA 2 ), as was described previously 19 and was then subjected to esterication with 1a and 1b in the presence of DMAP and DCC. Aer purication, we received products 5a in a 90% yield and 5b in a 58% yield. The formation of 5a-b was conrmed by the ESI-MS spectra, in which intensive signals at m/z 626.3824 for 5a and 656.3925 for 5b were detected. To determine their structures, 1D and 2D NMR experiments were performed (all data are presented in the Experimental section and all the spectra are in the ESI †). In the NMR spectra of 5a-b, all characteristic signals from the glycerol, choline, aromatic acid, and palmitic acid were identied. In the 1 H NMR spectra of 5a and 5b, signals from protons of the benzene ring (6.72-7.31 ppm) and olenic protons (d ¼ 6.23 and 7.47 for 5a and 6.20 and 7.42 ppm for 5b) were visible. The terminal methyl signal of palmitoyl residue at the sn-1 position was observed at d ¼ 0.64 (5a) and 0.62 (5b) as a triplet (J ¼ 6. 8 Hz), whereas the signals for methylene groups in the palmitic acid residue were observed in the range 0.95-1.09 ppm. The chemical shi of the multiplet of H-2 0 at 5.12 ppm in the spectra of 5a and 5b proved that the sn-2 position was esteried and PA-LPC was conjugated with aromatic acids. In the 13 C NMR spectra of 5a and 5b, two carbon atom signals from ester groups were identied at d ¼ 166.13 and 173.63 for phosphocholine 5a and at d ¼ 166.26 and 173.75 for compound 5b. Signals of C-2 and C-3 carbon atoms were observed at 116.74 and 145.60 ppm for 5a and at 116.88 and 145.72 ppm for 5b. The 31 P NMR data conrmed the presence of a phosphocholine 5a and 5b as a singlet at À0.66 and À4.64 ppm, respectively.
For the preparation of lyso PC 9a-b, containing the CA and 3-OMe-CA in the sn-1 position, GPC was regioselectively acylated using a tin-mediated mono-functionalization method described by D'Arrigo previously. 19 First, the stanylene acetal 7 was prepared in the reaction of sn-glycerophosphocholine 6 with dibutyltin oxide (DBTO) and then subjected to acylation by cinnamoyl and 3-methoxycinnamoyl chlorides 8a-b obtained in situ, according to the method proposed by Mattson. 20 Lysophosphocholines 9a-b were obtained in good yields of 67% and 69%, respectively. In the 1 H NMR spectra of 9a-b, the characteristic for the -N + (CH 3 ) 3 group single peak at 3.01 ppm was detected. The multiplets of protons H-2 0 in the range 3.73-3.85 ppm proved that the sn-2 position was non-esteried. The peaks from protons of the aromatic ring appeared in the range 6.83-7.31 ppm and conrmed the conjugation of CA and 3-OMe-CA with GPC. The structures of products 9a-b were fully conrmed also by the 13 C, 31 P, correlation spectroscopy and ESI-MS spectra as well.
The heterosubstituted phospholipids 10a-b, in which CA and 3-OMe-CA occur in the sn-2 position and the sn-1 position is occupied by the palmitic acid, were synthesized from 9a-b. These lyso PCs were subjected to Steglich esterication with palmitic acid. Phosphocholines 10a-b were obtained in high yields of 90% and 85.5%, respectively. The 1 H NMR spectra of 10a-b displayed all the proton signals from CA/3-OMe-CA acid and GPC, such as signals for the hydrogen protons in aromatic rings at 6.72-7.36 ppm and the -N + (CH 3 ) 3 group at 2.98-3.01 ppm of the GPC fragment. The appearance of new peaks in the range 0.63-2.15 ppm conrmed successfully the incorporation of palmitic acid into the structure of the lyso PC used as a substrate. The 1 H NMR spectra of 10a and 10b showed a multiplet of proton H-2 0 at d ¼ 5.10 and 5.07, respectively. In comparison to the lysophosphatidylcholines 9a-b, the chemical shis of these signals were shied to a lower frequency, which proved that the sn-2 position was esteried. The structural assignments were also accomplished through extensive 2D NMR spectroscopy, and mass spectra (ESI-MS).
Antiproliferative activity in vitro toward selected cancer cell lines
Cinnamic acid (1a), 3-methoxycinnamic acid (1b), and all the synthesized phosphatidylcholines (3a-b, 5a-b, 9a-b, 10a-b) were assessed for their antiproliferative activity against a panel of six cancer cell lines: MV4-11 (leukaemia), A549 (lung cancer), MCF-7 (breast cancer), LoVo (colon cancer), LoVo/DX (doxorubicinresistant colon cancer), HepG2 (liver cancer), and normal mice broblast cells BALB/3T3. The selection of cancer cell lines was based on the literature data indicating the antiproliferative activity of benzoic and cinnamic acids and their methoxy derivatives. 2,4,5,8,9 Biological studies were carried out using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or sulforhodamine B (SRB) assays. Two commercial anticancer drugs, i.e., cisplatin and doxorubicin, were used as the positive control. The results are shown in Table 1. Almost all the tested conjugates showed much stronger antiproliferative activity than free aromatic acids (which was statistically significant in comparison to the CA or 3-OMe-CA acids, p < 0.05). Except for the HepG2 line, asymmetrically substituted phosphatidylcholines 5a, 5b, and 10a were signicantly more toxic toward the studied cancer lines than normal mice broblast. Among the tested compounds, 1-palmitoyl-2-cinnamoyl-sn-3glycero-phosphocholine 5a appeared to be the most effective and promising chemoprevention agent active toward the wide range of studied cancer lines. Analyzing the correlations between the activity of the mentioned derivatives and their chemical structures, the position of the aromatic fragment in the skeleton of glycerol seemed to be signicant. Higher cytotoxic activities exhibited asymmetrically substituted phosphatidylcholines containing CA or 3-OMe-CA in the sn-2 position than those with aromatic acids in the sn-1 position. Lysophosphatidylcholines 9a and 9b were characterized by their selectivity of action and they effectively inhibited only the proliferation of leukaemia cells. Their determined IC 50 values were respectively 8-and 11-fold lower than those reported for free cinnamic and 3-methoxycinnamic acids, being at the same time denitely less toxic toward normal cells. It is also worth noting that in the group of symmetrically substituted phosphatidycholines, 1,2-di(3-methoxycinnamoyl)-sn-glycero-3phosphocholine (3b) was the most active derivative toward the doxorubicin-resistant line of colon cancer. This compound was effective in a concentration of 32.1 mM against LoVo/DX, while free acid 1b was not active at the tested range of concentrations (IC 50 > 625 mM).
A comparison of the activities of phospholipid derivatives of CA and 3-OMe-CA showed that homosubstituted PC with 3-OMe-CA 3b was a little more active than homosubstituted PC with CA 3a. Moreover, for heterosubstituted PC with CA 5a and 10a and heterosubstituted PC with 3-OMe-CA 5b and 10b, the opposite correlations were observed. Phosphatidylcholines 5a and 10a were signicantly more active than 5b and 10b.
The results of the antiproliferative activity of the previously reported phosphatidylcholines containing anisic/veratric acids 18 and those with cinnamic/3-methoxycinnamic acids presented here conrmed that some carcinoma cell lines, such as MV4-11, MCF-7, and LoVo, seemed to be more sensitive to the studied conjugates than the other ones, like HepG2. Based on our results, it can be concluded that phospholipids with Omethylated derivatives of benzoic acid are more active in the form of 1-acyl-LPC than phosphatidylcholines with CA and 3-OMe-CA residues mainly in the form of heterosubstituted PC. However, it was difficult to determine the impact of the presence and the position of methoxy group in the benzene ring on the activity of the novel phospholipids. For this purpose, more PC derivatives should be synthesized and tested.
The phospholipid derivatives of cinnamic and 3-methoxycinnamic acids had also high antiproliferative activity against the doxorubicin-resistant LoVo/DX cell line. Resistance index (RI) values were calculated and the data are presented in Table 2. All of the obtained phosphatidylcholines were able to overcome drug resistance, especially 3b, and only 9a had a moderate ability to overcome drug resistance.
The effect of compound 5a on the cell cycle of the MV4-11 cells In the next step of the study, the most active compound 1-palmitoyl-2-cinnamoyl-sn-glycero-3-phosphocholine (5a) was chosen. The cell cycle of leukemia MV4-11 cells was analyzed aer 72 h treatment of compound 5a in the concentration 50 mM (Fig. 1).
This compound arrested cell cycle in the G2/M phase (which was statistically signicant in comparison to the control cells, p < 0.05) and lowered the percentage of cells in G0/G1 and S phase (which was statistically not signicant in comparison to the control cells).
The effect of derivative 5a on the mitochondrial membrane potential and cell death of the MV4-11 cells The changes of mitochondrial membrane potential (DJ m ) and induction of cell death of MV4-11 cells were analyzed aer treatment of 5a (50 mM) compound. Aer 72 h of treatment, a high inuence on the mitochondrial membrane potential (Fig. 2) was observed, with about 45% of cells having a signicantly lowered J m (which was statistically signicant in comparison to the control cells). Analysis of cell death with using Annexin-V and PI staining showed a lack of induction of early apoptotic (An-V+/PIÀ), apoptotic (An-V+/PI+), or necrotic (An-VÀ/PI+) cell death (data not shown).
Compound 5a was used in a concentration about its IC 50 in all types of analyses: cell cycle distribution, mitochondrial membrane potential, and apoptosis. Based on these results, we can observe that compound 5a was able to arrest cell cycle in the G2/M phase, leading to a decrease in the subpopulation in the S and G0/G1 phases and was able to decrease mitochondrial potential, but did not induce apoptosis. The blockage of the cells exposed to 5a at G2/M implied an inhibition of mitosis and resulted in cell proliferation inhibition. The lack of apoptosis or necrosis induction may suggest that compound 5a acted rather as a cytostatic agent. On the other hand, the decreased J m may suggest a mitochondrial autophagy. 22,23 However, further studies are needed to explore the exact mechanisms of action of the tested compound.
Analysis
Column chromatography was performed on silica gel 60 with 0.1% of CA (230-400 mesh ASTM, Merck) using a solvent mixture of CHCl 3 /CH 3 , v/v/v) as the developing system. Products were detected by spraying the plates with a solution of 10 g of Ce(SO 4 ) 2 and 20 g of phosphoromolibdenic acid in 1 L of 10% H 2 SO 4 followed by heating or 0.05% primuline solution acetone/H 2 O (8 : 2, v/v) followed by UV (365 nm) visualization. All the NMR spectra were recorded using a Bruker Avance II 600 MHz spectrometer (Brüker, Billerica, MA, USA). High-resolution mass spectra (HRMS) were obtained using an electron spray ionization (ESI) technique on a Waters ESI-Q-TOF Premier XE spectrometer.
Chemical synthesis
The cadmium chloride complex (GPC Â CdCl 2 ) was obtained according to the procedure previously described. 24,25 Synthesis of symmetrically substituted phosphocholines (3ab) containing the CA and 3-OMe-CA residues. The synthesis of the structured phospholipids (3a-b) was carried out according to the procedure described earlier. 18 A mixture of GPC Â CdCl 2 (0.23 mmol), aromatic acid (0.92 mmol), 4-(N,N-dimethylamino) pyridine (DMAP) (0.46 mmol), and N,N 0 -dicyclohexylcarbodiimide (DCC) (0.97 mmol) was stirred for 72 h at 40 C in anhydrous CH 2 Cl 2 (12 mL), under nitrogen and protection from light. The reaction progress was monitored by TLC. When the reaction was completed, the precipitate formed during the reaction course was ltered off and Dowex® 50WX8 H + form was added to the mixture. The solution was stirred for 30 min and then the resin was ltered off under reduced pressure and the solvent was evaporated in vacuo. The crude PC was puried by column chromatography (CHCl 3 /CH 3 Synthesis of asymmetrically substituted phosphocholines (5a-b) containing the CA and 3-OMe-CA residues in the sn-2 position. Aromatic acid (0.604 mmol) was added to a CH 2 Cl 2 solution (8 mL) containing 1-palmitoyl-2-hydroxy-sn-3-glycerophosphocholine (0.302 mmol) and DMAP (0.6 mmol). Next the DCC (1.3 mmol) dissolved in CH 2 Cl 2 (4 mL) was added to the mixture. The reaction was carried out for 72 h in a nitrogen atmosphere in the dark at 40 C. The product was extracted and puried according to the procedure described for compounds 3a and 3b.
Twenty-four hours before addition of the tested compounds, the cells were plated in 96-well plates (Sarstedt, USA) at a density of 10 4 cells per well. All cell lines were exposed to each tested phosphatidylcholines for 72 h. Cells were also exposed to the commercially available drugs cisplatin and doxorubicin. Cell lines were also exposed to the solvent used for the tested compounds (DMSO) at concentrations corresponding to those present in the tested agents' dilutions. For adherent cells, a SRB assay was performed and an MTT assay was performed for leukaemia cells.
SRB assays. The cytotoxicity assay was performed aer 72 h exposure of the cultured cells to varying concentrations (from 5 to 625 mM) of the tested agents. The cells attached to the plastic were xed in situ by gently adding 50 mL per well of cold 50% TCA (trichloroacetic acid). The plates were incubated at 4 C for 1 h and then washed ve times with tap water. The background optical density was measured in the wells lled with culture medium, without the cells. The cellular material xed with TCA was stained with 50 mL of 0.4% sulforhodamine B (SRB) dissolved in 1% acetic acid for 30 min. Unbound dye was removed by rinsing (4Â) with 1% acetic acid. The protein-bound dye was extracted with 10 mM unbuffered Tris base for determination of the optical density (at 540 nm) in a computer-interfaced 96-well microtiter plate reader Multiskan RC photometer (Labsystems, Helsinki, Finland). Each compound in the given concentration was tested in triplicate in each experiment, which was repeated 3-5 times.
MTT assays. This technique was applied for the cytotoxicity screening against leukemia cells. An assay was performed aer 72 h exposure to varying concentrations (from 5 to 625 mM) of the tested agents. For the last 3-4 hours of incubation. 20 mL of MTT solution was added to each well (MTT: 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; stock solution: 5 mg mL À1 , Sigma, Germany). The mitochondria of viable cells reduce the pale yellow MTT to a navy blue formazan: the more viable cells are present in a well, the more MTT will be reduced to formazan. When the incubation time was completed, 80 mL of the lysing mixture was added to each well (lysing mixture: 225 mL dimethylformamide, 67.5 g sodium dodecyl sulphate, and 275 mL of distilled water). Aer 24 h, when formazan crystals had been dissolved, the optical densities of the samples were read with a Multiskan RC photometer at 570 nm wavelength. Each compound in a given concentration was tested in triplicate in each experiment, which was repeated 3-5 times.
The results of the cytotoxic activity in vitro were expressed as IC 50 -the concentration of compound (in mM) that inhibits the proliferation rate of the tumor cells by 50% as compared to control untreated cells.
Cell cycle analysis. For cell cycle analysis, the cells MV4-11 were treated with the tested compound 5a dissolved in DMSO at a concentration of 50 mM. The cells were treated also only by the DMSO for comparison. Aer 72 h of incubation, the cells (1 Â 10 6 ) were trypsinized and rinsed with cold PBS. Washed cell pellets were xed for 24 h in 70% ethanol at À20 C. Aer xation, the cells were washed twice with PBS once again and resuspended in PBS. Next, RNAse (8 mg mL À1 , Fermentas, Germany) was added and the cells were incubated for 1 h at 37 C. The cells were then stained with propidium iodide (50 mg mL À1 , Sigma Aldrich, Germany) at 4 C for 30 min and the cellular DNA content was analyzed by ow cytometry using a BD LSRFortessa cytometer (BD Bioscience, San Jose, USA).
Compounds at each concentration were tested at least three times independently. The control in the assay was the cells exposed to DMSO at a concentration of 0.05%. Data were analyzed using ModFit 3.2 soware (Verity Soware, USA).
Determination of the mitochondrial membrane potential. The loss of mitochondrial membrane potential was estimated using the uorescent probe JC-1 (Sigma Aldrich, Germany). This dye binds to nucleic acids and emits red uorescence, with a spectral overlap with the orange uorescence of JC-1 aggregates. The cells were exposed to the tested compound 5a at a concentration of 50 mM. The MV4-11 cells (5 Â 10 5 ) were washed in phosphate-buffered saline (PBS) containing 2% FBS. The pelleted cells were resuspended in 100 mL of the warm cultured medium with the addition of 10 mL of JC-1 (the nal concentration of JC-1 was 3 mg mL À1 ) and were then incubated for 30 min at 37 C. Next, the cells were washed with 1 mL of PBS with 2% FBS and resuspended in 300 mL of PBS with 2% FBS.
The mitochondrial membrane potential was analyzed by ow cytometry using BD LSRFortessa cytometry. Compounds at each concentration were tested at least three times independently. The control in the assay was the cells exposed to DMSO at a concentrations of 0.05%. Data were analyzed using Flowing soware 2.5.1.
Cell death determination by Annexin V and PI staining. The MV4-11 cells were exposed to the test compound 5a at a concentration of 50 mM for 72 h. Aer incubation, the cells (2 Â 10 5 ) were washed twice with PBS. APC-Annexin V (BD Pharmingen) was dissolved to the concentration of 1 mg mL À1 in a binding buffer (HEPES buffer: 10 mM HEPES/NaOH), pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , (IIET, Poland) and the cells were suspended in 200 mL of this 1 mg mL À1 solution (freshly prepared each time). Aer the incubation in the dark (15 min) at room temperature, the solution (1 mg mL À1 ) of propidium iodide (PI) was added prior to the analysis to give a nal concentration of 0.1 mg mL À1 . Data acquisition was performed by ow cytometry using a BD LSRFortessa cytometer. Compound 5a was tested at least three times independently. The control in the assay was the cells exposed to DMSO at a concentration of 0.05%. Results were analyzed using Flowing soware 2.5.1. The data were displayed as a two-color dot plot with APC-Annexin V vs. PI. Double-negative cells were live cells, PI+/Annexin V+ were late apoptotic or necrotic cells, and PI-/Annexin V+ were early apoptotic.
Statistical analysis. Statistical analysis was performed in Statso Statistica 10. All datasets were analyzed using t-test. p-Values lower than 0.05 were considered as statistically signicant.
Conclusions
In summary, we successfully synthesized eight new phosphatidylcholines as potential chemotherapeutics in the treatment of cancers. We evaluated the antiproliferative activities of these conjugates against six human cancer cell lines. We identied that some phosphatidylcholines posses high in vitro anticancer activity and at estimated doses are not toxic toward normal BALB/3T3 cells. The highest antiproliferative activity was observed for 1-palmitoyl-2-cinnamoyl-sn-glycero-3phosphocholine (5a). Mechanistic studies showed that 5a caused cell cycle arrest in the G2/M phase, decreasing the mitochondrial membrane potential, but with no induction of apoptosis induction. Our studies suggest that the production of phospholipid derivatives containing aromatic acid residues is a convenient method to obtain more active products that can play a role as new antitumor therapeutics.
Conflicts of interest
There are no conicts to declare. | v3-fos-license |
2020-10-19T18:10:35.785Z | 2020-09-23T00:00:00.000 | 224977254 | {
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} | pes2o/s2orc | Biopellet properties of agathis wood fortified with its peeled-off bark
The agathis bark is expected to increase the energy content and durability of the resulting biopellet. The present research was aimed to analyze chemical components of agathis and to determine the quality of agathis wood biopellet fortified with it loosen bark. The klason lignin, extractives, holocellulose and α-cellulose content of wood and bark of the agathis were determined following the standard procedures of TAPPI T 222 0m-88, TAPPI T 204 0m-88, and Browning (1967), respectively. Biopellets of 15 mm diameter were prepared with pelletizing pressure of 1500 psi. The moisture content, ash content, density, durability, and calorific value of the resulting biopellet were compared to those required by DIN EN 14961-2 and 51731 standards. The α-cellulose and holocellulose content of the agathis bark were found lower than these of its wood. However, its klason lignin, extractive, and ash content were higher than these of its wood. The resulting biopellet retained the density of 0.61-0.71 g/cm3, moisture content of 0.42-4.52%, ash content of 0.79-2.73%, durability of 21.26-44.59%, and calorific value of 4,524-4,628 kcal/kg. Except for that of the biopellet density and durability, all parameters satisfied the requirements of DIN EN standards. Thermal analysis of biopellet showed that water loss occurred at 36-100°C and significant mass decomposition between 100 up to 475°C with the weight loss of 77.34-80.51%. No further decomposition was found at above 480°C.
Introduction
The reserve of fossil-based energy such as petroleum, natural gas, and coal were predicted will last only for 40, 60, and 200 years to come, respectively [1]. Therefore, the search for fossil fuel alternatives is paramount. Biomass could be the most appropriate source for a renewable and environmentally friendly energy of the future. Biomass based energy is considered free of green house gases and CO2 emission [2,3]. Its renewable nature will ascertain the sustainability of energy reserve.
In the 2012, consumption of biomass-based fuels in Indonesia was about 56.12 million tones, which was higher than that of coal with about 28.97 million tones [4]. The source of biomass energy can be from wood, kenaf, bamboo, and the waste of sawmill, forest industries, agriculture, paper products, sugar cane, and other lignocellulosic materials [5].
Biomass can be converted into gas, liquid and solid bio-fuels. The most common form of solid biofuel are briquette, biopellet, and cubes. Biopellet, which is prepared by pelletizing wood meals, is gaining popularity mainly in the European countries [6]. Biopellet demand in Sweden for instance, increased to 240% from 1997 to 2006 with a 45% increase in its price [7].
Chemicals and proximate analysis
The chemical contents of wood and bark were determined in accordance with TAPPI [16] T 204 om-88 (EtOH-Benzene extractives), TAPPI T 222 om-88 (Klason lignin), and Browning (1967) for α-cellulose and hemicellulose content. Proximate analysis was done following the ASTM E-871 [17] for the determination of moisture content, and ASTM D-1102 [18] for the determination of ash content, volatile content, and fixed carbon.
The preparation of biopellet
Biopellets with a diameter of 15 mm were prepared in a pelletizer using pelletizing pressure of 10.34 MPa. Pelletizing temperature varied at 130, 160, and 190 o C following those applied previously by Lee et al. [13] Pelletizing process was carried out for 6 min consisting of 3 min pre-heating and 3 min of heating at the predetermined pelleting temperatures. Upon pelletizing, the biopellets were conditioned for one day before the determination of the biopellets moisture content, density, ash content, durability, and the calorific value. TG-DTA of the raw materials (wood and bark) and selected biopellet (biopellet with bark content of 30% was also conducted to evaluate their thermal behavior.
The measurement of biopellet quality 2.2.3.1. Moisture content
Moisture content was calculated as a ratio between moisture weight in a sample and oven dry weight of the biopellet. Oven dry weight of biopellet was procured by oven drying at 103±2 0 C to a constant weight. DIN EN 14961-2 [19] requires the biopellets moisture content of 10%. The moisture content was calculated by the following formulae: Moisture Content (%) = Green weight (g)−Oven dried weight (g) Oven dried weight (g) x 100 (1)
Density
The density of biopellet was determined in accordance with the standard of DIN EN 51731 [20], which requires the biopellets density of 1.00-1.40 g/cm 3 . The density of biopellet was calculated by the following formulae: (2)
Ash content
Ash content (in percent) is the residue of sample combustion at 600 0 C for 4 hours expressed as percentage. DIN EN 14961-2 [19] requires the ash content of less than or equal to 3%. The ash content was calculated by the following formulae: Ash content (%) = The weight of ash (g) Oven dry weight of sample (g) x 100 (3)
Durability
A durability tester was used to determine the durability of the biopellet. In which, 100 g of biopellet were shaken at 50 rpm for 10 min. Upon shaking, the biopellets were sieved in a 20 mesh screen. The biopellets remained on the 20 mesh screen were then weighed. DIN EN 14961-2 [19] requires the minimum durability of 96.5%. The durability of the biopellet was calculated following the formulae of: The weight of biopellet retained on 20 mesh sieve (g) 100 (g) x 100 (4)
The calorific value
Bomb Calorimeter Parr 6400 was used to determine the calorific value of the biopellet. The analysis was involving the use of oxygen (450 psi), nitrogen (80 psi) and benzoic acid. The calorific value of the biopellets was calculated following the formulae of: where: W = equivalent energy value of benzoic acid t = temperature increase differential ( 0 C) e1 = chrom nickel wire correction e2 = sulphur correction e3 = nitric acid correction m = sampel mass (gram) 2.2.4. Thermal analysis Thermographimetric analysis (TGA) and differential thermal analysis (DTA) using Thermal Gravimetric/ Differential Thermal Analyzer (TG/DTA) SII EXTAR 7300 (Hitachi High-Tech Science Corporation, Tokyo) were carried out to analyze the thermal behavior of the biopellet and its raw materials. Approximately 3-5 mg of sample was put in the sample container made of Alumina/Pt, and the analysis was done with the air flow rate of 50 cm 3 /min and the heating rate of 10 0 C/min until the temperature of 1200 0 C.
Data analysis
Data were analized using a factorial completely randomized design with 3 factors, i.e. particle size (A) with 2 levels (20-40 mesh and 40-60 mesh), percentage of peeled off bark (B) with 5 levels (0, 10, 20, 30, and 100% of bark content), and pelletizing temperature (C) with 3 levels (130, 160, and 190 o C). All experimentation was done in three replicates. The general odel of the design used to analyze the quality of biopellet such as density, moisture content, ash content, and durability was: : the score of the ith factor A (particle size), the jth factor B (percentage of peeled-off bark), and the kth factor C (peletizing temperature) μ
Yijk = μ + Ai + Bj + Ck + (AB)ij + (AC)ik + (BC)jk + (ABC)ijk + εijk
: Calorific value was analyzed with completely randomized factorial design with one factor A (the percentage of peeled-off bark in biopellet) at four levels (0, 10, 20, 30, and 100 %). The general model used was: where: Yi : the score of the ith level factor A (the percentage of peeled-off bark) μ : mean value Bi : effect of the ith level factor A εi : the error associated with Yi P-value was used as the test criteria. P-value < 0.05 indicates that the effect of treatment is significant at the level of confidence of 95% and P-value > 0.05 indicates that the effect of treatments is insignificant at the level of confidence of 95%. Duncan's multiple range test was carried out to evaluate the differences among treatments.
Chemical components of agathis wood
The content and types of the chemical components of wood influence its energy characteristics. Table 1 indicates the chemical compositions and proximate analysis of agathis wood and its bark. It can be seen (from Table 1) that Klason lignin, extractives, and fixed carbon of the bark are higher than those of wood are. The Klason lignin and extractives content of the peeled-off bark of agathis is higher than the lignin and extractives content found in the bark of Pinus sylvestris and Picea abies by Miranda et al. [21] that was 26.8-32.9 % and 5.0-5.3%, respectively.
Carbon content of biomass determines its calorific value, in which increasing carbon content tends to increase the calorific value. Lignin is a phenolic compound consist of phenylpropane units with a relatively high content of carbon. Extractives such as wax, protein, sugar, pectin, resin, terpene, starches, and saponin are also carbaneous compound [22]. Ma et al. [23] reported that the carbon content of lignin, cellulose, and non-structural cellulose was 63-66%, 44%, and 44%, respectively. Therefore, calorific value of lignin and extractives is commonly higher than that of cellulose and hemicellulose. The calorific value of cellulose, lignin and extractives is of 4150-4350 kkal/kg, 6100 kcal/kg dan 8500 kcal/kg, respectively [24]. Based on the content of its lignin, extractives, and fixed carbon, the peeled-off bark of agathis should be a good fortifier for increasing the calorific value of its wood biopellet. However, the benefit of higher lignin content of the bark to the calorific value of biopellet could be counterweighed by its higher ash and moisture content. Ash and moisture content adversely influence the calorific value of biomass. Table 2 indicates that biopellet with the highest moisture content was found on the biopellet produced from bark with 40 mesh particle size and peletized at 130 o C, while the lowest was from biopellet prepared from wood with particle size of 60 mesh peletized at 190 o C. All biopellet fulfiled the DIN EN 14961-2 [19] standard that require the moisture content of equal or less than 10%.
Analysis of variance (Table 3) indicates that particle size, bark content, pelletizing temperature and all interaction of factors significantly influenced the biopellet moisture content. Further evaluation using mean range test of Duncan indicates that the moisture content of biopellet produced from raw material with particle size of 60 mesh, 20% bark content and pelletized at 130 o C was significantly lower than that produce with the same particle size and bark content, but pelletized at 160 o C and 190 o C. The moisture content of biopellet with 160 o C and 190 o C pelletizing temperature was insignificantly different. Therefore, biopellet produced from particle size of 60 mesh, 20% bark content and pelletized at 130 o C is the most acceptable in term of moisture content, despite all pelletizing temperature applied fulfilled the DIN EN 14961-2 [19] standard requirement. It seemed that producing biopellet with smaller particle size and pelletizing at higher temperature tended to lower the moisture content. The moisture content of biopellet prepared from teak, acacia and sengon wood also decreased with increasing pelletizing temperature [14].
Density
Biomass is bulky and relatively low in energy content base on its volume. These properties bring about the biomass transportation and storage is inefficient. Densification or pelletizing will increase the density and the energy content of biomass. The density of biomass influences the density of the resulting biopellet. The density of agathis wood is in the range of 0.47-0.48 g/cm 3 [10]. In the present works, the density of the produced biopellet was of 0.61-0.71 g/cm 3 . It is far below the DIN EN 51731 standard [20] requirement of 1.00-1.40 g/cm 3 .
The influence of particle size, bark content, pelletizing temperature and interaction of factors can be seen in Table 3. The effect of bark content on the density of biopellet reported by several researchers is opposing, somehow. The present research indicates that bark tended to increase biopellet density; however, Filbakk et al. [11] found that bark insignificantly influenced the density of pine wood biopellet. The highest density of biopellet was resulted from biopellet made of 100% bark with particle size of 60 mesh pelletized at 160 o C, and the lowest density was resulted from biopellet made of 100% bark with ICFP 2020 IOP Conf. Series: Materials Science and Engineering 935 (2020) 012047 IOP Publishing doi:10.1088/1757-899X/935/1/012047 7 particle size of 40 mesh pelletized at 130 o C. Higher lignin content of bark and higher surface area of smaller particle size could be the origin of this occurrence. Duncan MRT indicated that the biopellet density using 20% and 30% bark content with particle size of 60 mesh pelletized at 130 o C was insignificantly different. Therefore, considering the relatively limited amount of bark, using 20% bark as fortifier is more efficient than that using 30% bark. Furthermore, using lower pelletizing temperature will be less costly in term of energy input during biopellet production processes. Refining process and pelletizing at high temperature are the most energy consuming in the production of biopellet. Data in Table 3 also indicates that temperature in interaction with bark content and particle size tends to be more influential on the density of biopellet at higher bark content. Table 3 shows that the R-squared for biopellet density is 69%. This implies that in addition to particle size, bark content, and pelletizing temperature, other factors (at the level of 31%) also influenced the density of the biopellet. Pelletizing pressure is an important influencing factor that determines biopellet
Ash content
Ash reduces the quality of biopellet. The ash content of the presently produced biopellet was in the range of 0.79-2.73 % (Table 2), well below the requirement of DIN EN 14961-2 standard [19] that requires an ash content of less than 3%. The ash content was mainly influenced by the bark content used in the preparation of the biopellet. Increasing bark content increased the ash content of the resulted biopellet. Using a 100% bark to produce biopellet increased ash content nearly to the same level of the ash content of bark (2.7%). The relatively low ash content of the presently produced biopellet could be a new source of renewable and environmentally friendly solid fuel. It is a better solid fuel compared to coal-based fuel [2], where coal combustion emits 18.1-19.2 % ash of its weight [25].
Analysis of variance (Table 3) idicates that pelletizing temperature insignificantly influenced the ash content of biopellet. Biopellet with 30% bark content made of 60 mesh particle size at pelletizing temperature of 130 o C was higher than that with 20% bark content prepared under similar particle sice and pelletizing temperature. Therefore, using 20 bark content will be a better choice in prpeparing biopellet from agathis wood. Refining the raw material to a higher degree also significantly increased the ash content. Biopellet made from particle size of 60 resulted in a higher biopellet ash content compared to that of biopellet made from particle size of 40. Similar results were reported by Miranda et al. [21] in the production of biopellet with Pinus sylvestris and Picea abies woods. These authors concluded that ash is biopellet commonly related to the presence of nitrogen, kalsium and potasium in the raw materials.
Durability
Durability is indicative of the biopellet resistance to an external impact during transportation process. Biopellet durability is very important in storage and for long distance transportation. The durability of the presently produced biopellet was in the range of 21.26-44.59% (Table 2), which was far below the requirement of DIN EN 14961-2 standard [19] that required the durability of 96.5% or higher. Biopellet durability is related to pelletizing pressure and density. A 10.34 MPa pressure used in the present works brought about a low density and consequently low biopellet durability. The pelletizing pressure used was assumed inadequate to form a good interparticle bonding in the biopellet. Table 3 shows that particle size, bark content, pelletizing temperature, and interaction of factors significantly influenced the durability of biopellet. The durability of biopellet prepared with 60 mesh particle size was higher than that prepared from particle size of 40 mesh, which is in accordance with ICFP 2020 IOP Conf. Series: Materials Science and Engineering 935 (2020) 012047 IOP Publishing doi:10.1088/1757-899X/935/1/012047 9 the previous finding of Lee et al. [13]. Higher durability of biopellet produced with 60 mesh particle size is possibly caused by the higher surface area of the finer particle that enable the particle to form a better interparticle bonding. Biopellet with 20% bark content pelletized at 130 o C is the most preferable in term of its durability. The glass transition (Tg) temperature of lignin is slightly varied among lignocellulosic materials. The Tg of eucalypt and pinewoods for instant was found in the range of 120-140 o C [26]. Pelletizing at 130 o C might be the right temperature to melt lignin without degrading its polymer structure, thus it forms a good adhesive for interparticle bonding. Possible degradation of lignin structure at higher pelletizing temperature could be the origin of the reduced biopellet durability. Table 5 shows that agathis wood biopellet with an acceptable calorific value can be produced by the addition of 10-30 % bark, particle size of 60 mesh, and pelletizing temperature of 190 o C. Even though a very high calorific value can be acquired by preparing biopellet purely with bark, the availability of the bark would prevent the sustainability of the biopellet production. Table 5 that biopellet with 20% bark content is preferred for biopellet production, despite its calorific value is lower than that of biopellet prepared merely of bark (100% bark content). As stated previously, the relatively limited quantity of bark produce by the agathis stands hinders the production of biopellet merely made of the barks. The increase of calorific value by increasing bark content in the biopellet has been well known. Bark contains a higher amount of lignin and extractives compared to that of the wood. Lignin and extractives are carbonous substances with relatively high calorific value. Therefore, calorific value of biopellet is positively correlated to the lignin Klason and extractives content [27].
Thermal analysis
Thermal analysis was done for wood biopellet (0% bark content/B0), biopellet with 30% bark content (B30) and biopellet of bark (100% bark content/B100). The thermogravimetric of the B0, B30, and B100 is depicted in Figure 2, 3, and 4, respectively. Degradation pattern of biopellet follows the commonly occurred for biomass that consists of three steps [28][29][30]. Evaporation of water (moisture loss) occurred at the first step, and followed by the decomposition of cellulose, hemicellulose and lignin in the second step. The third step is the residual combustion (burnout residue) characterized by a constant mass loss. Decomposition in the second step consists of decomposition I (volatilization) and decomposition II (burning) [28]. Thermogravimetric in Figure 2, 3, and 4 and Table 6 indicate that water loss occurred at 36-100 o C. That was a little lower than 125 o C as reported by several previous works [28][29][30][31]. The peak temperature of B100 was higher than these of B0 and B30, which could be brought about by the higher moisture content of biopellet made of 100% bark. Decomposition of biopellet occurred at 100-476 o C with total mass loss of 77.34-80.15%. Table 6 shows that mass loss at the first decomposition was of 45.90-50.58% and at the second decomposition the mass loss was of 27.33-34.40%. The mass loss temperature at the first decomposition occurred at 100-359 o C, where all of carbohydrate decomposed. Hemicellulose decomposes at the temperature of below 275 o C and cellulose at 200-340 o C and lignin at 250-360 o C [30]. Mass loss of B100 at the first was the lowest due to its lowest carbohydrate content. At the second mass loss that occurred at 350-474 o C, lignin is much decomposed. Because of the lignin content of B100 is higher than these of B0 and B30, the highest mass loss was experienced by B100. Mass loss pattern in the present results is similar than that found by Gil et al. [29] for pinewood, eucalypt, chestnut, and cellulose residue which was of 60-70% at the first decomposition and of 25-30% at the second decomposition. Mass loss of biomass generally occurred up to 500 o C [31]. In the present results, a constant mass loss occurred at 468-476 o C. Considering the temperature of mass loss, pelletizing temperature should be carried out at the temperature of below 200 o C in order to increase energy density and avoid significant mass loss.
Conclusion
The peeled-off bark of agathis wood can be used to fortify agathis wood biopellet with an acceptable quality and calorific value at 20% bark content. Except for the biopellet density and durability, all biopellet quality parameters satisfied the requirement of DIN EN 14961-2 and 51731 standard. Considering the bark resources and eficiency of pelletizing temperature, the most acceptable biopellet quality could be prepared from particle size of 40-60 mesh with 20% of bark content and pelletizing teperature of 130 o C. Based on thermal properties of the bark, wood and biopellet of the agathis wood, pelletizing temperature should be approximatly at 140 o C. | v3-fos-license |
2014-10-01T00:00:00.000Z | 2001-01-01T00:00:00.000 | 17069677 | {
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} | pes2o/s2orc | A Simple Method for Synthesis of Active Esters of Isonicotinic and Picolinic Acids
A method for preparation of the p-nitrophenyl-, N-hydroxysuccinimidyl- and pentafluorophenyl esters of isonicotinic and picolinic acids from the corresponding acids is reported.
Introduction
In connection with ongoing synthetic work, we needed access to good acylating reagents based on isonicotinic and picolinic acids. Acylations with isonicotinoyl and picolinoyl chlorides were found to be troublesome, because the acid chlorides are only prepared or available as the corresponding hydrochlorides, which are only sparingly soluble in inert solvents. A further complication was, that upon reaction with polyamines such as poly(propyleneimine) dendrimers incomplete reactions occurred due to extensive salt formation. Therefore we turned our attention to the so called active esters [1].
Results and Discussion
Searching the literature we found references to the p-nitrophenyl esters of isonicotinic [2,3] and picolinic acid [2,3,4], which have been used for the synthesis of potent antagonists of the luteinizing hormone-releasing hormone (LHRH) [4,5]. The only other ester reported was the pentachlorophenyl ester of isonicotinic acid [6], however in the latter case, no experimental details were given. All the procedures were based on the DCC coupling of the acids with the corresponding phenol, however especially in the case of picolinic acid the coupling reaction was hampered by the well known rearrangement of the intermediate O-acylureas to N-acylureas [2]. This problem could be solved by performing the coupling reaction in CH 2 Cl 2 , but again no experimental details were given [2]. We were primarily interested in obtaining the more reactive [1] N-hydroxysuccinimidyl and pentafluorophenyl esters, and since DCC-coupling mainly gave the corresponding N-acyl-N,N'-dicyclohexylureas, the present method was developed. Reaction of the corresponding pyridinecarboxylic acid with SOCl 2 catalyzed by DMF [7] at room temperature gives the acid chlorides as their hydrochlorides. These were reacted with 4-nitrophenol, pentafluorphenol and N-hydroxysuccinimide in THF with triethylamine as base to give the corresponding active esters. The compounds prepared are shown Table 1.
Conclusions
A convenient method has been developed for the synthesis of a variety of active esters of isonicotinic and picolinic acids.
Experimental
General 1 H-and 13 C-NMR spectra were obtained using a Varian Gemini 300 NMR or a Varian Unity 400 NMR. Mass spectra were obtained using a Jeol JMS-HX 110 A Tandem Mass Spectrometer. IRspectra were recorded on a Perkin Elmer 1760 X. All melting points are uncorrected. The elemental analyses were performed by Mrs. Karin Linthoe, Department of Organic Chemistry, University of Copenhagen. All chemicals were used as received.
Isonicotinoylchloride hydrochloride
Thionylchloride (60 mL) was added carefully to a stirred mixture of isonicotinic acid (24.6 g; 0.2 mol) and DMF (1 mL). A lively gas evolution started, and after 30 minutes all of the acid had gone into solution and the temperature had risen to about 40°C. Excess thionyl chloride was removed in vacuo, and diethyl ether (200 mL) was stirred into the residue. The crude product was filtered, washed with diethyl ether and dried in vacuo at 40°C. Yield: 35.0 g (98%) of white crystals sufficiently pure for the preparation of the activated esters.
Isonicotinic acid pentafluorophenyl ester
To a stirred suspension of isonicotinoylchloride hydrochloride (8.9 g; 0.05 mol) and pentafluorophenol (9.2 g; 0.05 mol) in THF (100 mL) was added triethylamine (20 mL; 0.14 mol) over 10 minutes. The suspension was stirred at room temperature for 12 hrs, filtered and concentrated in vacuo. The residue was dissolved in hexane (300 mL), treated with activated carbon and filtered. The hexane was removed to give the pentafluorophenyl ester as a slightly brownish oil, which crystallized to give a creme colored solid melting 52-54°C. Yield: 14.0 g (97%); 1
Isonicotinic acid N-hydroxysuccinimidyl ester
Prepared according to the general procedure with the exception that the crude product was crystallized from 2-propanol to give the ester in 84% yield. Mp. 140-141°C. 1 | v3-fos-license |
2020-11-20T14:07:47.850Z | 2020-11-19T00:00:00.000 | 227068216 | {
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} | pes2o/s2orc | Maltol prevents the progression of osteoarthritis by targeting PI3K/Akt/NF‐κB pathway: In vitro and in vivo studies
Abstract Osteoarthritis (OA), a prevalent degenerative arthritis disease, principle characterized by the destruction of cartilage and associated with the inflammatory response. Maltol, a product formed during the processing of red ginseng (Panax ginseng, CA Meyer), has been reported to have the potential effect of anti‐inflammatory. However, its specific mechanisms are not demonstrated. We investigated the protective effect of maltol in the progression of OA both in vitro and in vivo experiments. Human chondrocytes were pre‐treated with maltol (0, 20, 40, 60 μM, 24 hours) and incubated with IL‐1β (10 ng/mL, 24 hours) in vitro. Expression of PGE2, TNF‐α and NO was measured by the ELISA and Griess reaction. The expression of iNOs, COX‐2, aggrecan, ADAMTS‐5, MMP‐13, IκB‐α, p65, P‐AKT, AKT, PI3K and P‐PI3K was analysed by Western blotting. The expression of collagen II and p65‐active protein was detected by immunofluorescence. Moreover, the serious level of OA was evaluated by histological analysis in vivo. We identified that maltol could suppress the IL‐1β‐stimulated generation of PGE2 and NO. Besides, maltol not only suppressed the production of COX‐2, iNOs, TNF‐α, IL‐6, ADAMTS‐5, MMP‐13, but also attenuated the degradation of collagen II and aggrecan. Furthermore, maltol remarkably suppressed the phosphorylation of PI3K/AKT and NF‐κB induced by IL‐1β in human OA chondrocytes. Moreover, maltol could block the cartilage destroy in OA mice in vivo. To date, all data indicate maltol is a potential therapeutic agent by inhibiting inflammatory response via the regulation of NF‐κB signalling for OA.
found a series of factors influence the development of osteoarthritis including ageing, trauma, obesity, inflammation, joint malformation and osteoporosis. 4 However, the pathogenesis of OA is still murky. It is confirmed that inflammation and oxidative stress are vital risk factors in the progression of OA. 5 Many factors of OA factors also can imbalance the oxidant-antioxidant levels and then stimulates chondrocytes to produce inflammatory cytokines. [6][7][8] Inflammation cytokines including tumour necrosis factor-α (TNFα) and interleukin-6 (IL-6) are involved in the pathogenesis of OA. 9 IL-1β boosts the degradation of extracellular matrix (ECM) in the way of inducing the release of pro-inflammatory mediators, such as nitric oxide (NO), prostaglandin E2 (PGE2), matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase thrombospondin motifs (ADAMTS). 10,11 Therefore, anti-inflammatory has been demonstrated as a valid therapeutic strategy for attenuating the development of OA.
The NF-κB signalling pathway has been demonstrated to play a core role in catabolism and inflammatory response. 12 When initiated by certain stimuli such as IL-1β, the action of IκBα is caused by a succession of membrane-proximal events. 13 NF-κB p65 translocates from the cytoplasm to the nucleus, which stimulates the production of inflammatory genes, including TNF-α, iNOS, PGE2, NO, COX-2, IL-6, ADAMTS and MMPs. 14 Many studies found that inhibition of NF-κB could attenuate the development of OA by suppressing the activity of the PI3K/Akt signalling pathway, which is one of the most significant upstream factors of the NF-κB signalling pathway. 15,16 They all related to the degradation of the extracellular matrix.
Consequently, the PI3K/AKT/NF-κB signalling pathway is a potential therapeutic option in the progression of OA.
Maltol (3-hydroxy-2-methyl-4-pyrone), a safe and reliable flavour enhancer, is a product of the Maillard reaction of heated-processed ginseng. 17 It is also existed in the roasted Korean ginseng root. 18 In these years, it is recognized that maltol prevents renal injuries by suppressing PI3K/AKT signalling pathway. 19 What's more, maltol has the protective property for oxidative stress such as glaucoma and attenuates liver apoptosis inflammation by inhibiting the NF-κB signalling pathway. 20 Nevertheless, the protective effect of maltol in OA is still obscure. In our research, we demonstrated whether maltol exerted a protective role in OA progression and explored the potential mechanism both in vitro and vivo.
| Primary human chondrocyte isolation and culture
Human articular cartilage tissue collection with OA was performed by the Medical Ethical Committee of the Second Affiliated Hospital Firstly, human cartilage tissues were procured from ten OA patients (age 54 ± 8 years, five men and five women) who had undergone total knee replacement surgery at the Second Affiliated Hospital of Wenzhou Medical University. Secondly, cartilage tissues were cut into 1 × 1 × 1 mm 3 pieces and washed with PBS for three times.
The pieces were in collagenase II (2 mg/mL) for 4 hours at 37°C.
After centrifuging at 800 rpm for 6 minutes, the cells were cultured in DMEM/F12 medium with 10% FBS under an atmosphere containing 5% CO2 at 37°C. The cells were changed every other day. The human cells were passaged using 0.25% trypsin-EDTA solution at 80 to 90% confluence.
| CCK-8 assay
The cytotoxicity of Maltol on human chondrocytes was assayed by the Cell Counting Kit-8 obeying the protocols of the manufacturer.
| Griess reaction and ELISAs
The cells (3 × 10 5 cells/mL) were cultured in 6-well plates, pretreated with maltol (0, 20, 40 or 60 μM). After 24 hours, they were added IL-1β (10 ng/mL) and then incubated for 24 hours. The content of NO in each well was appraised by using the Griess reaction. The concentration of PGE 2 , tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in each well was detected by ELISA kits.
| Western blotting
The expression level of protein was measured by Western blotting. The proteins were separated by using RIPA lysis buffer (1 mM PMSF) and then sonicated on ice for 10 minutes and then centrifuged at 4000 g at 4°C for 15 minutes. Then, the protein concentration was evaluated via the BCA protein assay kit (Beyotime).
After blocking with 5% non-fat milk for 3 hours, the obtained mem- and p-PI3K (1:2000). The following step was that incubating the secondary antibodies at room temperature for 2.5 hours. The blots were visualized via the Imaging System (Bio-Rad) followed by washing with TBST 3 times.
| Analysis of immunofluorescence
The cells rinsed with PBS and treated with 4% paraformaldehyde fixation for 15 minutes; then, the wells were washed with PBS 3 times again. Then, we treated with 0.1% Triton X-100 diluted in PBS for 15 minutes at indoor temperature. Next, human chondrocytes were blocked with 10% goat serum and incubation with primary antibodies against collagen II (1:300) and p65 (1:300) for the whole night at the temperature of 4°C. The next day, cells were exposed to Alexa Fluor ® 594 and Alexa Fluor ® 488-labelled conjugated secondary antibodies (1:400) for 1.5 hours. Finally, the cells were exposed to DAPI (Beyotime) for 1 minutes. Ultimately, cell samples were detected on the Olympus fluorescence microscope. The fluorescence intensity was observed by using Image J software.
| X-ray imaging assay
The mice took on the X-ray machine After 8 weeks of experiments.
We performed X-ray imaging on all of the mice to assess osteophyte formation, the joint space and superficial cartilage changes detected by a digital X-ray machine (KUB Technologies Inc.) with the following settings: 50 Kv and 160 μA.
| Mice OA models
Forty-five ten-week-old C57BL/6 male wild-type (WT) mice were After DMM, the maltol treatment group received a dose of maltol for 100 mg·kg −1 ·day −1 (dissolved in saline, administered by oral gavage every day for 8 weeks). 44 Simultaneously, mice in the DMM alone group received an equal amount of saline. All animals (15 mice/group) were killed 8 weeks after the surgery, and cartilage samples were collected for immunological and histological analysis.
| Histopathologic analysis
Safranin-O/Fast Green was used to evaluate the articular cartilage destruction. Then, we utilized a light microscope to assess the morphologic changes of mouse chondrocytes and surrounding tissues, and the Osteoarthritis Research Society International (OARSI) scoring system was used to assess the extent of cartilage destruction as described previously.
| Immunohistochemical assay
The Knee joints were fixed in 4% paraformaldehyde, decalcified, embedded in paraffin, and cut into 7-μm sections that were deparaffinized, rehydrated. Then, the sections were treated with 3% (v/v) hydrogen peroxide and 0.25% trypsin-EDTA solution at 37°C for 30 minutes. Next, the histological sections were incubated 10% bovine serum albumin for 60 minutes at 37°C. Treated with the primary antibody against Collagen II and P65 for 24 hours at 4°C. On the second day, the sections were incubated with HRP-conjugated secondary antibody for 1 hour at 4°C. Images were analysed by Image-Pro Plus software, version 6.0 (Media Cybernetics). Five sections from each group were used for quantitative analysis.
| Statistical analysis
The experiments were required to be at least performed five times. The data obtained are expressed as the mean ± standard deviation. Statistical analyses were performed using GraphPad Prism version 5.0 software (GraphPad software, San Diego, CA, USA). Inter-group comparisons were performed using a one-way ANOVA followed by the Tukey test.
Probability values of P < .05 were considered statistically significant.
| Effect of maltol on human chondrocytes viability
The chemical structure of maltol is shown in Figure 1A. Human chondrocytes were treated with various concentrations of maltol (0, 10, 20, 40, 60, 80, 100 µM) for 24 hours and 48 hours to measure the maltol cytotoxicity by CCK-8 assay. As shown in Figure 1B,C, no cytotoxic effect of maltol were found at the dose of 0-60 µM (P < .01).
Whereas the cell viability was significantly decreased at 80 µM at 24 hours. Therefore, a related lower concentration of maltol (0, 20, 40 or 60 μM) was utilized for the following experiments.
| Effect of maltol on the expression of PGE2, NO, IL-6, TNF-α, COX-2 and iNOS in IL-1β-induced human OA chondrocytes
We ascertained the effect of maltol on COX-2 and iNOs induced by IL-1β detected through qRT-PCR and Western blotting. IL-1β boosted the expression of COX-2 and iNOS at protein and mRNA level (Figure 2A,C,D). Moreover, IL-1β increased the generation of PGE2 and NO, whereas maltol could down-regulated these inflammation mediators in a dose-dependent manner (20, 40 and 60 μM).
In addition, the results of ELISA and qRT-PCR showed that maltol suppressed the generation of IL-6 and TNF-α in a dose-dependent manner, which was increased after IL-1β stimulation ( Figure 2B,F).
These results suggested that maltol could dramatically suppress the
| Effect of maltol on the ECM degradation induced by IL-1βin chondrocytes
To determine the maltol on IL-1β-induced ECM degradation, we investigated the effect of maltol on aggrecan, collagen II, ADAMTS-5 and MMP13 via using Western blotting analysis. As shown in Figure 3A,B, human chondrocytes exhibited the apparent downregulation of mRNA and protein expression of type II collagen and aggrecan following IL-1β-induced. On the contrary, IL-1β enhanced ADAMTS-5 and MMP-13 production ( Figure 3A,B). Nevertheless, treatment with maltol reserved the destructive effects of IL-1β treatment in a dose-dependent manner. Moreover, the immunofluorescence outcomes indicated that maltol attenuated the degradation of collagen II ( Figure 3C,D). Therefore, these data covered that maltol could prevent ECM degradation induced by IL-1β in human OA chondrocytes.
| Effect of maltol on IL-1β-induced NF-κB activation in chondrocytes
To elucidate the role of maltol in anti-inflammatory function, we utilized Western blotting to evaluate the effect of maltol on the NF-κB signalling pathway in human chondrocytes. We, respectively, assessed the protein levels of IκBα and p65 in human chondrocytes. As shown in Figure 4A,B, IL-1β stimulation triggered the descending of IκBα and the increase of p65, which resulted from p65 translocating from the cytoplasm into the nucleus. However, the maltol outstandingly inhibited the above action in a dose-dependent manner (20, 40, 60 μM). Moreover, we performed immunofluorescence staining of p65 and then the image indicated that maltol mitigated the nuclear translocation of p65 induced by IL-1β ( Figure 4C). In brief, these results illustrated that maltol has an inhibitory effect on NF-κB activation induced by IL-1β.
| Effect of maltol on IL-1β-induced PI3K and AKT phosphorylation
PI3K is involved in the inflammatory induced by IL-1β and plays a key role in the action of Akt. To determine the effects of maltol on the PI3K/Akt axis, Western blotting was adopted to evaluate PI3K and AKT phosphorylation induced by IL-1β. As Figure 5A,B described, IL-1β stimulation notably increased the phosphorylation of AKT and PI3K, whereas maltol treatment reversed the phosphorylation of PI3K/AKT induced by IL-1β.
| Maltol inhibits the degradation of cartilage in a mouse DMM model
We conducted a surgical operation to establish the OA mice model through destabilizing the medial meniscus. To evaluate whether maltol has the protective effects on OA progression in vivo, we used Safranin-O staining and X-ray to assess cartilage histological analysis of OA. Besides, the surgery increased cartilage surface density and narrowed the joint space. However, the maltol group improved the above injuries ( Figure 6A). As shown in Figure 6B, Figure 6C).
| Effect of maltol on MMP13 and Collagen II production in OA articular cartilage
To demonstrate the effects of maltol in vivo, we carried out immunohistochemical staining to detect the expression of MMP 13 and Collagen II. As illustrated in Figure 7A,B, the results of immunohistochemical staining manifested that the quantity of Collagen II in the DMM group was less than the sham group and the quantity of MMP 13 in the DMM group was more than the sham group. Fortunately, maltol treatment decayed the condition.
| D ISCUSS I ON
Various evidence has verified inflammation has a significant influence on OA. 2,22,23 Hence, many anti-inflammatory drugs had been used to retard the process of OA. Compared to non-steroidal antiinflammatory drugs that only ameliorate clinical symptoms with deleterious side effects, an effective and plant-derived compound with minor side effects have raised interest in the treatment of OA. 24,25 Maltol, a dainty food-flavouring agent, is notable for its medicinal properties, including anti-inflammatory, anti-oxidative and more effects. 26 In our study, we revealed that maltol could reduce inflammatory responses and inhibited the degradation of ECM (collagen II and aggrecan) in human chondrocytes. In addition, our study demonstrated that maltol dramatically blocks the NF-κB pathway regulated by the PI3K/AKT signalling pathway. Moreover, maltol improved articular cartilage injury and attenuated the development of OA in OA mice models.
F I G U R E 4
Maltol attenuated IL-1βinduced the NF-κB signalling pathway. The protein level and quantification analysis of p65 and IκBα in human chondrocytes were assayed by Western blotting (A-B). Immunofluorescence of active protein of p65 was detected by a fluorescence microscope (OLYMPUS) (Scale bar: 10 μm) (C). Data represented are the mean values ± SD ## P < .01, vs control group; *P < .05, **P < .01, vs IL-1β-alone treatment group, n = 5 The NF-κB signalling pathway, a classical pro-inflammation pathway, has been demonstrated play a key role in the regulation of inflammatory mediators associated with OA development, such as COX-2, iNOs and MMPs. [27][28][29] The protein of P65 (a unit protein of NF-κB) is inactive when localized in the cytoplasm combined with IκBα, an inhibitory protein. 30 When IL-1β stimulation, the P65 was activated and then translocated into the nucleus. Finally, the active-P65 up-regulates the production of inflammatory mediators. 31,32 Previous studies reported that NF-κB p65-specific siRNA inhibited the expression of NF-κB p65, blocking the expression of iNOS, COX-2 and MMP-9 in IL-1β-stimulated chondrocytes. 33,34 Hence, targeted down-regulation of NF-κB may be deemed to cure OA effectively. Besides, the NF-κB inhibitor could decrease the IL-1βinduced expression of MMP13 in human chondrocytes. 35 As a result, in the present study, we explored whether maltol had anti-inflammatory capability on chondrocytes with the NF-κB signalling pathway. The data covered that maltol could dramatically suppress the phosphorylation of p65 in IL-1β treatment in human chondrocytes.
Moreover, suppressed the activity of the NF-κB signalling pathway could down-regulate the production of inflammatory factors, such as NO, MMPs and PGE2, to ameliorate the progression in OA. Taken together, our data, in some way, indicated that the anti-inflammatory induced by IL-1β of maltol on OA development and its potential mechanism was involved with the NF-κB signalling pathway.
Accumulated evidence showed that PI3K/AKT signalling involved in the degradation of ECM and alterations of cellular in OA pathogenesis. [35][36][37] Besides, previous studies demonstrated that PI3K/AKT signalling as an upstream element to regulate the activation of the NF-κB pathway. 38 Akt is a pivotal downstream effector of PI3K, which is an intracellular phosphatidylinositol kinase. 16,39,40 The NF-κB was suppressed via Inhibiting the PI3K/ AKT pathway. 41,42 Afterwards, the NF-κB (p65) was inhibited and subsequently abolished the expression of MMPs and COX-2. 43 Our previous study proved that maltol could suppress the PI3K/ AKT pathway and reduced the production of inflammatory cytokine. Taken together, maltol may exert inhibitory effects in OA F I G U R E 8 Schematic revealed that maltol suppressed the PI3K/Akt/NF-κB pathway and potential protective effects in the progression of osteoarthritis by preventing the IL-1β-induced inflammation via the PI3K/AKT/ NF-κB signalling pathway. The underlying mechanism was shown specifically in Figure 8. The potential mechanism of maltol was present.
In this study, we found that DMM groups presented a narrow joint space, severe cartilage erosion, vast proteoglycan loss and degradation of the ECM compared with the sham group. All of the above phenomena were ameliorated by treatment with maltol. Besides, the OARSI grade was lower when treated with maltol in the DMM mice model. These results and the intro findings altogether provide evidence that maltol can mitigate the progression in OA.
In conclusion, we covered that maltol could alleviate inflammatory response and ECM degradation in human chondrocytes via inhibiting the activation of the PI3K/Akt/NF-κB pathway. Meanwhile, in surgically induced DMM mice model, treatment with maltol performed a significant role in OA progression. Taken together, our data suggest maltol exerts protective effects against OA.
CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.
DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of the study are available from the corresponding author upon request. | v3-fos-license |
2019-12-09T07:54:10.748Z | 2019-12-01T00:00:00.000 | 208870652 | {
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} | pes2o/s2orc | Rice Protein Exerts Anti-Inflammatory Effect in Growing and Adult Rats via Suppressing NF-κB Pathway
To elucidate the effect of rice protein (RP) on the depression of inflammation, growing and adult rats were fed with caseins and RP for 2 weeks. Compared with casein, RP reduced hepatic accumulations of reactive oxygen species (ROS) and nitro oxide (NO), and plasma activities of alanine transaminase (ALT) and aspartate transaminase (AST) in growing and adult rats. Intake of RP led to increased mRNA levels, and protein expressions of phosphoinositide 3 kinase (PI3K), protein kinase B (Akt), nuclear factor-κB 1 (NF-αB1), reticuloendotheliosis viral oncogene homolog A (RelA), tumor necrotic factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and monocyte chemoattractant protein-1 (MCP-1) were decreased, whereas hepatic expressions of interleukin-10 (IL-10) and heme oxygenase 1 (HO-1) were increased by RP. The activation of NF-κB was suppressed by RP through upregulation of inhibitory κB α (IκBα), resulting in decreased translocation of nuclear factor-κB 1 (p50) and RelA (p65) to the nucleus in RP groups. The present study demonstrates that RP exerts an anti-inflammatory effect to inhibit ROS-derived inflammation through suppression of the NF-κB pathway in growing and adult rats. Results suggest that the anti-inflammatory capacity of RP is independent of age.
Introduction
Inflammation, an important response to infection and injury, is implicated in various diseases, e.g., metabolic syndrome, etc. [1]. Oxidative stress, which can be caused by the overproduction of reactive oxygen species (ROS), is one of the most potent inducers of inflammation [2]. Thus, the suppression of oxidative stress is suggested to be very useful for preventing inflammatory diseases.
As a major plant protein, rice protein (RP) has numerous physiological functions [3][4][5][6], including induction of the antioxidant response [7][8][9][10]. Consequently, rice protein can reduce oxidative stress by scavenging nitric oxide (NO) radicals, ROS (e.g., superoxide radical, hydrogen peroxide), etc. [11][12][13], which have been suggested as the major causes of inflammation. Thus, there is evidence supporting the notion that rice protein might exert an antioxidant capacity to prevent inflammation. However, until now, a comprehensive understanding of the link between the suppression of oxidative stress-induced inflammation with the intake of rice protein has not been elucidated. Particularly, although it has been demonstrated that rice protein hydrolysates can inhibit the inflammatory response in mouse leukemia cells of monocyte macrophage (RAW264.7) [14], the precise molecular mechanism and signal pathway by which rice protein prevents ROS-derived inflammation in growing and adult rats are not fully established.
Age is a major factor in inflammation. NF-κB activity has been shown to be upregulated with age, suggesting that NF-κB signaling is a culprit of inflamm-aging [15,20]. Moreover, NF-κB signaling has been recognized as one of the targets of thee phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt) pathway, which can drive the aging process [21]. Accordingly, in this study, growing (G) and adult (A) rats were used to elucidate whether rice protein can exert an anti-inflammatory effect via suppression of the NF-κB pathway.
Plasma ALT and AST Activities
After 2 weeks feeding, compared with CAS-G (growing rats fed with casein) and CAS-A (adult rats fed with casein), the activities of alanine transaminase (ALT) (Figure 1) in plasma were significantly decreased by RP-G (growing rats fed with rice protein) to the degree of 20.47% in growing rats and decreased by RP-A (adult rats fed with rice protein) to the degree of 38.41% in adult rats (p < 0.05). Similarly, as shown in Figure 1, RP-G and RP-A significantly reduced plasma aspartate transaminase (AST) activities, accounting for a decrease of 27.77% in growing rats and decrease of 40.98% in adult rats (p < 0.05). Values are the means ± SEM (n = 6). Bars marked with * are significantly different between CAS-G and RP-G (p < 0.05). Bars marked with # are significantly different between CAS-A and RP-A (p < 0.05). ALT, alanine transaminase; AST, aspartate transferase; CAS-A, adult rats fed with casein; CAS-G, growing rats fed with casein; RP-A, adult rats fed with rice protein; RP-G, growing rats fed with rice protein. Values are the means ± SEM (n = 6). Bars marked with * are significantly different between CAS-G and RP-G (p < 0.05). Bars marked with # are significantly different between CAS-A and RP-A (p < 0.05). ALT, alanine transaminase; AST, aspartate transferase; CAS-A, adult rats fed with casein; CAS-G, growing rats fed with casein; RP-A, adult rats fed with rice protein; RP-G, growing rats fed with rice protein.
Hepatic NO Levels and iNOS Activity
Compared with CAS-G and CAS-A, RP-G and RP-A significantly reduced hepatic contents of NO in growing and adult rats (Figure 2A, p < 0.05). As illustrated in Figure 2B, RP-G and RP-A significantly different between CAS-G and RP-G (P < 0.05). Bars marked with # are significantly different between 20 CAS-A and RP-A (P < 0.05). Bars marked with * are significantly different between CAS-G and RP-G (p < 0.05). Bars marked with # are significantly different between CAS-A and RP-A (p < 0.05). CAS-A, adult rats fed with casein; CAS-G, growing rats fed with casein; iNOS, inducible nitric oxide synthase; NO, nitric oxide; RP-A, adult rats fed with rice protein; RP-G, growing rats fed with rice protein.
Hepatic ROS Accumulation
As shown in Figure 3, compared with CAS-G and CAS-A, hepatic contents of ROS were reduced by RP-G to the degree of 18.27% and reduced by RP-A to the degree of 24.71% (p < 0.05). The results suggest that hepatic ROS accumulation could be inhibited by rice protein.
Compared with CAS-G and CAS-A, RP-G and RP-A significantly reduced hepatic contents of NO in growing and adult rats (Figure 2A, p < 0.05). As illustrated in Figure 2B, RP-G and RP-A significantly reduced hepatic iNOS activities by 25.71% in growing rats and by 30.00% in adult rats (p < 0.05), further supporting the results that hepatic contents of NO could be reduced by rice protein feeding. Bars marked with * are significantly different between CAS-G and RP-G (p < 0.05). Bars marked with # are significantly different between CAS-A and RP-A (p < 0.05). CAS-A, adult rats fed with casein; CAS-G, growing rats fed with casein; iNOS, inducible nitric oxide synthase; NO, nitric oxide; RP-A, adult rats fed with rice protein; RP-G, growing rats fed with rice protein.
Hepatic ROS Accumulation
As shown in Figure 3, compared with CAS-G and CAS-A, hepatic contents of ROS were reduced by RP-G to the degree of 18.27% and reduced by RP-A to the degree of 24.71% (p < 0.05). The results suggest that hepatic ROS accumulation could be inhibited by rice protein. Values are the means ± SEM (n = 6). Bars marked with * are significantly different between CAS-G and RP-G (p < 0.05). Bars marked with # are significantly different between CAS-A and RP-A (p < 0.05). CAS-A, adult rats fed with casein; CAS-G, growing rats fed with casein; ROS, reactive oxygen species; RP-A, adult rats fed with rice protein; RP-G, growing rats fed with rice protein.
Expressions of PI3K and AKT
Compared with CAS-G and CAS-A, the protein expression and mRNA levels of PI3K ( Figure 4A) were significantly decreased by RP-G and RP-A in growing and adult rats (p < 0.05). Similarly, as illustrated in Figure 4B, RP-G and RP-A significantly reduced the protein expression and mRNA levels of Akt in growing and adult rats as compared to CAS-G and CAS-A (p < 0.05).
Expressions of PI3K and AKT
Compared with CAS-G and CAS-A, the protein expression and mRNA levels of PI3K ( Figure 4A) were significantly decreased by RP-G and RP-A in growing and adult rats (p < 0.05). Similarly, as illustrated in Figure 4B, RP-G and RP-A significantly reduced the protein expression and mRNA levels of Akt in growing and adult rats as compared to CAS-G and CAS-A (p < 0.05). Values are the means ± SEM (n = 6). Bars marked with * are significantly different between CAS-G and RP-G (p < 0.05). Bars marked with # are significantly different between CAS-A and RP-A (p < 0.05). AKT, protein kinase B; CAS-A, adult rats fed with casein; CAS-G, growing rats fed with casein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PI3K, phosphoinositide 3 kinase; RP-A, adult rats fed with rice protein; RP-G, growing rats fed with rice protein.
Expressions of NF-κB
After 2 weeks of feeding, the inhibition of PI3K/Akt led to significantly decreased mRNA levels of NF-κB1 ( Figure 5A) and RelA ( Figure 5B) by RP-G and RP-A, with respect to CAS-G and CAS-A. Similarly, RP-G and RP-A downregulated the protein expression of NF-κB1 ( Figure 5A) and RelA ( Figure 5B) as compared to CAS-G and CAS-A, accounting for decreases of 15.72% (NF-κB1) and 12.11% (RelA) in growing rats, as well decreases of 18.01% (NF-κB1) and 14.17% (RelA) in adult rats (p < 0.05), respectively. Values are the means ± SEM (n = 6). Bars marked with * are significantly different between CAS-G and RP-G (p < 0.05). Bars marked with # are significantly different between CAS-A and RP-A (p < 0.05). CAS-A, adult rats fed with casein; CAS-G, growing rats fed with casein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NF-κB1, nuclear factor-κB1; RelA, reticuloendotheliosis viral oncogene homolog A; RP-A, adult rats fed with rice protein; RP-G, growing rats fed with rice protein.
NF-κB Activation
In this study, the effects of rice protein on the activation of NF-κB were determined after 2 weeks feeding. . Bars marked with * are significantly different between CAS-G and RP-G (p < 0.05). Bars marked with # are significantly different between CAS-A and RP-A (p < 0.05). AKT, protein kinase B; CAS-A, adult rats fed with casein; CAS-G, growing rats fed with casein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PI3K, phosphoinositide 3 kinase; RP-A, adult rats fed with rice protein; RP-G, growing rats fed with rice protein.
Expressions of NF-κB
After 2 weeks of feeding, the inhibition of PI3K/Akt led to significantly decreased mRNA levels of NF-κB1 ( Figure 5A) and RelA ( Figure 5B) by RP-G and RP-A, with respect to CAS-G and CAS-A. Similarly, RP-G and RP-A downregulated the protein expression of NF-κB1 ( Figure 5A) and RelA ( Figure 5B) as compared to CAS-G and CAS-A, accounting for decreases of 15.72% (NF-κB1) and 12.11% (RelA) in growing rats, as well decreases of 18.01% (NF-κB1) and 14.17% (RelA) in adult rats (p < 0.05), respectively. Values are the means ± SEM (n = 6). Bars marked with * are significantly different between CAS-G and RP-G (p < 0.05). Bars marked with # are significantly different between CAS-A and RP-A (p < 0.05). CAS-A, adult rats fed with casein; CAS-G, growing rats fed with casein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NF-κB1, nuclear factor-κB1; RelA, reticuloendotheliosis viral oncogene homolog A; RP-A, adult rats fed with rice protein; RP-G, growing rats fed with rice protein.
NF-κB Activation
In this study, the effects of rice protein on the activation of NF-κB were determined after 2 weeks feeding.
Herein, we show that the inhibitory effect of rice protein in the expression levels of NF-κB can be attributed to the upregulation of both mRNA and protein levels of IκBα ( Figure 6A). As a result, a decrease in the nuclear proportion of p50 was produced by RP-G to the degree of 15.02% in growing rats and by RP-A to the degree of 18.27% in adult rats ( Figure 6B, p < 0.05). Similarly, RP-G and RP-A decreased the nuclear proportion of p65 by 12.86% in growing rats and by 14.26% in adult rats as compared to CAS-G and CAS-A ( Figure 6C, p < 0.05). Also, RP-G and RP-A respectively reduced protein expression of cytosolic p50 ( Figure 6B) and p65 ( Figure 6C) as compared to CAS-G and CAS-A. These results suggest that rice protein could suppress NF-κB activation. 121 122 123 124 Bars marked with * are significantly different between CAS-G and RP-G (p < 0.05). Bars marked with # are significantly different between CAS-A and RP-A (p < 0.05). CAS-A, adult rats fed with casein; CAS-G, growing rats fed with casein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H1.2, histone cluster 1; IκBα, inhibitory κB α; p50, nuclear factor-κB 1; p65, reticuloendotheliosis viral oncogene homolog A; RP-A, adult rats fed with rice protein; RP-G, growing rats fed with rice protein.
mediators, were markedly reduced by RP-G and RP-A, as compared to CAS-G and CAS-A (P < 0.05).
Expressions of Anti-Inflammatory Mediators
In addition to the downregulation of inflammatory mediators, after 2 weeks of feeding, the protein and gene expression of anti-inflammatory mediators, such as interleukin-10 (IL-10) and heme oxygenase 1 (HO-1), were also regulated by rice protein feeding.
As illustrated in Figure 8A, the mRNA levels and protein expression of IL-10 were significantly stimulated by rice protein feeding, accounting for enhancements of 18.26% (RP-G) to 20.05% (RP-A) in mRNA levels and 16.90% (RP-G) to 20.31% (RP-A) in protein expression with respect to CAS-G and CAS-A (p < 0.05). As shown in Figure 8B, compared with CAS-G and CAS-A, RP-G and RP-A significantly increased hepatic mRNA levels of HO-1 by 45.58% in growing rats and by 59.64% in adult rats (p < 0.05), while RP-G and RP-A upregulated the protein expression of HO-1 to the degree of 12.81% in growing rats and 17.26% in adult rats (p < 0.05). These results further supported the findings that rice protein could prevent inflammation.
Expressions of Anti-Inflammatory Mediators
In addition to the downregulation of inflammatory mediators, after 2 weeks of feeding, the protein and gene expression of anti-inflammatory mediators, such as interleukin-10 (IL-10) and heme oxygenase 1 (HO-1), were also regulated by rice protein feeding.
As illustrated in Figure 8A, the mRNA levels and protein expression of IL-10 were significantly stimulated by rice protein feeding, accounting for enhancements of 18.26% (RP-G) to 20.05% (RP-A) in mRNA levels and 16.90% (RP-G) to 20.31% (RP-A) in protein expression with respect to CAS-G and CAS-A (p < 0.05). As shown in Figure 8B, compared with CAS-G and CAS-A, RP-G and RP-A significantly increased hepatic mRNA levels of HO-1 by 45.58% in growing rats and by 59.64% in adult rats (p < 0.05), while RP-G and RP-A upregulated the protein expression of HO-1 to the degree of 12.81% in growing rats and 17.26% in adult rats (p < 0.05). These results further supported the findings that rice protein could prevent inflammation.
Discussion
The present study demonstrates that rice protein can prevent inflammation in growing and adult rats. The anti-inflammatory effects of rice protein are attributable to suppression of the activation of NF-κB.
As a crucial regulator of chronic or acute inflammatory responses in inflammation, NO can be catalyzed via iNOS [1,22,23]. Thus, the anti-inflammatory effect is generally attributed to the decrease in NO production via suppression of iNOS activity and downregulation of iNOS expression. To support this view, in this study, the generations of NO were markedly reduced by RP-G and RP-A in growing and adult rats, reflecting that the status of inflammatory damage could be suppressed by rice proteins. These results were in agreement with our in vitro studies that rice protein could scavenge NO radicals [12,13]. Accordingly, hepatic iNOS activities and the expressions of iNOS were significantly reduced by RP-G and RP-A in growing and adult rats. On the other hand, as major hepatic inflammatory biomarkers, the activities of ALT and AST were significantly reduced by RP-G and RP-A, suggesting that rice protein could efficaciously improve hepatic inflammation in growing and adult rats. Taken together, in this study, it was evident that rice protein could inhibit inflammation in growing and adult rats.
The inflammatory process can be initiated by ROS-derived oxidative stress, suggesting that the overproduction of ROS may be a major contributor to the inflammation [1]. In light of this view, the anti-inflammatory response induced by rice protein should be focused on ROS-scavenging activity. In this study, the inhibitory effect of rice protein on hepatic ROS accumulation was observed in both
Discussion
The present study demonstrates that rice protein can prevent inflammation in growing and adult rats. The anti-inflammatory effects of rice protein are attributable to suppression of the activation of NF-κB.
As a crucial regulator of chronic or acute inflammatory responses in inflammation, NO can be catalyzed via iNOS [1,22,23]. Thus, the anti-inflammatory effect is generally attributed to the decrease in NO production via suppression of iNOS activity and downregulation of iNOS expression. To support this view, in this study, the generations of NO were markedly reduced by RP-G and RP-A in growing and adult rats, reflecting that the status of inflammatory damage could be suppressed by rice proteins. These results were in agreement with our in vitro studies that rice protein could scavenge NO radicals [12,13]. Accordingly, hepatic iNOS activities and the expressions of iNOS were significantly reduced by RP-G and RP-A in growing and adult rats. On the other hand, as major hepatic inflammatory biomarkers, the activities of ALT and AST were significantly reduced by RP-G and RP-A, suggesting that rice protein could efficaciously improve hepatic inflammation in growing and adult rats. Taken together, in this study, it was evident that rice protein could inhibit inflammation in growing and adult rats.
The inflammatory process can be initiated by ROS-derived oxidative stress, suggesting that the overproduction of ROS may be a major contributor to the inflammation [1]. In light of this view, the anti-inflammatory response induced by rice protein should be focused on ROS-scavenging activity.
In this study, the inhibitory effect of rice protein on hepatic ROS accumulation was observed in both growing and adult rats. These results were consistent with our previous findings that rice protein could scavenge ROS, including superoxide radical, hydrogen peroxide, etc. [11,12,24], which are the major cause of inflammation. In support, the findings observed in this study showed significant positive correlations between hepatic ROS content and ALT activity (r = 0.7888, p < 0.05), as well as AST activity (r = 0.8068, p < 0.05), suggesting that the decreased activity of ALT/AST was attributed to the reduced hepatic accumulation of ROS. Therefore, it is possible that the stronger ROS-scavenging capacity of rice protein might be closely linked with inhibition of the inflammatory process, further suggesting that rice protein can inhibit ROS-derived inflammation in growing and adult rats.
To elucidate the molecular mechanism by which rice protein induced an anti-inflammatory action, the influence of rice protein on the NF-κB pathway was assessed in this study. After 2 weeks of feeding, we could show that rice protein effectively and significantly inhibited NF-κB activation, although the activity of NF-κB was not directly determined in this study. It is clear that the activity of NF-κB can be primarily regulated by the interaction of IκB protein. Accordingly, the regulation of NF-κB-IκB interaction was particularly emphasized in this study as the key step for controlling NF-κB activity. As a master regulator of the inflammatory process, NF-κB is negatively regulated by IκB protein, e.g., IκBα. As stimulated by ROS-induced oxidative stress, IκBα is degraded and NF-κB is translocated into the nucleus and an inflammatory response can be induced. Subsequently, the inflammatory response is induced. Hence, NF-κB plays a crucial role in initiating inflammation [1,15]. With regard to this, the major finding in this study was that rice protein could enhance the expression of IκBα, suggesting that rice protein could augment the stabilization of the NF-κB-inhibitor. Consequently, the interaction of NF-κB-IκBα with rice protein was clearly observed in this study, implying that the activity of NF-κB could be suppressed by rice protein feeding. Supportably, RP-G and RP-A significantly inhibited the translocations of p50 and p65 into the nucleus, which were the major complexes of NF-κB. Results showed significant positive correlations between hepatic ROS and nuclear contents of NF-κB (p50, r = 0.8726, p < 0.05; p65, r = 0.9011, p < 0.05). Furthermore, it is clear that the canonical NF-κB pathway is characterized by activation of p50 and p65, suggesting that the inflammatory response is dependent on the nuclear translocations of p50 and p65 [15]. In this study, there were significant positive correlations between nuclear NF-κB and ALT (p50, r = 0.8100, p < 0.05; p65, r = 0.8107, p < 0.05), as well as AST (p50, r = 0.8397, p < 0.05; p65, r = 0.8478, p < 0.05), supporting the view that the downregulation of p50 and p65 could inhibit inflammatory action. In support of this notion, a significant negative correlation was observed between p50 and IL-10 (r = −0.8630, p < 0.05), as well as p65 and IL-10 (r = −0.8715, p < 0.05), in which IL-10 is an important anti-inflammatory mediator [25]. The present study, therefore, provides clear evidence that the suppression of NF-κB activation might be one of main anti-inflammatory mechanisms exerted by rice protein.
Upon suppression of NF-κB, downregulation of hepatic expressions in TNF-α, IL-1β, IL-6, iNOS, COX-2, and MCP-1, which are major inflammatory mediators regulated by NF-κB, were also observed in growing and adult rats fed with RP-G and RP-A. These results were consistent with an in vitro study reported by Wen et al., which indicated that rice protein hydrolysates inhibit the lipopolysaccharide-stimulated inflammatory response in RAW264.7 macrophages by inhibiting the release of NO and decreasing the expressions of TNF-α, iNOS, IL-6, and IL-1β, etc. [14]. More significantly, in this study, the results showed significant positive correlations between hepatic ROS and the expressions of TNF-α (r = 0.8858, p < 0.05), IL-1β (r = 0.9107, p < 0.05), IL-6 (r = 0.8465, p < 0.05), iNOS (r = 0.8620, p < 0.05), COX-2 (r = 0.8959, p < 0.05), and MCP-1 (r = 0.9235, p < 0.05). Thus, our findings further support the view that the ROS-induced inflammatory process could be prevented by rice protein in growing and adult rats.
Here, the question might arise of how rice protein can suppress NF-κB activation after 2 weeks of feeding. In order to identify the mechanism behind the rice protein-induced NF-κB inhibition, we investigated the PI3K/Akt signaling pathway in this study. NF-κB signaling has been recognized as one of the targets of the PI3K/Akt pathway [21,26]. Accordingly, the suppression of NF-κB activation might be a consequenc of downregulation of PI3K/Akt. To support this view, we found that RP-G and RP-A respectively decreased the gene and protein expressions of PI3K and Akt in growing and adult rats. More significantly, the results showed significant positive correlations between the expressions of PI3K/Akt and nuclear contents of p50 (PI3K, r = 0.8751, p < 0.05; Akt, r = 0.8491, p < 0.05), as well as nuclear p65 (PI3K, r = 0.9032, p < 0.05; Akt, r = 0.9477, p < 0.05). To support our findings, Madrid et al. suggested that Akt could stimulate the transactivation potential of the RelA/p65 subunit of NF-κB [27]. Therefore, it is not surprising that the downregulation of PI3K/Akt might be a switch to the suppression of NF-κB activation involving the anti-inflammatory action exerted by rice protein.
Now, another question might also emerge of why this anti-inflammatory response could be induced by rice protein. To explain this phenomenon, the view that the inflammation can be prevented by dietary antioxidants should be taken into account in this study [28]. It has been demonstrated that nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) can modulate the anti-inflammatory response through the inhibition of NF-κB and upregulate antioxidant responsive element (ARE)-dependent gene expressions, such as HO-1 [11,[29][30][31]. In light of this view, a satisfactory explanation could be drawn from our previous studies, in which rice protein could exert an antioxidant capacity via activation of Nrf2 and upregulation of HO-1 expression in adult rats [11]. Consistent with our previous studies, in this study, RP-G and RP-A effectively enhanced the expressions of HO-1, which is an important anti-inflammatory mediator, in growing and adult rats. Moreover, as another important anti-inflammatory mediator, the gene and protein expressions of IL-10 were also enhanced by RP-G and RP-A, further confirming the fact that rice protein could exert an anti-inflammatory effect. On the other hand, it has been demonstrated that the biological utilization of dietary protein is primarily dependent on its amino acid composition [7,8]. With regard to this, the fact that rice protein is rich in arginine particularly attracts our attention [5,9,10]. Some studies suggest that arginine can prevent or control the excessive inflammatory response through modulation of the immune system [32,33]. More importantly, our recent study showed that arginine could augment the expressions of HO-1 via activation of the Nrf2-ARE pathway [29]. In support, in this study, there were significant positive correlations between arginine intake and expressions of HO-1 (r = 0.9217, p < 0.05), as well as IL-10 (r = 0.8560, p < 0.05). Thus, the role of arginine in the stimulation of the anti-inflammatory response of rice protein should be emphasized in this study. The results showed a significant positive correlation between arginine intake and IκBα expression (r = 0.8620, p < 0.05), whereas there were the significantly negative correlations between arginine intake and expressions of PI3K (r = −0.8952, p < 0.05), as well as Akt (r = −0.9284, p < 0.05). Consequently, the significantly negative correlations between arginine consumption and nuclear contents of NF-κB (p50, r = −0.8765, p < 0.05; p65, r = −0.9471, p < 0.05) were clearly observed in this study. Thus, this study provides an insight that higher levels of arginine in rice protein (RP, 88.17 µg/mg; CAS, 33.33 µg/mg) might be a contributor to the anti-inflammatory effect of rice protein via suppression of NF-κB activation. In addition to arginine, rice protein is also rich in sulfur-containing amino acids (methionine and cystine), glycine, and glutamic acid, which could stimulate endogenous antioxidant activity to inhibit the inflammatory response.
NF-κB signaling is the molecular culprit of inflamm-aging, suggesting that the inflammatory process can be activated and accelerated with age [15,20]. However, under the present experimental condition, of interest was the finding that the increase of age did not cause a weakened anti-inflammatory capacity of rice protein in adult rats in comparison with growing rats. To explain this interesting phenomenon, the view that p53 and Nrf2 negatively regulates NF-κB signaling pathways in the aging process should be noted [34]. As longevity factors, both p53 and Nrf2 can protect inflamm-aging via inhibition of the NF-κB activation [21,34,35]. Recently, Liang et al. reported that rice protein could suppress DNA damage via activation of the ataxia-telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2)-p53 and Nrf2-Keap1 (Kelch-like ECH-associated protein 1) pathways in growing and adult rats [24], implying that rice protein might protect DNA damage-dependent inflamm-aging. Furthermore, in this study, increased age could not enhance the expressions of PI3K/Akt, which could drive the aging process [21], in adult rats fed with RP-A. Thus, in light of these facts, a satisfactory explanation that anti-inflammatory action via inhibition of NF-κB activation exerted by rice protein might be independent of age is convincing. Namely, this anti-inflammatory effect might be dependent on the amino acids profile of native rice protein, in which some amino acids, e.g., arginine, might play a key role in inducing the anti-inflammatory response to prevent inflammation. Clearly, additional studies are required to confirm this view.
Protein Sources
Rice protein (RP) from Oryza sativa L. cv. Longjing 20 (Rice Research Institute of Heilongjiang Academy of Agricultural Sciences, Jiamusi, China) and casein (CAS) (Gansu Hualing Industrial Group, Gansu, China) were used as dietary protein sources in the present study.
Animals Experiments
According to our previous studies [6][7][8]11,24,29,30], animal studies (SCXK2012-0001, 8 February 2012) were approved and performed in conformity with the Guidelines of the Committee for the Experimental Animals of Harbin Institute of Technology (Harbin, China). Briefly, growing male Wistar rats (body weight 180-200 g) and adult male Wistar rats (body weight 390-410 g) were purchased from the Vital River Laboratories (Beijing Vital River Laboratory Animal Technology Co. Ltd., Beijing, China) and individually housed in metabolic cages in a room maintained at 22 ± 2 • C under a 12-h light-dark cycle (07:00-19:00 for light). In this study, four groups, consisting of six animals per group, were used for the investigation. Growing rats were respectively fed casein (CAS-G) and rice protein (RP-G) with a dietary protein level of 20% (as crude protein, CP) for 2 weeks, according to the formula recommended by American Institute of Nutrition for growth (AIN-93G) [36]. Adult rats were respectively fed 14% (as CP) dietary proteins of casein (CAS-A) and rice protein (RP-A) for 2 weeks, according to the formula recommended by American Institute of Nutrition for adult maintenance (AIN-93M) [36].
Measurement of Plasma Enzyme Activity
The activities of plasma ALT and AST, the major inflammatory biomarkers, were determined using the methods described in the kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
Analyses of Hepatic NO Level and iNOS Activity
The contents of NO in the liver and hepatic activity of iNOS were measured using the commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Determination of Hepatic ROS
The analysis of ROS levels in the liver was according to our previous studies [11,24,29,30]. Briefly, ROS was determined by fluorescence of 2 , 7 -dichlorofluorescin diacetate (DCF-DA) as described by the manufacturer's protocol of the commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The fluorescence intensity was measured at the 485-nm excitation wavelength and 530-nm emission wavelength. Data are expressed as an arbitrary unit of fluorescent intensity per µg protein.
Quantitative Real-Time PCR
Quantitative real-time PCR was measured in this study, according to our previous studies [24,29,30]. Briefly, using the TRIzol reagent kit (Invitrogen, Carlsbad, CA, USA) and the PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio. Inc., Otsu, Shiga, Japan), total RNA was extracted from the livers and cDNA was reverse transcribed. The mRNA level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was treated as a normalization. The primers sequences used are shown in Table 1. In this study, the relative mRNA level in group CAS-G and CAS-A was respectively set as 1.00.
Statistical Analysis
The statistical analysis was in accordance with our previous studies [6][7][8]11,24,29,30]. Briefly, data are expressed as the mean ± SEM. Differences between groups were examined for statistical significance using one-way analysis of variance (ANOVA) followed by the least significant difference test. The criterion for significance was p < 0.05.
Conclusions
The present study is the first to demonstrate that rice protein exerts anti-inflammatory effects in growing and adult rats. The results indicate that the anti-inflammatory response induced by rice protein is primarily attributed to suppression of the NF-κB pathway ( Figure 9). Significantly, the study confirms a link between the inhibition of ROS-derived inflammation with the intake of rice protein in growing and adult rats. The novel finding observed in this study is that the anti-inflammatory activity of rice protein cannot be attenuated by increased age. Clearly, more detailed investigations are needed to explore the precise anti-inflammatory mechanisms exerted by rice protein in further study. The green "T" represents for the inhibitory interaction; the red arrow shows the up-regulatory effect; the blue arrow indicates that rice protein can suppress the inflammatory action; the "(+)" represents the activation; the "(−)" represents the inhibition. Akt, protein kinase B; COX-2, cyclooxygenase-2; HO-1, heme oxygenase-1; IκBα, inhibitory κB α; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-10, interleukin-1β; iNOS, inducible nitric oxide synthase; MCP-1, monocyte chemoattractant protein-1; NF-κB, nuclear factor-κB; NO, nitric oxide; PI3K, phosphoinositide 3 kinase; p50, nuclear factor-κB1; p65, reticuloendotheliosis viral oncogene homolog A; ROS, reactive oxygen species; TNF-α, tumor necrotic factor α.
Author Contributions: L.Y. designed the study and wrote the paper. Z.W., M.L., H.L. and L.C. carried out the study. L.Y. supervised the entire study and had primary responsibility for final content. All authors read and approved the final manuscript.
Funding: This work was supported by National Natural Science Foundation of China (31371755). | v3-fos-license |
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} | pes2o/s2orc | Protective effects of epicatechin on the oxidation and N-nitrosamine formation of oxidatively stressed myofibrillar protein
ABSTRACT The present study investigated the protective effects of the polyphenol epicatechin on the protein structural changes, gel properties and N-nitrosodiethylamine (NDEA) formation of oxidatively stressed myofibrillar protein (MP). Results showed that oxidation treatments to MP significantly increased their carbonyl content and surface hydrophobicity as compared to the control groups (p < 0.05). Epicatechin inhibited protein carbonyl content and surface hydrophobicity, especially treated with 2 mM epicatechin. Under oxidative conditions, the fluorescence intensity of tryptophan gradually decreased with the increase of epicatechin concentrations from 0 to 2 mM. The storage modulus (G’) was increased gradually after oxidation and epicatechin treatment. Epicatechin reduced the loss of P 22 and water holding capacity (WHC), promoted the hydration of the proteins. Under oxidative conditions, epicatechin had a concentration dependent effect on the formation of NDEA; low concentrations of epicatechin promoted NDEA formation, while high concentrations inhibited NDEA production. In conclusion, an appropriate quantity of polyphenols can inhibit the oxidation of MP and formation of N-nitrosamines.
Introduction
Meat is a substance that is rich in protein and lipid, and is rather susceptible to oxidation. During events such as meat processing (i.e., storing, cooking and drying), protein oxidation can occur frequently. As a result, the quality of meat products is reduced through phenomena such as the loss of juiciness and reduced cooking yield. [1,2] Therefore, it is of great importance to control protein oxidation and reduce lipid oxidation in meat products.
In the past, synthetic antioxidants have traditionally been used to prevent the oxidation of proteins and lipids in food products. However, recent consumer concerns have been focused on the potential of synthetic antioxidants in being carcinogenic. As a result, interest has developed in the use of natural antioxidants in food products and garnered attention from the scientific community. Among various natural antioxidants, plant-derived antioxidants have been widely used in recent years. [3,4] Antioxidants found in plant extracts are primarily phenolic compounds. These extracts provide promising antioxidant properties through their capacity to regulate intra-cellular hydrogen supplies and metal-ion chelation potentials. [5] Polyphenols have widely been used to inhibit the oxidant modification of meat products. However, while the potential of polyphenols to act as antioxidants in meat products has proven to be effective in delaying lipid oxidation, the same is not true concerning protein oxidation. In some studies, phenolic acids have been found to accelerate carbonylation and sulfhydryl loss. For example, green tea extracts increased thiol loss and protein polymerization in Bologna-type sausages prepared from UV-irradiated pork. [6] Furthermore, beef patties treated with polyphenol-rich willowherb extracts resulted in reduced lipid oxidation, but accelerated protein carbonylation and discoloration. [7] Rosemary and oregano essential oils were found to retard thiol loss, whereas garlic essential oil promoted thiol loss and myosin cross-linking formation in pork patties during chill storage. [8] In protein-rich foods, phenolic substances can interact with proteins through covalent bonds and non-covalent bonds, thereby altering the structure of proteins, leading to functional changes. [9] The oxidation of processed muscle foods was inevitable due to loss of tissue integrity during meat processing and through endogenous prooxidants in fresh muscles. When these phenolic compounds are oxidized, they produce quinones when added to the meat product formulations. Quinones may be associated with the oxidation of proteins during meat processing or cooking. [10] N-nitrosamines (NAs) are potentially carcinogenic, mutagenic and teratogenic effects to various animals and humans. [11] There are three primary ways of human interaction with NAs: foods, other environmental sources and internal endogenous synthesis. NAs are formed from primary, secondary amines and nitrosating agents in foods such as fish and meat. Therefore, it is essential to reduce or completely inhibit the formation of NAs. The degradation and oxidation of proteins into small molecules may be the reason for the formation of NAs. [12,13] The chemical changes (oxidation, nitrosation and proteolysis) of meat proteins during digestion lead to a decrease in their nutritional value. [14] To clarify the mechanisms of NAs formations, the model system has commonly been used to investigate the different processing parameters or precursors. Although meat proteins are an important reactant in NAs formation, the potential effects of protein oxidation on NAs formation has rarely been reported.
Although there have been some studies on the use of phenolic compounds to inhibit MP oxidation, few reports have studied the effects of phenolic compounds on NAs in the oxidation systems. In general, plant polyphenols include catechins, epicatechin, procyanidins, chlorogenic acid and caffeic acid. Epicatechin alias: 2-(3,4-dihydroxyphenyl)-2,3,4-trihydro-3,5,7-trihydroxychromene, has two hydroxyl groups at the ortho position of the aromatic ring. Epicatechin is a phenolic compound with the potential to protect products against oxidative/nitrative stress. The purpose of this investigation was to study the protective effects of epicatechin on the protein structure characteristics, water distribution of protein gel, and NAs formation.
Materials
Fresh pork meat from the Longissimus muscle of pig carcasses (24-hours postmortem) was bought from a local supermarket (Suguo, Nanjing, Jiangsu, China). Individual loin chops (weighing~100 g) were packaged and stored in a freezer (−18ºC) until use within one month. In the present study, the chemicals were of reagent grade, and purchased from Aladdin Industrial Corp. or Sigma-Aldrich Co.
MP extraction and sample preparation
MP extraction: MP was prepared according to methods of Park & Xiong [15] and Feng et al. [16] with some modifications. About 400 g of meat was chopped into small sections. The isolation buffer containing 20 mM of sodium phosphate, 0.1 M of NaCl, 2 mM of MgCl 2 , and 2 mM of EGTA at pH 7.0 was used for the MP extraction. Then 50 g of meat and 200 g of isolation buffer were homogenized using an Ultra Turrax dispersing instrument (T18-Digital, IKA, Staufen, Germany) in an ice bath. After filtration through four layers of gauze, the homogenate was spun twice at 2000 × g for 15 min at 4°C. Subsequently, the pellets were washed twice with 4 L of a 0.1 M NaCl solution. Afterwards, the collected pellets were washed again with 4 L of 20 mM PBS (pH 7.0). The concentration of the obtained protein was measured by the Biuret method.
Oxidative treatments with epicatechin: MP suspensions were prepared with 15 mM piperazine-N,N´bis (2-ethanesulfonic acid) (PIPES) buffer containing 0.6 M of NaCl at pH 6.25. Six different reaction mixtures (protein concentration, 40 mg/mL) were prepared with epicatechin (0, 0.1, 0.2, 0.5, 1 and 2 mM). Samples with or without epicatechin were oxidized at 4ºC for 12 hours using a hydroxyl radical generation system (10 μM of FeCl 3 , 100 μM of ascorbic acid, and 1 mM of H 2 O 2 ). [17] Oxidation was stopped by adding a Trolox C/EDTA mixture (1 mM). A nonoxidized and epicatechin-free MP suspension was used as the control.
Changes of protein side chain structure
Sample carbonyl contents were analyzed using the 2,4-dinitrophenylhydrazine (DNPH) colorimetric method. [18] Briefly, the carbonyl groups were reacted with DNPH to produce protein hydrazones in acidic conditions. The absorbance was measured at 370 nm with an absorption coefficient of 22,000 M −1 cm −1 in order to calculate the carbonyl content.
Total sulfydryl content and free sulfydryl content were measured using absorbance at 412 nm with 5,5ʹ -dithio-bis (2-nitrobenzoic acid) (DTNB) used as an indicator. [19] The total sulfhydryl group was measured with a solution containing urea (8 M urea, 0.086 M Tris, 0.09 M glycine, 4 mM EDTA, pH 8.0), and the free sulfhydryl group was determined using a solution without urea(0.086 M Tris, 0.09 M glycine, 4 mM EDTA, pH 8.0). A molar extinction coefficient of 13,600 M −1 cm −1 was used to calculate sulfydryl content.
Surface hydrophobicity was measured using data obtained from absorbance at 595 nm with the bromophenol blue (BPB) binding method. [20] Tryptophan fluorescence was determined using an F-4600 fluorescence spectrophotometer (Hitachi, Japan). MP suspensions (0.1 mg/mL) were excited at 283 nm, with the emission spectra recorded from 300 to 400 nm.
Gelation properties
MP suspensions (5 g, 40 mg/mL in 15 mM PIPES) were transferred into glass vials. These were covered with aluminum foil, heated in a water bath from 20ºC to 80ºC at 1ºC/min increments, and were then maintained at 80ºC for 20 minutes. After heating, the formed gels were immediately chilled in an ice bath for 30 minutes and then the juice was removed from the surface of the gels. Cooking yield was calculated using the following formula, in which W gel and W sol are the weight of the gel and the original solution, respectively.
The water holding capacity (WHC) of the samples was measured using the method described by Kocher and Foegeding. [21] Exactly 5 g of gel was placed in a centrifuge tube and centrifuged at 10,000 × g for 10 minutes at 4ºC. Then, WHC was calculated as the percentage of the retained weight of a gel after centrifugation relative to its initial weight. Gel whiteness was determined using a CR-400 chroma meter (Konica Minolta, Osaka, Japan). Color analyses were performed using the CIE coordinates (L*, a*, and b*). Whiteness value was calculated as: Dynamic rheological testing was measured during the heat-induced gelation with a model AR1000 rheometer (TA Instruments, West Sussex, U.K.) in an oscillatory mode equipped with parallel plate geometry (40 mm diameter). Gelation was induced by heating the protein emulsion from 20 to 90°C at a rate of 2°C/min. Changes in the storage modulus (G') were monitored continuously.
NDEA in an oxidized protein solution
The formation of NDEA was measured on according to the report with slight modifications. [13,24] MP suspensions (10 mg/mL), diethylamine (50 mM) and sodium nitrite (5 mM) were mixed into a 50 mM phosphate buffer (pH 3.0, a simulated gastric acid system) (all concentration was final). Two series reactions were carried out in stoppered conical flasks. The reactions of one group were set for 4 h at 37°C, while the other group was set for 1 h at 80°C. After the reaction, the mixtures were mixed with 5 mM of sulfanilic acid to stop the reaction, and centrifuged at 3,000 × g for 5 min at 4°C . The clear liquid on the upper section of the tube was immediately subjected to HPLC analysis for nitrosamines, targeting NDEA.
An HPLC unit (Agilent 1260, CA, USA) equipped with an Agilent UV detector was employed. Liquid chromatography separation was carried out on a ZORBAX SB-C18 column (150 cm × 4.6 mm, 5 μm) and peaks were detected at 230 nm. An elution program was used with a mixture of 35% methanol and 65% water at a flow rate of 1 mL per minute. A linear relationship was obtained over a range of the concentration between 0.1 and 10.0 μg/mL. The obtained correlation coefficients (R 2 ) were greater than 0.99.
Statistical analyses
The entire experiment was replicated three times on different occasions. Statistical treatments were performed with SPSS version 18.0 (SPSS Inc., Chicago, IL). One way analysis of variance (ANOVA) was applied to assess the significance by means of Duncan's multiple range tests. The statistical significance was set at p < 0.05. All results were calculated as the means and standard deviations.
Carbonyl content
Carbonyl levels are a well-known index for the level of protein oxidation in meat products during processing. [2,25] The carbonyl content of the control MP was 1.08 nmol/mg protein (Table 1) which was close to the report of freshly prepared MP as described by Cao and Xiong. [26] Oxidation significantly increased the carbonyl content of samples (3.63 nmol/mg protein) when compared to the control MP (p < 0.05). The presence of epicatechin significantly inhibited the formation of the carbonyl, especially at a large epicatechin dose of 2 mM (1.14 nmol/mg protein), which was near to the control MP. Epicatechin was oxidized into a quinine derivative in this oxidation system. This study was in accordance with those previous reports in which phenolic compounds inhibited protein carbonyl content, such as quercetin [27] ，rosmarinic acid [28] and EGCG. [29,30] Total and free sulfydryl groups MP is rich in sulfydryl groups, which are susceptible to attack by reactive oxygen species and can then convert to intra-and intermolecular disulfide bond linkages. [10] As shown in Table 1, the total sulfydryl group content of control MP was 109.63 nmol/mg protein. About 6.6% total sulfydryl group (p > 0.05) was lost when MP samples were exposed to hydroxyl groups. Compared to the control MP, the total sulfydryl group contents were significantly lower in the absence of epicatechin. Furthermore, the total sulfydryl group levels exhibited an epicatechin dose dependent decrease. Compared to the control MP, about 44% total sulfydryl group (p < 0.05) was lost when MP samples were exposed to hydroxyl groups and 2 mM epicatechin. The free sulfydryl group levels in oxidized MP had the same transformation tendency as the total sulfydryl group levels. This could be explained by the formation of thiol-quinone adducts. These observed results are in accordance with previous reports. Cao & Xiong observed that chlorogenic acid had no protection on thiol groups, and high chlorogenic acid revealed the lowest sulfydryl content. [26] Surface hydrophobicity It is generally believed that the amount of protein binding BPB can be used as an estimator of protein hydrophobicity estimation. The surface hydrophobicity of the oxidized MP was significantly higher (p < 0.05) than that of the control ( Table 1). The surface hydrophobicity of the oxidized MP significantly decreased in the presence of EC (p < 0.05), especially in a large dose (2 mM). This decrease of surface hydrophobicity with increased epicatechin may be due to protein aggregation caused by the addition of epicatechin. The decrease was consistent with the change trend of carbonyl content. This study was consistent with other previous reports which indicate that phenolic compounds inhibit the surface hydrophobicity. [29] It is also shown that phenolic compounds can protect protein side-chain groups.
Intrinsic tryptophan fluorescence
As shown in Figure 1, when compared with the control MP, the intensity of tryptophan fluorescence from oxidized MP was significantly decreased indicating that the protein was denatured. The inclusion of epicatechin promoted the loss of intrinsic fluorescence, especially at the concentration of 2 mM epicatechin. This was consistent with the change seen in surface hydrophobicity, indicating that oxidation leads to further expansion and possible interactions of protein side chains and phenolic compounds. The fluorescence characteristic of tryptophan was particularly sensitive to the polarity of tryptophan microenvironment. It was widely used as a measure index of protein tertiary structure. Similar results were obtained when binding chlorogenic acid to MP [26] or epigallocatechin (EGC) to β-lactoglobulin. [31] In the process of protein oxidation, non-covalent and covalent interactions involving proteins, as well as possible quinone-protein interactions, eventually lead to modifications of proteins in primary, secondary and tertiary structures.
Gel properties
The cooking yield, WHC and whiteness of myofibrillar protein gels are summarized in Table 1.
Cooking yield was slightly reduced when treated with epicatechin, but this had no significant effect on the cooking yield with or without epicatechin in the hydroxyl radical generating system (p > 0.05). The results of this study may be influenced by the amount of epicatechin addition. In the present study, the amount of epicatechin utilized was low. The WHC of the MP gels increased significantly (p < 0.05) as the epicatechin concentration increased from 0 to 2 mM. This indicates that epicatechin can increase the WHC of the gel, allowing it to be used in meat processing for improving the quality of meat products. The addition of epicatechin at 0.1 mM had no effect on gel whiteness (p > 0.05), while epicatechin from 0.2 to 2 mM reduced gel whiteness (p < 0.05). The reduction in whiteness was possibly due to the chelation of ferric iron and epicatechin oxidation. The storage modulus (G') are used to represent the amount of recoverable energy stored in the elastic gel ( Figure 2). The oxidized MP had a final G' significantly higher than the control, suggesting that oxidation promoted protein interactions. The G' of the MP emulsion gel treated with 2 mM epicatechin was higher than 0 mM epicatechin, suggesting that epicatechin did not prevent the crosslinking induced by heating under oxidative stress. This was consistent with previous research on MP treated with EGCG. [29] The LF-NMR technique is an alternative method for assessing water distribution and mobility during protein gel formation. Figure 3 shows the T 2 relaxation time distribution of MP gels. The calculated relaxation times, T 21 , T 22 and T 23 , correspond to the combined water, immobilized water, and free water in the gel, respectively. [32] Table 2 shows the effect of epicatechin induced modification on the proportions of P 21 , P 22 and P 23 of MP gels. The proportions of P 21 were not significantly (p > 0.05) affected by oxidation and epicatechin. The oxidation treatment resulted in a lower P 22 when compared with the control MP gels (p < 0.05). P 22 was slightly increased with increasing epicatechin concentration from 0 to 2 mM, corresponding to the results of WHC in this study. Tea polyphenols may increase the exposure of sensitive side chain groups and enhance the reaction between proteins and phenolic compounds. This hastened the formation of protein gelation, which is retained in the MP network. [33] For P 23 , the peak area was increased significantly (p < 0.05) by oxidation. The peak area was decreased with increasing epicatechin concentration from 0 to 2 mM (p > 0.05), thus indicating that phenolic compounds can protect the oxidized protein. These findings suggest that oxidation decreased the presence of immobilized water in the gel，while phenolic compounds have a tendency to increase the occurrence of immobilized water. In addition, compared to the oxidized MP, gels with high epicatechin had much higher immobilized water content. This proved that phenolic compounds can be good antioxidants and inhibit protein oxidation, reduce the loss of WHC, and improve the hydration of the proteins. [34] NDEA analyses Previous studies have shown that NAs can be formed in cured meat and fish: N-nitrosodimethylamine (NDMA), NDEA and the other NAs. [12,13,35] According to the classification of carcinogenic compounds, NDMA and NDEA fit in the group of probable carcinogens, whereas other NAs fall into the group of possible carcinogens. [36] Previous studies have also shown that only the level of NDEA was high in most samples, which is easy to measure by HPLC. [13,24] Therefore, NDEA was specifically measured in the present study.
The representative chromatogram of NDEA by HPLC was shown in Figure 4. NDEA can achieve a good symmetrical peak shape under test conditions with good accuracy and precision. The retention time of standard and sample was the same in this study. The levels of NDEA in the stimulated nitrite curing solution are shown in Figure 5. The content of NDEA in the solution for 4 h at 37°C increased when the epicatechin content increased from 0 to 1 mM, attaining the maximum quantity of NDEA (4.88 µg/mL). However, when epicatechin content was 2 mM, the amount of NDEA production decreased to zero. The content of NDEA in the solution for 1 h at 80°C solution increased when the epicatechin content increased from 0 to 0.5 mM, attaining the maximum quantity of NDEA (10.22 µg/mL). However, when epicatechin content was at 1 or 2 mM, the amount of NDEA production decreased near to zero. Overall, the amount of NDEA formation was first increased to a certain amount, and subsequently rapidly decreased. The formation trend of NDEA was consistent under different temperature treatments. This study also showed that epicatechin concentration higher than 2 mM can completely inhibit the formation of nitrosamines. Choi et al. reported that green tea had a strong inhibitory effect on the nitrosation of secondary amines under gastric conditions. [37] However, since they used green tea at high concentrations, and not at low concentrations, they were not able to observe the promoting effects of green tea on the formation of NAs. Other studies reported both the promoting and inhibiting effects of green tea on NAs formation in vitro, which was similar to that in the present study. [38] From these results, we considered that the promoting and inhibitory effects of epicatechin on the information NDEA might depend on the concentration of epicatechin and nitrite.
Furthermore, Nakamura and Kawabata also studied that the nitrosation reaction could be inhibited by other polyphenols, such as flavonols, flavones and isoflavones. [39] Masuda also investigated the effects of eight chemicals in catechins on the formation of nitrosomorpholine. Resorcinol and phloroglucinol, which have an A-ring model structure, accelerated the formation of nitrosomorpholine at a low concentration, and inhibited it at a high concentration. [38] A conceivable mechanism was for the catalysis of nitrosation by some of phenolic conpounds. Nitroso derivatives may be formed by further reaction with the nitrite to generate a more powerful nitrosating agent, which could be a nitrosoquinone oxime derivative. Its reaction with the nitrosatable substrate produces the nitroso compounds. This mechanism indicates that increasing amounts of catalytically active phenolic compounds lead to higher concentrations of nitroso intermediates. This mechanism could explain the optimum ratio for nitrite to phenolic compound. Accordingly, a large excess of phenolic compounds would inhibit nitrosation, because the nitrosating agent would be completely used up in forming the catalyst agent and none would be left to continue the catalytic reaction. Although we confirmed that the A-ring structure in catechins affected the formation of NAs, the mechanisms have yet to be clearly clarified.
In the present study, the heating temperature at 37°C was representative of gastrointestinal temperature. Meanwhile, the heating temperature at 80°C was representative of the processing for most instant meat products. However, further studies are needed to determine whether high temperatures (100 degrees or higher) could increase the contents of NAs or form other types of NAs.
Correlation coefficients between NDEA and protein oxidation variables
The correlation coefficients between NDEA, protein oxidation variables and gel properties are shown in Table 3. NDEA was significantly correlated with free sulfhydryl content, which is consistent with the results reported by Sun. [40] In addition, carbonyl production or total sulfhydryl reduction was significantly correlated (p < 0.01 or p < 0.05) to that of surface hydrophobicity, suggesting that MP fraction oxidation processes might affect each other. WHC and whiteness were extremely and significantly correlated with free and total sulfhydryl content (p < 0.01), suggesting that protein oxidation affects gel properties. Overall, the significant correlation between the content of NDEA and protein oxidation systems when reacted with nitrite suggests that the oxidative formation of carbonyls and oxidative breakage of sulfhydryls with possible deamination contributed to the production of the secondary amine compounds in the manufacture of cured meat.
Conclusion
In the present study, epicatechin influenced oxidation-induced changes through modifying the structure of proteins. This action was demonstrated to be concentration-dependent, in which carbonyl content, sulfydryl content, surface hydrophobicity, and intrinsic tryptophan fluorescence decreased when epicatechin increased from 0 to 2.0 mM. LF-NMR analysis showed that weak hydration of MP induced by oxidation could be improved by the addition of epicatechin. We also demonstrated that the formation of NDEA was inhibited by high epicatechin concentration, and was accelerated by low epicatechin concentration. In conclusion, these findings demonstrate potential benefits of the antioxidant treatments for meat products, such as through reducing the production of unwanted secondary amines by inhibiting the oxidation of proteins. | v3-fos-license |
2018-09-16T02:56:27.533Z | 2018-09-01T00:00:00.000 | 52151820 | {
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} | pes2o/s2orc | Production, Absorption, and Blood Flow Dynamics of Short-Chain Fatty Acids Produced by Fermentation in Piglet Hindgut during the Suckling–Weaning Period
Luminal short-chain fatty acids (SCFA) are rapidly absorbed from the intestine and subsequently utilized by the host as substrate for metabolic energy production. In pigs, the energy contribution of SCFA is thought to be 30–76%. However, since absorption and blood flow dynamics of SCFA in pigs, particularly during the suckling–weaning period, remain unclear, we aimed to elucidate these phenomena. Thirty-two piglets were used in the present work. Cecal vein blood and digesta, and portal and abdominal vein blood were sampled from suckling (7-, 14-, 21- and 28-day-old) and weaned (weaning at 21 and 28 days of age) piglets. Four piglets from each group were euthanized. SCFA concentrations in blood samples were analyzed by a highly sensitive gas chromatography-mass spectrometry technique. Age at weaning tended to affect SCFA absorption. For example, acetate and propionate concentrations in the cecal vein tended to be higher in piglets weaned at day 21 than at day 28. SCFA concentrations in the abdominal vein tended to differ from those in other veins. Mucosal gene expression analysis suggested that monocarboxylate transporter 1 and occludin were associated in absorption of SCFA from the lumen into the blood of piglets.
Introduction
Short-chain fatty acids (SCFA), particularly acetate, propionate and n-butyrate, are major end-products of gut microbiota [1]. SCFA production depends not only on substrates flowing into the large intestine [1], but also on the organ size and the population and composition of luminal bacteria [2]. Therefore, the large intestine is the major production site of SCFA in hindgut fermenters such as humans and pigs [2]. It is well established that luminal SCFA are rapidly absorbed from the intestine, and subsequently utilized by the host as substrate for metabolic energy production [3]. The energy contribution of SCFA to the basal metabolic rate is thought to be 30-76% [3] and 10% [4] in pigs and humans, respectively. Furthermore, SCFA are useful to the host not only for maintenance of the gut morphology and function [5,6] but also for reduction of appetite and diet-induced obesity [7,8].
During suckling, newborn mammals are fed only maternal milk. In humans [9] and pigs [10], maternal milk contains large amounts of disaccharides and oligosaccharides and thus, hindgut fermentation starts merely a few days after delivery [11]. When hindgut fermentation starts, the concentrations of SCFA in infant feces are approximately 60 mmol/kg [11]. Weaning, which occurs for all newborn mammals, can be a very stressful event. For example, it has been observed that the intestinal structure and function of piglets are drastically affected by weaning [12,13]. Moreover, during weaning, the transition from maternal milk to solid food affects not only the structure and function of the intestine but also the components of the luminal environment such as the intestinal microbiota and its metabolites. In pigs, it has been reported that weaning affects the luminal SCFA concentrations and composition in the large intestine [14,15]. Although the absorption process and blood flow dynamics of SCFA in weaning piglets remain unclear, most SCFA (95%) produced by the luminal microbiota is thought to be quickly absorbed from the mucosa, while only 5% is excreted in the feces [16]. van Beers-Schreurs et al. [17] compared the concentrations of SCFA in the portal and peripheral blood with those in the large intestinal digesta in weaning piglets. They reported that while giving a solid diet during weaning increased the SCFA concentrations in portal blood, the concentrations of SCFA remained unchanged in the intestinal lumen [17]. These data suggest that the concentrations of luminal SCFA do not always reflect the concentrations of absorbed SCFA.
Age at weaning age is one of the important factors for a good post-weaning development of pigs. In our previous study, early weaning at 14 days of age caused functional and morphologic ateliosis in piglets [12,13]. It is worth noting that marked maturation of the mucosal structure and function took place in 14-and 21-day-old suckling piglets [12]. Therefore, it is likely that age at weaning may also affect the ability of the intestine to absorb SCFA.
The aim of the present study was to evaluate the production and influx of SCFA in the cecum of pigs from suckling to weaning using a highly sensitive gas chromatography-mass spectrometry (GC-MS) technique [18]. Gene expression of SCFA transporters and molecules related to the tight junction were also assessed to elucidate the mechanism of SCFA absorption from the lumen into blood of piglets.
Animals
The 32 piglets used in the present study are shown in Table 1. The crossbred (Landrace × Large white × Duroc) piglets used in the present experiment were the same as those described in a previous study [13], except for piglets weaned at 14 days of age, which were omitted in the present work because we demonstrated that weaning at such age is not commercially practical for the pig industry [12,13]. All piglets were raised at the Toyohashi Feed Mills Technical Center (Shinshiro, Aichi, Japan). Suckling piglets were fed only maternal milk and weaning piglets were given a typical commercial weaning diet (JustOne Sprout; Toyohashi Feed Mills, Aichi, Japan). The nutrient composition of the diet was as follows (g/kg): crude protein, 214; crude fat, 75; crude fiber, 3; and crude ash, 60. All diets and water were given ad libitum. The animals were handled in accordance with the guidelines for animal studies of the Experimental Animal Committee of Kyoto Prefectural University (approval number KPU240410).
Dissection and Sampling
Ad libitum feeding was maintained until just before the dissection in all piglets. At the dissection, pigs were intraperitoneally anesthetized with sodium pentobarbital (Somnopentyl; Kyoritsu, Tokyo, Japan). All dissections started at 11:00 a.m. and collection of blood samples from all location of a piglet were finished within 5 min after confirmation of deep anesthesia. Briefly, the abdominal wall of pigs was incised along midline, blood was quickly collected from the cecal, portal, and abdominal veins, and the animals were euthanized by exsanguination. Afterward, the entire intestine was removed, the large intestine separated, and the cecal digesta collected. The cecum was washed several times with sterilized saline, and its middle section of mucosa soaked in RNA-later ® solution (Sigma, Tokyo, Japan), and stored first at 4 • C for 24 h, then at −80 • C until use. Blood samples were centrifuged at 1750× g for 10 min at 4 • C and serum was collected. Serum and digesta samples were stored at −80 • C until use.
Short Chain Fatty Acid Analysis by Ion-Exclusion High-Perfornance Liquid Chromatography
The concentrations of SCFA in the cecal digesta were measured by ion-exclusion high-performance liquid chromatography (HPLC) as previously described [18].
High-Sensitivity Detection of Short Chain Fatty Acid by Gas Chromatography-Mass Spectrometry
SCFA in serum samples were analyzed by GC-MS using a high-sensitivity detection method as previously described [18].
Gene Expression Analyses Using Real-Time Polymerase Chain Reaction
Total RNA extraction from cecal mucosa was conducted as described elsewhere [19]. cDNA synthesis and real-time polymerase chain reaction (PCR) were conducted as previously described by Inoue et al. [20]. Gene expressions of monocarboxylate transporter 1 (MCT1), sodium monocarboxylate transporter 1 (SMCT1), and occludin were evaluated by ∆∆Ct methods with reference to glyceraldehyde 3-phosphate dehydrogenase (gapdh) as internal control [21]. The primers and Taqman probes used in the present study are listed in Table 2.
Statistical Analyses
Depending on the results of the Bartlett test, either a complete randomized design, one-way analysis of variance (ANOVA) or the Kruskal-Wallis test was used to analyze the differences in each variable between S7, S14, S21, S28, W21p7, W21p14, W28p7, and W28p14. Tukey-Kramer post hoc (parametric or non-parametric) methods were used for multiple comparisons as needed. Correlation coefficient and its probability between parameters were analyzed by the Pearson's correlation coefficient test. Differences between means were considered significant at p < 0.05. Values are given as the means ± standard errors. All data were analyzed using STATCEL4 (OMS, Saitama, Japan), an add-in package for Excel ® (Microsoft Corp., Redmond, WA, USA).
Concentrations of Short-Chain Fatty Acids in the Cecal Digesta
Acetate, propionate and n-butyrate concentrations were found to increase in the cecal digesta of piglets (Figure 1a-c), from day 7 to day 28 after birth, but only the concentration of propionate in S28 piglets was found to be significantly higher than that in S7 piglets (Figure 1b). Age at weaning tended to affect SCFA concentrations in the cecal digesta. For example, although the concentrations of acetate and propionate were unaffected after weaning at day 21, they tended to be affected after weaning at day 28. The concentrations of acetate and propionate detected at weaning at day 28 temporarily decreased within the next seven days (W28p7), before recovering at day 14 post-weaning (W28p14). Nonetheless, these changes in concentrations were not significantly different.
Concentrations of Short-Chain Fatty Acids in the Cecal Vein
In serum in the cecal vein, SCFA concentrations did not change from day 7 to day 28 after birth, although weaning tended to increase the concentrations of acetate, propionate, and n-butyrate (Figure 1d-f). Particularly, acetate and propionate concentrations in W21p14 piglets increased significantly than those in S14 piglets.
Concentrations of Short-Chain Fatty Acids in the Portal Vein
In serum in the portal vein, changes in SCFA concentration were not detected (Figure 1g,h,j), due to the wide range of individual values.
Concentrations of Short-Chain Fatty Acids in the Abdominal Vein
The concentrations of SCFA in serum in the abdominal vein tended to differ from those in other veins (Figure 1j-l). For example, the concentrations of acetate and propionate gradually increased from day 7 onward, being those detected at day 28 significantly higher than those at day 7. It is worth noting that the concentration of n-butyrate was barely detected from day 7 to day 28 after birth ( Figure 1l).
Age at weaning also tended to affect SCFA concentrations in the abdominal vein after weaning. For example, after weaning at day 21, the concentration of acetate initially tended to increase (W21p7), before showing a tendency to decrease at day 14 post-weaning (W21p14). In contrast, after weaning at day 28 a decrease in the concentration of acetate was detected, which by day 14 post-weaning (W28p14) became significant, when compared with S28 piglets. In addition, the concentration of propionate tended to decrease in piglets weaned at days 21 and 28. Regarding the concentration of n-butyrate, a non-significant increase was detected in all weaned piglets, regardless of age at weaning.
Gene Expressions of SCFA Transporters and Occludin in the Cecal Mucosa
Gene expression of SCFA transporters MCT1 and SMCT1, and occludin-a molecule related to the tight junction-is shown in Figure 2. While the gene expression of MCT1 decreased during suckling, it increased after weaning (Figure 2a). Indeed, there were significant differences between the gene expression of MCT1 observed at weaning days and after weaning (S21 vs. W21p7, W21p14, and W28p7). The highest expression of smct1 was detected in S14 piglets (Figure 2b). In addition, smct1 expression showed non-significant increases after weaning at days 21 and 28. Gene expression of occludin was the highest in S7 piglets, but it decreased over time (Figure 2c). While a significant difference was observed between S7 and S21 piglets, weaning did not affect the gene expression of occludin.
Correlation Analysis of Short-Chain Fatty Acids Concentration in the Cecal Vein
Correlations of the concentrations of SCFA in the cecal vein with those in other samples and gene expression associated with SCFA transporters and the tight junction are shown in Table 3. While acetate and propionate concentrations in the cecal vein positively correlated with those in cecal digesta (p < 0.05), n-butyrate concentration in the cecal vein did not. Moreover, while the concentration of acetate in the cecal vein also positively correlated with that in the portal vein (p < 0.05), the concentrations of propionate and n-butyrate did not. Finally, the concentrations of SCFA in the cecal and the abdominal veins did not correlate. Interestingly, all SCFA concentrations in the cecal vein significantly correlated with the gene expression of MCT1 and occludin (p < 0.05). However, while the correlation with MCT1 gene expression was positive, the correlation with occludin gene expression was negative.
Discussion
SCFA are produced mainly in the hindgut of non-ruminant mammals [22]. In pigs, the cecum contains larger concentrations of SCFA in comparison with those found in the colon and rectum [6,22]. Therefore, in the present work cecum was chosen as the organ to evaluate SCFA production.
SCFA were readily detected in the cecal digesta of piglets, and their concentrations were detected to increase in suckling piglets from day 7 to day 28 after birth, but significant difference was not detected in acetate and n-butyrate (Figure 1a-c). Regarding the acetate, however, significant difference was observed between S14 and S21 when statistical analysis was performed on the values for only suckling piglets. Due to piglets ingested only maternal milk during suckling, the increase in SCFA concentrations in the cecal digesta may have been caused mainly by changes in milk composition and consequently in microbiota composition. For example, Noblet & Etienne [23] reported that the lactose concentration in milk gradually increases after parturition. Therefore, carbohydrates flowing into the cecum at higher concentrations are likely to further stimulate fermentation. The data from our previous study seem to validate this hypothesis because we showed that the gut microbiota of neonatal piglets undergoes changes even during suckling [24]. It has been well documented that weaning is one of the most stressful events for newborn mammals in general [25]. Indeed, the transition from liquid to solid food during weaning drastically affects the morphology and functions of the intestine [12,13], which in turn causes the gut microbiota to undergo profound changes [24]. In that context, in our study it was unexpected that SCFA concentrations in the cecal digesta did not change with weaning (Figure 1a-c). One possible explanation for the lack of change in SCFA concentration may be the size of cecum, which naturally increases after weaning [26]. The enlargement of cecum likely concealed an increase in the total amount of SCFA occurring in cecal digesta. Another fact that may help explain this unexpected outcome may be an increase in SCFA absorption after weaning, which is further discussed below.
SCFA found in blood serum in the cecal vein are those absorbed from the lumen following a partial utilization by the mucosal cells [27]. In the present work, absorption of SCFA was relatively low during suckling, but increased after weaning (Figure 1d-f). By contrast, although the concentrations of SCFA in the cecal vein did not change during suckling, they gradually increased in the cecal digesta during the same period (Figure 1a-c). These contrasting results seem to indicate that some sort of impaired absorption of SCFA took place in the lumen of suckling piglets. Nonetheless, it was detected that after weaning, SCFA concentrations in the cecal vein were high, which may indicate that absorption from the hindgut mucosa substantially increased in this period. When the correlation coefficient between the concentrations of SCFA in the cecal digesta and cecal vein were evaluated, it was found that acetate and propionate correlated positively, but n-butyrate did not (Table 3). These results seem to indicate that a considerable amount of n-butyrate was not absorbed into the cecal vein but remained within the cecal mucosa, perhaps utilized by the cells, especially the epithelial cells. The fact that n-butyrate is the major energy source of the epithelial cells in the large intestine [28] seems to validate our results. Interestingly, unlike in the suckling period, during the post-weaning period a higher concentration of n-butyrate was detected in the cecal vein (Figure 1f), which suggests that in weaned piglets, more n-butyrate flowed into blood and reached the liver through the portal vein (Figure 1i). In comparison, n-butyrate and other SCFA are transported through the portal vein to the liver of humans [29], implying that the dynamics of n-butyrate may be a similar feature in both humans and pigs.
When the host absorbs SCFA from the lumen, two routes have been observed: a 'passive' diffusion and an 'active' transport, but their contribution rate remains unclear [3], particularly during the suckling-weaning period. Passive diffusion, also known as gut permeability, permits the entry not only of SCFA but also of pathogenic microorganisms [30], which is generally problematic for the pig farming industry if it occurs after weaning [31]. The active transport of SCFA is a carrier-mediated transport dependent on metabolic energy, and it is believed that some transporters expressed by the epithelial cells are involved in the active transport of SCFA [32]. Indeed, HCO 3 /monocarboxylate exchange proteins, MCT and SMCT have been suggested to be facilitators of the influx of SCFA in the large intestine [16]. However, the HCO 3 /monocarboxylate exchange proteins in the large intestine are yet to be fully identified. For the present study, we selected two well-known transporters; MCT1 and SMCT1 as possible candidates involved in the active transport of SCFA in the epithelial cells of the intestine [16]. In the present work, although expression of mct1 was induced by weaning (Figure 2a), that of smct1 was not (Figure 2b). Moreover, mct1 expression positively correlated with the concentrations of acetate, propionate and n-butyrate in the cecal vein ( Table 3), suggesting that MCT1 plays an important role as active transport in suckling-weaned SCFA absorption. With regard to the passive diffusion of SCFA, weaning seems to increase gut permeability, because the expression of genes associated with the tight junction such as occludin, zonula occludens protein-1 and claudin-1 are downregulated by weaning [30,31]. Indeed, a downregulated expression of these genes induces an increased passive diffusion in a lactulose/mannitol tolerance test [30]. In the present study, while gene expression of occludin decreased during suckling, the expression of this gene was unaffected by weaning ( Figure 2c). Furthermore, occludin expression negatively correlated with the concentrations of acetate, propionate and n-butyrate in the cecal vein ( Table 3), suggesting that passive diffusion may also contribute to SCFA absorption in developing piglets.
Although no significance was found, the dynamics of SCFA in the portal vein bore a resemblance to those in the cecal vein (Figure 1g-i). Conversely, the dynamics of the concentrations of acetate and propionate in the abdominal vein were different from those in other veins (Figure 1j,k). Acetate and propionate concentrations increased in the abdominal vein during suckling, but sharply decreased after weaning (Figure 1j,k). After flowing into the liver, SCFA are readily metabolized by hepatocytes [33]. For example, propionate is almost all metabolized to glucogenic and lipogenetic substrates in the liver, hence it is hardly detected in the abdominal vein afterward [33]. In the present study, however, a relatively high amount of propionate was still observed in the abdominal vein of 28-day-old suckling piglets (Figure 1k). Two scenarios can be proposed to explain this apparent discrepancy: (1) under-development prevents the liver from completely metabolizing propionate during suckling; and (2) additional acetate and propionate may have directly flown in from the lymph fluid via the thoracic duct. To corroborate this possibility, we confirmed that the lymph fluid of suckling piglets contained higher concentrations of acetate (125 µmol/L) and propionate (2.0 µmol/L) than did that of weaned piglets (acetate: 83 µmol/L; propionate: undetected). Therefore, it can be cautiously asserted that the concentrations of acetate and propionate in the lymph fluid likely contributed to an increase in the concentrations of these SCFA in the abdominal vein during suckling.
Conclusions
In the present study, the dynamics of SCFA production and influx in piglets were evaluated from suckling to weaning. It was observed that while that the concentrations of SCFA in the cecal digesta changed from day 7 to day 28 after birth (suckling), those in the cecal vein did not. Our results suggested that some sort of impaired absorption of SCFA took place during suckling. Moreover, while the concentrations of SCFA in the cecal digesta were unaffected by weaning, those in the cecal vein substantially increased after weaning. Based on these results, it can be hypothesized that absorption from the hindgut mucosa likely begins after weaning. Gene expression analysis seemed to support this hypothesis, as it was observed in the cecal mucosa that while SCFA transporter mct1 was upregulated, occuludin-a protein associated with the tight junction-was downregulated. It may be concluded that the present work demonstrated that age at weaning affected SCFA absorption, especially an earlier weaning at 21 days of age rather than a later weaning at 28 days of age. Nonetheless, because limited numbers of piglets were used in this study, further study is needed to determine our hypothesis regarding the effect of weaning. | v3-fos-license |
2020-01-01T15:20:47.617Z | 2019-12-26T00:00:00.000 | 209518604 | {
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} | pes2o/s2orc | Dietary Supplementation with Chitosan Oligosaccharides Alleviates Oxidative Stress in Rats Challenged with Hydrogen Peroxide
Simple Summary Oxidative stress adversely affects animal health and performance. Feed additives with antioxidant abilities supplementation can alleviate oxidative stress. The aim of this study was to evaluate the hypothesis that dietary supplementation with COS alleviates the damage caused by oxidative stress in Sprague Dawley rats challenged with hydrogen peroxide (H2O2). The results shown that COS exhibited better radical scavenging capacity of 1, 1-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion (O2−), H2O2, and ferric ion reducing antioxidant power (FRAP) than butylated hydroxy anisole (BHA), increasing activity of SOD, CAT, GSH-Px, GSH, and T-AOC, as well as decreasing MDA level in serum, liver, spleen, and kidney. Our results indicated that COS can protect Sprague Dawley rats from H2O2 challenge by reducing lipid peroxidation and restoring antioxidant capacity. Abstract Oxidative stress is induced by excessive oxidative radicals, which directly react with biomolecules, and damage lipids, proteins and DNA, leading to cell or organ injury. Supplementation of antioxidants to animals can be an effective way to modulate the antioxidant system. Chitosan oligosaccharides (COS) are the degraded products of chitosan or chitin, which has strong antioxidant, anti-inflammatory, and immune-enhancing competency. Therefore, the current study was conducted to evaluate the hypothesis that dietary supplementation with COS alleviates the damage caused by oxidative stress in Sprague Dawley rats challenged with hydrogen peroxide (H2O2). The rats were randomly divided into three groups: CON, control group, in which rats were fed a basal diet with normal drinking water; AS, H2O2 group, in which rats were fed the basal diet and 0.1% H2O2 in the drinking water; ASC, AS + COS group, in which rats were fed the basal diet with 200 mg/kg COS, and with 0.1% H2O2 in the drinking water. In vitro, COS exhibited better radical scavenging capacity of 1, 1-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion (O2−), H2O2, and ferric ion reducing antioxidant power (FRAP) than butylated hydroxy anisole (BHA). In vivo, dietary supplementation with COS alleviated the H2O2-induced oxidative damage, evidenced by comparatively increasing activity of SOD, CAT, GSH-Px, GSH, and T-AOC, and comparatively decreasing level of MDA in serum, liver, spleen, and kidney. COS also comparatively alleviated the H2O2-induced inflammation. In conclusion, COS supplementation reduced lipid peroxidation and restored antioxidant capacity in Sprague Dawley rats, which were challenged with H2O2.
Introduction
Oxidative stress is induced by excessive oxidative radicals, which directly react with biomolecules, damaging lipids, proteins, and DNA, and leading to cell or organ injury [1][2][3][4]. Moreover, oxidative the FRAP reagent consisted of acetate buffer (0.3 M, pH = 3.6), TPTZ (10 mM) in hydrochloric acid (40 mM), and ferric chloride (20 mM), at a ratio of 10:1:1. The FRAP reagent was prepared just before the reaction, then 1.5 mL of FRAP reagent was mixed with 150 µL distilled water and 50 µL sample solutions at 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 g/L. It was then incubated for 30 min at room temperature in the darkness and the absorbance was determined at 593 nm. The reducing power is presented as micromoles Fe/gram fresh weight.
Animals, Experiment Design, and Diets
The male Sprague Dawley rats (8-10 weeks, 178.39 ± 5.12 g) used in this study were obtained from the Beijing Administration Office of Laboratory Animals (Beijing, China). The rats were individually housed in polycarbonate cages with soft wood granulate floors, and kept at 24 • C, with a 12 h light-dark cycle. After a week of acclimatization, 30 rats were randomly divided into one of 3 groups with 10 rats in each group for this 10 day experiment: CON, control group, rats were fed basal diet and normal drinking water; AS, H 2 O 2 group, rats were fed basal diet with 0.1% H 2 O 2 in drinking water [29]; ASC, AS + COS group, rats were fed basal diet with 200 mg/kg COS, and with 0.1% H 2 O 2 in drinking water. The supplementation level of COS was based on our previous preliminary research. All rats had free access to water and diet. The composition of the basal diet is listed in Table 1, and was made according to the nutritional requirement recommendations by the American Institute of Nutrition-93 diet [30].
Plasma Collection and Tissue Preparation
On the last day of the experiment, after 12 h fasting, all rats were euthanized with diethyl ether. Blood was collected from the posterior vena orbitalis, then centrifuged at 3000× g × 10 min to collect the serum, and stored at −20 • C until analysis. The liver, kidney, and spleen were collected and a 10% homogenate was prepared in PBS and centrifuged at 3000× g × 10 min at 4 • C, the supernatant was used for further biochemical assays.
Antioxidant and Inflammatory Cytokines Assays
The content or the activity of MDA, SOD, CAT, GSH-Px, GSH, T-AOC, IL-1β, IL-6, IL-10, and TNF-α in serum, liver, kidney, and spleen were measured according to the manufacturer's instruction.
Statistical Analysis
Data were analyzed by ANOVA using the GLM procedures of SAS (V9.1, SAS Inst., Inc., Cary, NC, USA). Duncan's multiple range tests were done to check the differences among treatments, and p < 0.05 was considered significant.
Antioxidant and Inflammatory Cytokines Assays
The content or the activity of MDA, SOD, CAT, GSH-Px, GSH, T-AOC, IL-1β, IL-6, IL-10, and TNF-α in serum, liver, kidney, and spleen were measured according to the manufacturer's instruction.
Statistical Analysis
Data were analyzed by ANOVA using the GLM procedures of SAS (V9.1, SAS Inst., Inc., Cary, NC, USA). Duncan's multiple range tests were done to check the differences among treatments, and p < 0.05 was considered significant.
DPPH, O2 -, H2O2, and FRAP Scavenging Capacity
The DPPH, O2 -, H2O2, and FRAP scavenging capacity of COS and BHA is shown in Figure 1. The concentration ranges from 0.01 to 0.06 g/L, the scavenging activity of COS ranges from 50.19% to 61.77%, and the scavenging activity of BHA ranges from 24.52% to 36.10%. For O2 − scavenging capacity, the scavenging activity of COS ranges from 32.94% to 40.31%, and the scavenging activity of BHA ranges from 23.45% to 30.02%. For the H2O2 scavenging capacity, the scavenging activity of COS ranges from 61.65% to 86.21%, and the scavenging activity of BHA ranges from 38.07% to 49.10%. For ferric reducing antioxidant power (FRAP) scavenging capacity, the scavenging activity of COS ranges from 44.24% to 42.62%, and the scavenging activity of BHA ranges from 23.73% to 24.93%.
Effects of COS on Antioxidant Status in Serum
Administration of H2O2 in drinking water increased (p < 0.05) the content of MDA in serum (Figure 2A), and decreased (p < 0.05) the activity of SOD ( Figure 2B) and CAT ( Figure 2C) in the AS
Effects of COS on Antioxidant Status in Serum
Administration of H 2 O 2 in drinking water increased (p < 0.05) the content of MDA in serum (Figure 2A), and decreased (p < 0.05) the activity of SOD ( Figure 2B) and CAT ( Figure 2C) in the AS group compared with the CON group. No significant differences were observed on the activity of MDA, SOD, CAT, GSH-Px, GSH, or T-AOC.
Animals 2020, 10, x FOR PEER REVIEW 5 of 13 group compared with the CON group. No significant differences were observed on the activity of MDA, SOD, CAT, GSH-Px, GSH, or T-AOC.
Effects of COS on Antioxidant Status in the Liver
The activity of SOD and GSH in the AS group was significantly (p < 0.05) lower than that in the CON group ( Figures 3B,E) in the liver. The activity of CAT ( Figure 3C), GSH-Px ( Figure 3D), and T-AOC ( Figure 3F), and the content of MDA ( Figure 3A) were not significantly affected by exposure to H2O2 or supplementation with COS.
Effects of COS on Antioxidant Status in the Liver
The activity of SOD and GSH in the AS group was significantly (p < 0.05) lower than that in the CON group ( Figure group compared with the CON group. No significant differences were observed on the activity of MDA, SOD, CAT, GSH-Px, GSH, or T-AOC.
Effects of COS on Antioxidant Status in the Liver
The activity of SOD and GSH in the AS group was significantly (p < 0.05) lower than that in the CON group ( Figures
Effects of COS on Antioxidant Status in the Spleen
The activity of CAT and GSH-Px in the AS group was significantly (p < 0.05) lower than that in the CON group ( Figure
Effects of COS on Antioxidant Status in the Spleen
The activity of CAT and GSH-Px in the AS group was significantly (p < 0.05) lower than that in the CON group ( Figure
Effects of COS on Antioxidant Status in the Kidney
The activity of SOD, GSH-Px, GSH, and T-AOC in the AS group was significantly (p < 0.05) lower than that in the CON group ( Figure 5B-F, respectively), the activity of GSH and T-AOC in the AS group was also lower (p < 0.05) than that in the ASC group. The activity of CAT ( Figure 5C) and the content of MDA ( Figure 4A) in the kidney were not significantly affected by exposure to H2O2 or supplementation with COS.
Effects of COS on Antioxidant Status in the Kidney
The activity of SOD, GSH-Px, GSH, and T-AOC in the AS group was significantly (p < 0.05) lower than that in the CON group ( Figure 5B-F, respectively), the activity of GSH and T-AOC in the AS group was also lower (p < 0.05) than that in the ASC group. The activity of CAT ( Figure 5C) and the content of MDA ( Figure 4A) in the kidney were not significantly affected by exposure to H 2 O 2 or supplementation with COS.
Effects of COS on Inflammatory Cytokines in the Serum, Liver, Spleen, and Kidney
The content of IL-1β, IL-6, TNF-α, and IL-10 in the serum, liver, spleen, and kidney are presented in Figures 6-9, respectively. The content of IL-1β ( Figures 6A-9A), IL-6 ( Figures 6B-9B), IL-10 ( Figures 6C, 7C and 9C), and TNF-α ( Figures 6D-9D) in the serum, liver, spleen, and kidney did not differ among treatments, except the content of IL-10 ( Figure 8C) in the spleen. The amount of IL-10 was significantly lower (p < 0.05) in the AS group compared with the CON group.
Discussion
Increasing attention has been paid to the field of antioxidants in recent years. Food scientists have paid attention to antioxidants because of their ability to prevent fat from oxidative rancidity. Doctors are interested because of their ability to protect from oxidative injury. COS possess stronger antioxidant competency as evidenced by their reaction with unstable free radicals to form stable radicals [31]. In the present study, the effects of COS on antioxidant property were analyzed both in vitro and in vivo.
DPPH is a stable nitrogen radical, and is widely used to evaluate radical quenching capacities. The DPPH scavenging ability of COS was higher than BHA. H2O2, and O2 − act as signaling intermediates, and the over-production of H2O2 and O2 − is an indicator of oxidative stress [32]. DPPH reflects limited oxidation situations because it only exists in vitro; therefore, we further evaluated the H2O2 and O2 − scavenging capacity of COS. H2O2 can be synthesized and destroyed in response to external stimuli [33], and O2 − acts as a potential precursor to generate reactive radical species. Thus, evaluating the scavenging capacity of H2O2 and O2 − [34] is important for clarifying the antioxidant capacity. The results showed that the scavenging capacity of COS against H2O2 and O2 − was higher than BHA. There is an association between antioxidant capacity of antioxidants and their reducing power. We investigated the reducing power of COS using the FRAP assay. The results showed the scavenging capacity of COS against FRAP was higher than BHA. The antioxidant capacity of COS is related to its characteristic structure, including its deacetylation ratio, molecular weight, and the source of the material. The molecular weight and deacetylation ratio of COS also exerts some synergistic effects on the biological capacities, where, generally, the low molecular weight gives the stronger scavenging activity of DPPH, H2O2 and O2 − [35].
Many reports indicated that oxidative stress was related to animal health [36]. H2O2 is generally used as an oxidative stress stimulus, and studies have indicated that oral administration or intraperitoneal administration of H2O2 induces oxidative stress [37,38]. The detrimental effects of H2O2 depends on its conversation to hydroxy ions and other subsequent redox products. In this study, H2O2 challenge increased the content of MDA in the serum, liver, and kidney. Increasing content of MDA is an indicator of lipid peroxidation. COS supplementation resulted in a comparative
Discussion
Increasing attention has been paid to the field of antioxidants in recent years. Food scientists have paid attention to antioxidants because of their ability to prevent fat from oxidative rancidity. Doctors are interested because of their ability to protect from oxidative injury. COS possess stronger antioxidant competency as evidenced by their reaction with unstable free radicals to form stable radicals [31]. In the present study, the effects of COS on antioxidant property were analyzed both in vitro and in vivo.
DPPH is a stable nitrogen radical, and is widely used to evaluate radical quenching capacities. There is an association between antioxidant capacity of antioxidants and their reducing power. We investigated the reducing power of COS using the FRAP assay. The results showed the scavenging capacity of COS against FRAP was higher than BHA. The antioxidant capacity of COS is related to its characteristic structure, including its deacetylation ratio, molecular weight, and the source of the material. The molecular weight and deacetylation ratio of COS also exerts some synergistic effects on the biological capacities, where, generally, the low molecular weight gives the stronger scavenging activity of DPPH, H 2 O 2 and O 2 − [35].
Many reports indicated that oxidative stress was related to animal health [36]. H 2 O 2 is generally used as an oxidative stress stimulus, and studies have indicated that oral administration or intraperitoneal administration of H 2 O 2 induces oxidative stress [37,38]. The detrimental effects of H 2 O 2 depends on its conversation to hydroxy ions and other subsequent redox products. In this study, H 2 O 2 challenge increased the content of MDA in the serum, liver, and kidney. Increasing content of MDA is an indicator of lipid peroxidation. COS supplementation resulted in a comparative decrease in MDA content in the serum, liver, and kidney, indicating that COS had protective effects due to its antioxidant capacity [39].
Both SOD and CAT can scavenge superoxide ions and hydroxyl ions. The present study showed decreasing activity of SOD in the serum, liver and kidney, and decreasing activity of CAT in the serum and spleen of H 2 O 2 -exposed rats. H 2 O 2 induced a dramatic decrease in the activity of SOD and CAT, which may be related to the formation of reactive oxygen species (ROS) [29]. Dietary COS increased the activity of SOD and CAT, which can save the depletion of the two enzymes. GSH-Px is a glutathione-related enzyme, and there was a significant decrease in the activity of GSH-Px in the spleen and kidney of H 2 O 2 -exposed rats in this study. H 2 O 2 can efficiently be scavenged by GSH-Px; therefore, the decreasing activity of GSH-Px reflects perturbations in the normal oxidative balance by H 2 O 2 exposure. GSH is a typical non-enzyme antioxidant, which defends against reactive free radicals and other oxidant species in cellular defense systems. In this study, there was a significant decrease in the activity of GSH in the liver and kidney of H 2 O 2 -exposed rats, indicating that the depletion of GSH led to enhanced formation of ROS [40]. Meanwhile, COS supplementation increased the activity of GSH in the kidney, which suggests that COS maintained the redox balance of ROS through both the enzymatic and non-enzymatic antioxidant defense system. The value of T-AOC reflects the total antioxidant capacity [41]. In this study, the T-AOC value of the kidney decreased by H 2 O 2 exposure, but increased with COS supplementation, which suggests that COS supplementation can alleviate oxidative stress induced by H 2 O 2 through the non-enzymatic antioxidant system in the kidney. Both in vitro and in vivo experiments showed that COS has strong radical scavenging activity and antioxidant capacity. The radical scavenging activity of COS is associated with their proton donation ability [42]. Dietary supplementation with COS prevented H 2 O 2 -induced lipid peroxidation and reserved depletion of SOD, CAT, GSH-Px, and GSH activity, which was consistent with former reports indicating antioxidant and protective properties of COS [43][44][45].
Oxidative stress and inflammation are highly related [46]. Oxidative stress and excessive production of ROS is associated with inflammation, leading to the synthesis and release of pro-inflammatory cytokines. No significant differences were detected in the concentration of TNF-α, IL-1β, IL-6, and IL-10 in the serum, liver, spleen and kidney, except a significant decreasing concentration of IL-10 in the AS group compared to the CON group in the spleen. With COS supplementation, the level of TNF-α, IL-1β, and IL-6 in the ASC group was comparatively lower than that in the AS group in the serum, liver, spleen, and kidney, while the level of IL-10 in the ASC group was comparatively higher than that in the AS group, which may be due to the anti-inflammatory activity of COS [24].
Conclusions
In conclusion, COS had higher antioxidant activities than BHA when checked by DPPH, O 2 − , H 2 O 2 , and FRAP scavenging capacity in vitro. COS significantly increased the content of GSH and T-AOC in the kidney, and comparatively decreased the content of MDA in the serum, liver and kidney, which suggested COS had protective effects against H 2 O 2 -induced oxidative damage and can be used as a potential antioxidant in feed.
Author Contributions: R.L., L.A. and Z.Z. participated in the experiment design and manuscript writing. R.L. and Q.C. involved in feeding trail, sample and data collection, and analysis. All authors have read and agreed to the published version of the manuscript. | v3-fos-license |
2018-12-07T11:36:16.823Z | 2013-03-20T00:00:00.000 | 54598076 | {
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} | pes2o/s2orc | Adsorption of Waste Metal Cr ( VI ) with Composite Membranes ( Chitosan-Silica Rice Husks )
Chromium compounds are widely used in modern industry. Many of these compounds are dumped into the surrounding environment. Membrane technology is more efficient and effective than conventional methods for waste treatment. The research objective was to make a membrane separation process that can be applied to Cr(VI). Membranes were made from chitosan and silica rice husks. Variations of chitosan and silica rice husk used (g) are 2:1 (A1), 2:2 (A2), 3:1 (B1), and 3:2 (B2). The membrane was made by using an inverted phase technique. Results of SEM characterization of membranes showed that B2 has the largest pores at 2.58 μm. The FTIR characterization results indicate the presence of crosslinking between chitosan with silica rice husk with the appearance of Si-O adsorption band at wavelength 1122980/cm. A1 membrane, with the smallest pore size has the greatest rejection value towards Cr(VI) which is 70%. The result of this research showed that the composite membrane of silica rice husk was effective enough to adsorb metal Cr(VI) with an average adsorption capacity of 1665.85 mg/g.
Introduction
The increase in industrial activity in Indonesia has had an effect on increasing environmental pollution.Pollution of water, air, soil and the disposal of hazardous and toxic waste (B3) is a problem that must be faced by communities living around the industrial area.Heavy metals, a type of B3 waste, are toxic and carcinogenic [1].Metal Cr(VI) is a heavy metal that is commonly found in electroplating industrial waste and is hazardous to health and the environment.
Many methods have been used to process waste metal Cr(VI) including adsorption methods such as chromium with activated biomass sludge as an adsorbent [2], reduction methods with the compound KI as a reductor of Cr(VI) [3] and adsorption with straw [4].However, none of these waste treatment methods are effective in treating wastewater.The method now widely used to treat wastewater is membrane technology because the process is very simple, energy efficient, retains the material and does not use additional chemicals [5].In this study, composite membranes made of chitosan are mixed with silica rice husk.
Rice husk is an abundant material because Indonesia is a country that produces rice husks.The rice husks contain silica.Based on research conducted, the content of silica (SiO 2 ) in the rice husk is quite high, ranging from 87-90% [6].
Chitosan is a polymer that has been widely used as a membrane material.Chitosan has the ability to adsorb metals by forming complexes with metals so chitosan can be used to treat waste metal [7].Chitosan membrane has a dense structure so it has very small porosity.For that reason chitosan is combined with silica to form pores that are more effective in screening waste.Particles of silica are ideal porogens for producing porous chitosan membranes with controlled porosity and good mechanic properties [8].
The purpose of this research was to make membrane filtration from chitosan composited with silica rice husks.Furthermore, the membrane is tested for its ability to adsorb metal Cr(VI) from artificial waste.
Methods
The main materials used in this study were rice husk and chitosan.Rice husk is taken from the rice mills around IPB at Darmaga campus.Chitosan was purchased from the Fisheries Faculty of the Department of Technology, IPB, with 74.25% degree of deacetylation.
Making silica rice husk was conducted in three stages i.e. drying, incinerating and acidification Pre-dried rice husks were washed with distilled water and then dried at 190 o C for 1 h in a furnace.Then the husks were incinerated at a temperature of 300 o C for 30 minutes, and 600 o C for 1 h.Once charcoal had formed it was heated again at a temperature of 600 o C for 1 h to become ash.The next process is acidification with HCl (37%) and more heating at a temperature of 1000 o C for 6 hours [9].
Preparation of composite membranes.The inverse phase technique was used.Chitosan was dissolved in 50 ml of 2% acetic acid solution and sonicated for 1 h.Then, the silica particles were gradually added to the chitosan.The solution was further sonicated for 1 h to make a homogeneous solution.Sonication was performed at a frequency of 42 kHz with a Bronson 2510 which breaks down the molecules so that they dissolve faster.The solution was then filtered, then shaped in the membrane mold and dried at 50 o C for 12 h in an incubator.After drying, the membrane was soaked in a solution of NaOH (5%).Membranes were further heated for 2 h at a temperature of 80 o C to remove silica and produce a porous chitosan membrane.The membrane was washed again with distilled water to remove residual NaOH.The modification made from previous studies [9][10] was the use of chitosan and silica rice husk mass composition.Variations in composition were performed to make membranes with the best pores to filter waste.In making this membrane the various mass compositions (g) of chitosan and silica rice husk used are: 2:1 (A1), 2:2 (A2), 3:1 (B1) and 3:2 (B2).
Test of flux and membrane rejection.Determining water flux and waste rejection is obtained by measuring the amount of permeate volume that passes through a unit of membrane surface per unit area per unit time.The waste used is artificial wastewater obtained by dissolving K 2 Cr 2 O 7 in distilled water with a concentration of 300 ppm.The rejection coefficient (R) can be calculated using the following equation [11]: where C f ,, C p (mg/L) showed the value of feed concentration and permeation.
Results and Discussion
Characterization of silica rice husk using XRD and FTIR.Heat at a temperature of 1000 o C produces silica rice husk in the form of crystals.Figure 1 depicts two silica peaks (SiO 2 ) at an angle of 2θ at 22 o and 36 o with quite high intensity.Referring to the XRD analysis research by Handayani [9], peaks also appear with high intensity at 2θ at 22 o and 36 o .The degree of crystallinity formed was 75.98%.The type of crystal formed can be determined by calculating the parameters of the crystal lattice by a value of 2θ.Lattice parameters can be determined by determining the value of a, b, and c.The XRD results of silica rice husk in Figure 1 The IR spectra of silica rice husk in Figure 2 show the appearance of a sharp peak of Si-O functional group at wavelengths of around 1055/cm.Si-O functional group is a characteristic of the silica compound.Moreover, the groups of O-H, Si-C and Si-Cl also appear at wavelengths 3371/cm, 820/cm, and 471/cm respectively.
Analysis of functional groups membrane.Functional groups that appear in membranes A1, A2, B1 and B2 have transmittance intensity similar to the sharp peaks in the adsorption band of each group function.Morphological analysis of membrane.The membrane formed is porous.The porous membrane is formed due to the influence of the silica composition.The pore shapes formed area symmetrical and the pore size is not homogeneous, see Figure 4.
Pore size of the membrane that forms varies depending on the composition of chitosan and silica (Table 1).A2 membrane has a larger pore size than membrane A1 because A2 added as much as 2 grams of silica while A1 has only 1 gram of silica.Similarly, for the B membranes, B1 has bigger sized pores compared to B2 which as more silica than B1.So the addition of silica will increase thepore size of the membrane formed.The results are consistent with previous research by Liu et al. [10] that states that silica serves as a porogen, meaning the particles of silica form pores in the chitosan membrane.This research revealed that the mass ratio of silica/chitosan is 10:1 (g), the membrane pore size on average is 25-35 μm.
Flux membrane.In Figure 5 it can be seen that in minute10 the flux sharply declined until minute 22.If screening is continued the flux values continue to fall until they almost reach zero.Decline in the flux value is due to membrane fouling.The direct effect of fouling causes a decrease in permeated flux, while the longterm effect can cause irreversible fouling of the membrane material and reduce its lifetime [12].In this condition the membrane begins to clog up, thus affecting the amount that permeates the membrane.Membrane rejection.In the A1 membrane, as can be seen in Figure 6, the rejection value does not vary significantly from the screening waste process.The average value of membrane rejection for A1 is 70%.
The average concentration of the final solution of Cr(VI) after being filtered by membrane A1 is 90.12 ppm.The average rejection value of membrane A2 is 66.18%, which is less than membrane A1.The final concentration of Cr(VI) after being filtered by membrane A2 is 101.47 ppm.The average rejection of B1 membrane is 67.55%.The average final concentration of Cr(VI) after being filtered by membrane B1 is 97.35 ppm, while after being filtered by membrane B2 it is 101.48 ppm.
In Figure 6 it can also be seen that the chitosan membrane without silica is capable of holding metal Cr(VI) to a maximum of 41.37%.Chitosan can act as an adsorbent as it is able to bind the metal.However, the rejection value is still small because the chitosan membrane without silica is dense so its pores are very small compared to if it has the addition of silica.So, of the four membranes that have been tested, based on rejection ability the average is 70%, 66.18%, 67.55%, 55.32% and 30.50% for membranes A1, A2, B1, B2 and chitosan for separation within 22 minutes.A1 membrane with smaller pore sizes than membranes A2, B1 and B2 has the highest rejection value of metal Cr(VI) which is an average of 70%, meaning that only 30% of the metal membrane Cr(VI) can escape.This value is greater than the metal uptake of Cr(VI) with the ion exchange membrane cell method at the amount of 30% for 300 minutes [13].The results of research by Devaprasath et al. [14] using spicigeraprosopis plant leaves also has a Cr(VI) percentage adsorption of about 69.4%.
The mechanism of metal adsorption that occurs between the membrane composite of chitosan with silica rice husk is supported by the nature of chitosan as a polycationic polymer.Chitosan is a chelating polymer t (minute) Flux (Lm-2 Hours -1 derived from natural ingredients so that chitosan is able to bind metals to form a metal-chitosan complex.Chitosan is an excellent ion chelating agent.Electrons of nitrogen contained in the amine group can form covalent bonds with transitory metal ions, including chromium.Chitosan acts as an electron pair donor in the transition of metal ions in the formation of a metalchitosan complex.While silica, in addition to forming pores in the membrane is also thought to act as a metal adsorbent of Cr(VI).This is evidenced by the increased rejection value of Cr(VI) on the chitosan membrane composited with silica compared with the chitosan membrane without the addition of silica rice husk.
Average adsorption capacity of chitosan-silica rice husk composite membrane is 1665.85mg/g.When compared with the results of research by Vijaya et al. [15], the adsorption ability of chitosan with silica rice husk composite is greater than adsorbent chitosan coated with a commercial silica which is 294.1 mg/g.Kinetic adsorption.Determining the reaction rate (k) is calculated using the first-order and second-order kinetic model [16].According to Table 2 the adsorption of Cr(VI) with chitosan-silica rice husk membrane followed the second order because the value of R 2 is close to 1 (linear).The k values obtained in this study illustrate that the greater the reaction rate constant is the faster is the depletion of reactants.
Conclusions
This study has created a composite membrane with chitosan and silica rice husk.Composite membrane can be used as membrane filtration for separating Cr(VI) metal.Based on the results of this study it can be concluded that the addition of silica rice husk in the manufacture of chitosan membrane affects the pore size of the membrane.Chitosan and silica rice husk form cross-links as indicated by the appearance of the Si-O adsorption b and at wavelength 1122-980/cm.SEM characterization results show that membrane B2 with 3 gram of chitosan and 2 grams of silica has the greatest pore size.A1 membrane with mass ratio 2:1 produces a smaller pore size and more effectively eliminates the metal Cr(VI) compared to other membranes with a rejection value of 70%.The average adsorption capacity of chitosan-silica rice husk membrane is equal to 1665.85 mg/g with the adsorption rate following the second-order kinetics model.
Figure 1.X-rayDifraction (XRD) Analysis of Silica Rice Husk at a Heating Temperature of 1000 o C Figure 3 shows the emergence of functional groups on the Si-O wavelength of around 1055 and 1122-980/cm.The Si-O adsorption band of the membrane appears around the wavelength range 1122-980/cm indicating a cross-link between chitosan and silica rice husk.Si-O wave length that appears in membranes A1 (1110/cm), A2 (1092/cm), B1 (1122/cm) and B2 (1118/cm) is greater than the value of the Si-O wavelength on silica rice husk (1055/cm.)This indicates a shift in the Si-O adsorption band due to a hydrogen bond between the functional groups of Si-O silica rice husk with the O-H chitosan group.The hydrogen strengthens the link between silica rice husk with chitosan, but causes the Si-O bond to be shortened so that the Si-O bond becomes stronger and the vibrational energy bond becomes greater.Based on the plank equation (E=h.c.ν), the wave length is proportional to the energy (E), so the increase in energy will make the adsorption band shift towards a greater wavelength (ν).
Figure 5 .
Figure 5. Flux Value of Membranes; A1 (■), A2 ( ), B1 (•), B2 (♦) The flux test results showed that membrane A1 produces a maximum flux value of about 50,000 Lm/m 2 hour in 300 ppm potassium dichromate (artificial waste).The value decreases within creasing flux time.At 10 minute the flux value is almost constant until 20 minute.This indicates clogging in the membrane called fouling.The maximum flux value of membrane A2 in artificial waste is around 60,000 Lm/m 2 hour.This flux value is higher than the A1 membrane.This indicates that membrane A2 has larger pore sizes.The A2 membrane has a chitosan:silica composition of 2:1 whereas the A1 membrane composition is 2:2.An increased amount of silica in the membrane causes larger pore size and consequently a greater flux value.Likemembrane A1, fouling of A2 membrane begins at minute10.Flux values of membranes B1 and B2 are not too different from membranes A1 and A2 as shown in Figure 3. Fouling also occurred in 10 minute marked by an increase of flux values that almost fell to zero at minute twenty.The average values of the artificial waste flux on membranes are in sequence 9,192.46Lm/m 2 hour; 8,788.74Lm/m 2 hour; 8,460.25 Lm/m 2 hour, and 2,403.77Lm/m 2 hour respectively for membranes A2, A1, B1 and B2.Membrane flux values are also influenced by the degree of membrane swelling.When the feed concentration permeates membrane A2 it opens pores thereby increasing pore size thus causing swelling. | v3-fos-license |
2020-11-19T09:08:58.041Z | 2020-11-16T00:00:00.000 | 228832866 | {
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} | pes2o/s2orc | Condensed Phase Guerbet Reactions of Ethanol / Isoamyl Alcohol Mixtures
: The self-condensation and cross-condensation reactions of ethanol and isoamyl alcohol are examined to better understand the potential routes to value-added byproducts from fuel ethanol production. Reactions have been carried out in both batch autoclave and continuous condensed-phase reactors using a lanthanum-promoted, alumina-supported nickel catalyst at near-critical condensed phase conditions. Analysis of multiple candidate kinetic models led to a Langmuir–Hinshelwood rate expression that is first-order in alcohol with water as the strongly adsorbed species. This model provides the best fit of data from both batch and continuous reactor experiments. Activation energies for primary condensation reactions increase as carbon chain lengths increase. Selectivities to higher alcohols of 94% and 87% for ethanol and isoamyl alcohol, respectively, were observed at di ff erent operating conditions. D.J.M. and I.N.; investigation, D.J.M. and I.N.; resources, D.J.M. and I.N.; data curation, D.J.M. and I.N.; writing—original draft preparation, D.J.M. and I.N.; writing—review and editing, D.J.M. and I.N.; and
Introduction
The predominant route for the conversion of hexose sugars (sucrose, glucose, maltose, etc.) to ethanol (EtOH) with yeast of the Saccharomyces family is the Embden Meyerhof pathway [1,2]. In this pathway, the main product (~99%) is EtOH, with the byproducts formed typically referred to as "fusel" oils or alcohols [3,4]. Fusel alcohols consist primarily of isoamyl alcohol (IAOH) (3-methyl-1-butanol) along with n-propanol, isobutanol, and optically active amyl alcohol [5]. Whether formed as byproducts of EtOH fermentation [6] or from fermentation-derived amino acids [7], these compounds have essential applications as aroma and flavoring agents in the food and beverage industry [3,[8][9][10][11]. For instance, isoamyl alcohol is used to produce isoamyl acetate, widely used in the food industry for its banana flavor [12,13]. Further, mixtures of fusel alcohols can be used as solvents or cleaners, and as reagents with various organic acids to make mixed esters that have desirable properties as solvents or as fuel additives [14][15][16]. But presently, the most common end use of fusel alcohols involves blending with purified EtOH as fuel for internal combustion engines, where their high energy density and compatibility with hydrocarbons make them attractive additives [12,[17][18][19].
Fusel alcohols contain at least one hydrogen atom on the β-position of their carbon backbones, and so can participate in Guerbet condensation reactions, in which higher alcohols are formed via one of several postulated condensation mechanisms. Because they are produced along with EtOH and require substantial processing to be recovered in pure form, the opportunity exists to efficiently produce additional higher alcohols via reaction of partially purified fusel alcohols with EtOH and with each other. With current U.S. EtOH production at approximately 15 billion gallons annually and fusel oils constituting 0.3 wt% to 0.7 wt% (~200 million kg/y) of total alcohols produced, the potential exists to produce fusel oil-based chemicals priced at least an order of magnitude higher than ethanol. This impact would enhance profitability of existing EtOH production, could help facilitate more economical cellulosic-based EtOH production, and would contribute to reducing the selling price of other chemicals derived from EtOH [20][21][22].
Despite the comprehensive analysis of the Guerbet condensation of various alcohols via process, catalytic, thermodynamic, and mechanistic aspects [23][24][25][26][27][28], Guerbet reactions of fusel alcohols have been the subject of only a few studies. Matsu-ura et al. studied the conversion of fusel alcohols over a homogeneous Ir-based catalyst at 120 • C and atmospheric pressure and obtained yields of as high as 98% for the self-coupling of C 5 and C 6 alcohols, and 86% for the self-coupling of C 12 alcohol [29]. They also studied isoamyl alcohol as the reagent and were able to achieve 50% yield of C 10 alcohol at the same reaction conditions. Later, Busch et al. confirmed the feasibility of the synthesis of branched C 10 alcohols through the Guerbet reaction of isoamyl alcohols at 180 • C and elevated pressure ranges (1.4-4.6 bar) using a Pd/C-based homogeneous catalyst [30]. Unfortunately, no analytical data were reported.
To date, no studies have been reported for Guerbet condensation reactions of fusel oil components with EtOH. We report here the reaction of isoamyl alcohol (IAOH), the primary constituent of fusel alcohols, with EtOH and with itself at condensed-phase conditions over supported nickel catalyst. Both batch reactions with different initial compositions and continuous, fixed bed reactions at different temperatures and reactor space velocities have been carried out. Higher alcohol yields and selectivities have been determined, and a kinetic model is developed to ascertain the relative rates of condensation of different alcohol species.
Batch Reactor Experiments
Batch reactions were performed in a 300 mL Parr reactor (Model 4842, Parr Instruments, Chicago, IL, USA). The Parr reactor was equipped with an Omega 1/8" stainless steel Type J thermocouple to measure reaction temperature within ±1 • C. Calibrations for achieving this accuracy were conducted in previous work [32,33]. Reaction pressure was measured using an electronic pressure transducer (maximum pressure 200 atm) that was calibrated against a 100 atm mechanical gauge with increments of 0.7 atm. The mechanical stirrer was set at 1000 rpm during the reaction.
After adding alcohols and catalyst, the reactor was sealed and purged with nitrogen at 1.0 atm overpressure. The reactions were carried out at autogenous pressure. An initial liquid sample was taken after nitrogen purging and before heating to the reaction temperature to verify initial composition. At the end of the reaction, the reactor was cooled and then depressurized. The total quantity of liquid products formed was taken as the sum of liquid sample masses collected and residual liquid in the reactor following depressurization. A sample of this residual liquid was weighed and then analyzed by gas chromatography as described below. The total quantity of gaseous products formed during reaction was determined by measuring the change in cooled reactor mass over the course of depressurization and collecting the gas in a sample bag. By determining the volume of gas collected via water displacement, the average molecular weight of the gas product was determined.
Continuous Reactor Experiments
A 1.91 cm external diameter (1.57 cm ID) by 76 cm length jacketed 316 Stainless Steel up-flow packed bed reactor was used for continuous experiments. Approximately 30 g of catalyst were placed in the reactor. The temperature profile during reaction was measured with a Type K thermocouple inside a 3 mm OD internal thermowell located on the center axis of the reactor. The reactor temperature was controlled by circulating silicon oil through the jacket using a Julabo (Model SE-6) heating circulator. A Tescom (Model 26-1764-24) back-pressure regulator was used to control the reactor pressure at 100 bar and reduce the effluent pressure to near atmospheric. To preheat the feed mixture to the reactor temperature, silicon carbide (SiC, 20-50 mesh) was placed upstream of the catalyst bed. Stainless steel rod fillers were also used before and after the reaction zone to reduce dead space in the reactor.
A feed composition of 79 mol% EtOH and 21 mol% IAOH was used in continuous flow experiments. Reactor temperature was varied from 210 • C to 250 • C, and the liquid feed flow rate was varied from 0.5 mL/min to 1.3 mL/min, corresponding to a weight hourly space velocity (WHSV) of 0.8 h −1 to 2.1 h −1 or a superficial residence time (τ) of 0.96 to 0.25 m 3 reactor/(m 3 feed/h), respectively. Once steady state operation of the reactor was achieved, condensable liquid products were collected in an ice/water trap over a period of time ranging from 30 to 90 min depending on the feed rate. The liquid product collected was analyzed using gas chromatography as discussed in the following section. The gaseous products were collected in a gas bag located downstream of the ice/water trap and quantified by the measurement of effluent gas rate at several time points during the product collection period.
Analytical Methods
The analytical methods and instruments used in this study are the same as those described in previous studies [22,[31][32][33]. Liquid product samples were diluted 10-fold in acetonitrile and analyzed using a Varian 450 gas chromatograph (GC) with a flame ionization detector. A 30 m SolGel-Wax column (0.53 mm ID, 1 mm film thickness) was used with the following temperature program: initial temperature 37 • C for 4 min; ramp at 10 • C/min to 90 • C, and hold at 90 • C for 3 min; ramp at 10 • C/min to 150 • C; ramp at 30 • C/min to 230 • C and hold for 2 min. Butyl hexanoate 1% solution was used as an internal standard in liquid product GC analyses. Multi-point calibration curves were used to determine the response factor of each known product. Unidentified liquid product peaks were quantified for carbon recovery calculations by using an average molecular weight and response factor based on values from adjacent known peaks in the chromatogram.
The number of moles of gas formed in reaction was determined volumetrically as described above. From the average molecular weight of gas byproducts, which ranged from 18 to 22 g/mol in all experiments, it was assumed that on average one mole of carbon was present in each mole of gas formed. Selected gas samples were analyzed by gas chromatography, as presented in previous works [22,[31][32][33][34], to support this assumption. The typical composition of gas byproducts is shown in Figure S1a of Supporting Information.
An in-house Excel spreadsheet was used to convert the species concentrations from GC analyses to EtOH and IAOH conversion, selectivity to each higher alcohol formed from a particular feed alcohol (mol feed alcohol to product/mol feed alcohol converted), overall selectivity to liquid byproducts (mol C in liquid byproducts/mol C in EtOH + IA converted), overall selectivity to gas byproducts (mol C in gas byproducts/mol C in EtOH + IA converted), and overall carbon recovery (mol C in reactor effluent/mol C in feed alcohols). Overall carbon recovery is the carbon balance that reflects uncertainty in experimental methods and analysis, and therefore exceeds 100% in some cases. Selectivity to liquid byproducts was normalized to make total product selectivity sum to 100% in cases where total carbon recovery exceeded 100%. Selectivity to liquid byproducts and gas byproducts is presented on a carbon basis based on both feed alcohols taken together, as it was not possible to distinguish between the two alcohols as the source of some liquid and gas byproducts in mixed alcohol feed experiments.
To further characterize the product composition, Karl Fischer titration was carried out in triplicate to determine water content of each sample. This value was used as a check of the overall molar balances for the reaction; the quantity of water formed was usually 25-30% greater than that predicted by the extent of alcohol condensations. The excess water formation arises from side reactions to form gas and liquid byproducts.
Experimental Results
Guerbet reactions with two alcohols lead to a significantly wider variety of product species than for a single alcohol. The key higher alcohol products of the mixed IAOH and EtOH experiments are shown on the right side of Figure 1; the alcohols responsible for forming the products are shown on the left side of Figure 1. byproducts is presented on a carbon basis based on both feed alcohols taken together, as it was not possible to distinguish between the two alcohols as the source of some liquid and gas byproducts in mixed alcohol feed experiments.
To further characterize the product composition, Karl Fischer titration was carried out in triplicate to determine water content of each sample. This value was used as a check of the overall molar balances for the reaction; the quantity of water formed was usually 25-30% greater than that predicted by the extent of alcohol condensations. The excess water formation arises from side reactions to form gas and liquid byproducts.
Experimental Results
Guerbet reactions with two alcohols lead to a significantly wider variety of product species than for a single alcohol. The key higher alcohol products of the mixed IAOH and EtOH experiments are shown on the right side of Figure 1; the alcohols responsible for forming the products are shown on the left side of Figure 1. In addition to desired higher alcohol products, liquid byproducts are formed at the reaction conditions examined. The typical composition of these liquid byproducts for experiments with pure EtOH and IAOH is given in Figure S1b,c of the Supporting Information. Additional peaks that likely represent liquid byproducts of reactions between EtOH and IAOH or their reaction intermediates were observed in chromatograms of liquid samples. No attempt was made to identify these byproducts of cross alcohol reactions, although they were included in the overall carbon balance for experiments by estimating the carbon number of each product based on its location in the chromatogram.
Batch Experiments
Experiments were conducted at 230 • C with 4.85 g of catalyst and 120 g of feed alcohols of composition varying from 100% EtOH to 100% IAOH. Reactions B2-B6 were run for 24 h, and Reaction B7 was run for 51 h. Results obtained from batch reaction studies are summarized in Table 1.
Continuous Experiments
Condensed-phase continuous reactions were carried out at 100 bar over 8 wt% Ni/9 wt% La 2 O 3 /Al 2 O 3 catalyst with a feed composition of 79 mol% EtOH and 21 mol% IAOH. Results of continuous flow experiments are given in Table 2. Prior to these experiments, control studied were performed with the same feed composition and catalyst at ambient conditions and without any catalyst at reaction conditions to ensure that the system does not contain any leaks and the reactor material does not provide any reactivity to the feed material. Tables 1 and 2 show EtOH selectivity toward higher alcohols ranging from 70-85% through both self-condensation and cross-condensation reaction with IAOH. The conversion rate of IAOH is lower than that of EtOH; very little C 10 product, the direct condensation product of IAOH, is formed. Nearly all IAOH reaction to higher alcohols takes place with EtOH to form the C 7 alcohol, even at lower initial EtOH/IAOH molar ratios (Exp. B4). The composition of IAOH liquid byproducts, shown in Figure S1c, indicates that formation of the initial aldehyde intermediate (3-methyl-butanal) of IAOH is significant, but the subsequent condensation of the intermediate to the C 10 product alcohol is slow. Steric hindrance resulting from the longer carbon chain length likely plays a role in condensation to form the C 10 product. The conversion rate of IAOH to liquid and gas byproducts, based on outlet concentrations of species in Figure S1, is similar to that of EtOH.
Kinetic Model Development
A kinetic model has been developed, based on prior studies of condensed-phase ethanol conversion [22,[31][32][33], to characterize reaction rates in the EtOH/IAOH reaction system. The following Guerbet (condensation) reactions are considered: In addition to these Guerbet reactions, two additional reactions (assumed to be first-order in alcohols) are required to account for conversion of EtOH and IAOH to gas and liquid byproducts. Including these reactions is necessary to maintain the correct feed alcohol concentration profile in the reactor.
EtOH side reactions : C 2 H 5 OH Since the selectivities to C 8 and C 9 alcohols formed in continuous reactor experiments were low, the formation of C 8 product alcohols is combined with C 6 alcohol formation, and the formation of C 9 product alcohols is combined with C 7 alcohol formation in the kinetic model. This is warranted because C 8 and C 9 alcohols are formed via further reaction of C 6 and C 7 alcohols with EtOH, respectively, as opposed to reactions of higher alcohols (C 4 + C 4 to C 8 , for example). EtOH is both more reactive than higher alcohols and EtOH is present in much higher concentration than other alcohols. The low selectivities to C 8 and C 9 products, and associated uncertainty with their low concentrations in the product mix, does not warrant the additional complexity of another rate expression and rate constant for each in the kinetic model.
Initially, kinetic rate expressions for R1-R4 were assumed to be second-order in alcohols. However, this simple second-order model did not provide a satisfactory fit of either batch or continuous reaction data. Analysis of the "indirect" mechanism [27,31,[35][36][37][38] of EtOH condensation to BuOH (see Supplementary Information) [31], where EtOH dehydrogenates to acetaldehyde (AA), AA undergoes aldol condensation to crotonaldehyde (CA), and CA hydrogenates to BuOH, shows that ethanol condensation rate is first-order in EtOH if (1) local H 2 concentration is assumed to be equal to AA concentration, and (2) either EtOH dehydrogenation or AA condensation are rate-limiting.
Prior work [33] showed that the presence of water (W), present initially or produced in reaction, inhibits the rate of EtOH condensation to BuOH. Other studies also have shown that the presence of water can limit the extent of dissociative H 2 adsorption on Ni [39,40] and other metal [41,42] surfaces, thus affecting initial alcohol dehydrogenation in the Guerbet reaction system. Therefore, a Langmuir-Hinshelwood (L-H) rate expression can be written for EtOH condensation to BuOH (R1): A similar rate expression can be written for IAOH condensation to C 10 alcohols, if only IAOH is present.
For reactions involving EtOH/IAOH mixtures, generating rate expressions based on rigorous analysis of the indirect mechanism is challenging (see Supplementary Information), because H 2 is produced both by EtOH and IAOH dehydrogenation. The rates are thus coupled, and the resulting rate expressions are complex. However, because in this study EtOH is present in larger molar quantities than IAOH, and EtOH reaction rate is faster than that of IAOH, the assumption can be made that H 2 forms predominantly from EtOH. With this assumption, the condensation reaction of IAOH (R3), and the cross-condensation reactions of EtOH with IAOH (R2) and with BuOH (R4) simplify to be first-order only in IAOH and BuOH, respectively. Including the L-H water adsorption term gives the final form of the rate expression for R2-R6. Details of the model development and the explicit rate expressions for R1-R6 are given in Supplementary Information.
The rate expressions described above have been applied to experimental data to determine the rate constants; the process to fit the kinetic model to data is given in the sections below. To ensure that these rate expressions best fit the experimental data, several alternate kinetic models were evaluated. In addition to the simple second-order rate expression, L-H rate expressions with water adsorption and the numerator second-order in alcohols were examined. Similar L-H rate expressions with EtOH, IAOH, or BuOH as the dominant adsorbing species were also examined. None of these kinetic models provided as good a fit to experimental data as the model that is first-order in alcohols with water as the dominant adsorbing species.
Continuous Reactor Modeling
The experimental data from the continuous reactor were used to determine the values of the rate constants for R1-R6. This was accomplished by writing the differential molar balances for each species in the reaction system (Equations (1)-(8) below) and numerically integrating them (using Euler's method in Microsoft Excel) to determine outlet concentrations from the reactor at each residence time. Reactor feed concentrations were calculated from densities of EtOH and IAOH at each reaction temperature and are given in Table S2 of the Supporting Information; the inlet concentrations vary significantly over the temperature range from 210 • C to 250 • C because the feed is an expanded liquid at these conditions close to the critical temperatures of each alcohol.
For brevity in the following equations, EtOH (C 2 H 5 OH) and IAOH (C 5 H 11 OH) are represented as E and IA, BuOH (n-C 4 H 9 OH) and C 10 H 21 OH are represented as the C 4 and C 10 condensation products of EtOH and IAOH, respectively. The terms C 6 , C 7 , C 8 , and C 9 refer to multiple cross-condensation alcohol products with the designated carbon number (Figure 1); identification of the structure of each individual alcohol with these carbon numbers was not attempted. Reaction rates r i in Equations (1)-(8) refer to Reactions 1-6 above.
Rate Constant Determination from Continuous Reactor Data
Evaluation of the observable modulus ηϕ 2 at the reactor inlet at 250 • C (Supporting Information) shows that there are, in the worst case, modest intraparticle mass transfer resistances in the reactions studied. Values of the six rate constants and the adsorption equilibrium constant for water (K W ) were adjusted independently at each reaction temperature to minimize the objective function, taken as the sum of the square of differences between experimental and modeled outlet concentrations ((C i Exp − C i Mod ) 2 ) at each residence time for pertinent species in each reaction.
Rate constants for the reactions of EtOH (k 5 ) and IAOH (k 6 ) to byproducts were determined by setting the quantity of byproducts formed equal to the quantity of each feed alcohol reacted that was not converted to higher alcohol product, thus maintaining the correct feed alcohol concentration profile through the reactor. Depending on the conditions, the fraction of EtOH or IAOH converted to byproducts ranged from 12% to 33% in the continuous reactor.
From the optimized rate constant values at each reaction temperature (210 • C, 230 • C, and 250 • C), an Arrhenius plot for each rate constant was generated to determine the activation energy and pre-exponential factor. The Arrhenius plots are shown in Figure S2 of the Supporting Information. Values of activation energies and pre-exponential factors for each reaction, along with the value of the rate constant at 230 • C, are given in Table 3 below. Table S3 of Supporting Information.
A comparison of the rate constants at 230 • C in Table 3 for the various condensation reactions shows a decrease in value as alcohol chain lengths increase from C 2 -C 2 (k 1 ) to C 2 -C 5 (k 2 ) to C 5 -C 5 (k 3 ). The activation energy for these three condensation reactions also increases as the chain length increases, in accordance with the expected steric effects associated with the longer carbon backbone of the alcohols. Interestingly, the rate constant (k 4 ) for (C 6 + C 8 ) formation from C 2 plus C 4 is larger than that of C 2 -C 2 condensation, and the activation energy for R4 is lower. It is possible that the kinetic model does not capture the formation pathway of (C 6 + C 8 ) alcohols correctly (e.g., perhaps there is a direct route to C 6 formation that does not involve C 4 explicitly), or that C 4 and C 6 alcohols, once formed, have a locally higher concentration within the catalyst that results in locally higher reaction rates and thus a larger rate constant. A comparison of the rate constants at 230 °C in Table 3 for the various condensation reactions shows a decrease in value as alcohol chain lengths increase from C2-C2 (k1) to C2-C5 (k2) to C5-C5 (k3). The activation energy for these three condensation reactions also increases as the chain length increases, in accordance with the expected steric effects associated with the longer carbon backbone of the alcohols. Interestingly, the rate constant (k4) for (C6 + C8) formation from C2 plus C4 is larger than that of C2-C2 condensation, and the activation energy for R4 is lower. It is possible that the kinetic model does not capture the formation pathway of (C6 + C8) alcohols correctly (e.g., perhaps there is a direct route to C6 formation that does not involve C4 explicitly), or that C4 and C6 alcohols, once formed, have a locally higher concentration within the catalyst that results in locally higher reaction rates and thus a larger rate constant.
Comparison of Batch Experimental Data with Kinetic Model Simulation
The kinetic model developed above was applied to batch reactions conducted by writing the molar balance for each species "i" in the batch reaction system: The reaction rates for each species are identical to the right-hand side of Equations (1)-(8) above for the continuous reactor system. The molar balances were integrated over the batch reaction time for each experiment using the Euler's method in Microsoft Excel. The rate constants determined from the continuous reactor data at 230 °C (Table 3) were used in the rate expressions. Initial concentrations of EtOH and IAOH for each experiment are given in Table S4 of Supporting Information. A comparison of experimental and simulated concentrations at the end of each batch experiment for key species in the reaction system is given in Figure 3a-d below. Given that the kinetic model is derived entirely from continuous reactor data, the agreement between experimental and simulated batch results is good. The complete comparison of batch experimental and simulated species concentrations is given in Table S5 of the Supporting Information.
Comparison of Batch Experimental Data with Kinetic Model Simulation
The kinetic model developed above was applied to batch reactions conducted by writing the molar balance for each species "i" in the batch reaction system: The reaction rates for each species are identical to the right-hand side of Equations (1)-(8) above for the continuous reactor system. The molar balances were integrated over the batch reaction time for each experiment using the Euler's method in Microsoft Excel. The rate constants determined from the continuous reactor data at 230 • C (Table 3) were used in the rate expressions. Initial concentrations of EtOH and IAOH for each experiment are given in Table S4 of Supporting Information. A comparison of experimental and simulated concentrations at the end of each batch experiment for key species in the reaction system is given in Figure 3a-d below. Given that the kinetic model is derived entirely from continuous reactor data, the agreement between experimental and simulated batch results is good. The complete comparison of batch experimental and simulated species concentrations is given in Table S5 of the Supporting Information. Reactions 2020, 3, x FOR PEER REVIEW 10 of 13
Conclusions
Guerbet condensation reactions of EtOH and IAOH mixtures have been carried out over a lanthanum-promoted nickel on alumina catalyst in both batch and continuous fixed bed flow reactor. The EtOH selectivity toward C4+ alcohols of 94% at 21% conversion, and IAOH selectivity of 87% to C7+ alcohols (mainly cross-condensation products with EtOH) at 12% conversion was achieved. A kinetic model has been developed with a Langmuir-Hinshelwood rate expression with product water as the dominant adsorbed species that inhibits reaction, and rate constants have been determined based on the outlet species concentrations from the continuous reactor. The kinetic model developed with continuous reactor data predicts the batch reactor behavior reasonably well.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Development of Kinetic Model from "Indirect" Guerbet Reaction Mechanism. Table S1: Rate expressions for Reactions (R1)-(R6) in kinetic model, Evaluation of Intraparticle Mass Transport Resistances. Figure S1: Typical molar compositions of liquid and gas byproducts formed in Guerbet reactions of EtOH and IAOH. Figure S2: Arrhenius plots for rate constants in kinetic model. Table S2: Inlet concentrations of EtOH (E) and IAOH (IA) in continuous reactor. Table S3: Comparison of experimental and simulated outlet concentrations from continuous flow reactor. Table S4: Initial concentrations of EtOH (E) and IAOH (IA) in batch reactions (230 °C). Table S5
Conclusions
Guerbet condensation reactions of EtOH and IAOH mixtures have been carried out over a lanthanum-promoted nickel on alumina catalyst in both batch and continuous fixed bed flow reactor. The EtOH selectivity toward C 4+ alcohols of 94% at 21% conversion, and IAOH selectivity of 87% to C 7+ alcohols (mainly cross-condensation products with EtOH) at 12% conversion was achieved. A kinetic model has been developed with a Langmuir-Hinshelwood rate expression with product water as the dominant adsorbed species that inhibits reaction, and rate constants have been determined based on the outlet species concentrations from the continuous reactor. The kinetic model developed with continuous reactor data predicts the batch reactor behavior reasonably well. | v3-fos-license |
2014-10-01T00:00:00.000Z | 2007-02-05T00:00:00.000 | 18765701 | {
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} | pes2o/s2orc | Antibacterial Activity of Long Chain Fatty Alcohols Against Staphylococcus aureus. Molecules 2007
The antibacterial activity against Staphylococcus aureus of long-chain fatty alcohols was investigated, with a focus on normal alcohols. The antibacterial activity varied with the length of the aliphatic carbon chain and not with the water/octanol partition coefficient. 1-Nonanol, 1-decanol and 1-undecanol had bactericidal activity and membrane-damaging activity. 1-Dodecanol and 1-tridecanol had the highest antibacterial activity among the long-chain fatty alcohols tested, but had no membrane-damaging activity. Consequently, it appears that not only the antibacterial activity but also the mode of action of long-chain fatty alcohols might be determined by the length of the aliphatic carbon chain.
Introduction
Staphylococcus aureus is a pathogenic microorganism that is responsible for serious problems, in particular in medical facilities, such as nosocomial infection and resistance to antibiotics.Novel compounds with antibacterial activity are needed to solve these problems.We have studied the antibacterial activities of plant-derived compounds, such as essential oils, against S. aureus [1][2][3][4][5] and have reported that two sesquiterpenes with an aliphatic carbon chain, namely, farnesol and nerolidol, have effective activity [3].Methodical investigation of anti-staphylococcus activity of terpene alcohols suggested that mono-and diterpenes with an aliphatic carbon chain have lower activity [3].Our results indicated that there might be a relationship between antibacterial activity and chemical structure.In this study, we selected the length of the carbon chain as the characteristic of the chemical structure and used fatty alcohols with a carbon chain of various lengths.
In an effort to investigate the antibacterial activities of long-chain fatty alcohols against S. aureus in detail, we used a broth dilution with shaking (BDS) method, a time-kill assay and an assay of the leakage of K + ions from cell.The BDS method was an unique method to investigate the antibacterial activity, such as minimum inhibitory concentration (MIC), of hydrophobic compounds.The time kill assay was carried out to observe the antibacterial activity in detail in combination with the BDS method.Leakage of K + ions from cells reflects the cell membrane damage activity.Since farnesol and nerolidol showed effective antibacterial activity and also cell membrane damaging activity, it might be important to measure the leakage of K + ions from cells in response to reagents.
Growth-inhibitory activity
Table 1 summarizes the antimicrobial activities of long-chain fatty alcohols on the growth of S. aureus FDA209P, as determined by two different methods.Long-chain fatty alcohols with aliphatic carbon chains with fewer than seven (data not shown) and eight carbon atoms (Table 1) had no antimicrobial activity.Long-chain fatty alcohols with an aliphatic carbon chain with more than 17 carbon atoms had hardly any antimicrobial activity.The results obtained by the broth dilution method indicated that the most effective number of carbon atoms in long-chain fatty alcohols, in terms of growth-inhibitory activity, ranged from 13 to 15.We obtained similar results when we determined the bactericidal activity by the broth dilution method.The BDS method indicated that long-chain fatty alcohols with an aliphatic carbon chain of 12 or 13 carbons had the greatest growth-inhibitory activity (Figure 1).The highest bactericidal activity was observed with an aliphatic carbon chain of 11 carbon atoms.The growth-inhibitory and bactericidal activities depended on concentration (Figure 2).The viable cell count barely changed over 24 h in the presence of C 8 -OH, while C 9 -OH and C 12 -OH decreased the viable cell count from 10 7 to approximately 10 4 colony forming units (cfu) mL -1 within 4 h.Contact with C 12 -OH decreased the viable cell count to beneath the limit of detection within 24 h.In the presence of C 10 -OH and C 11 -OH, the viable cell count decreased rapidly.The viable cell count fell below the limit of detection within only 2 and 4 h in the presence of C 10 -OH and C 11 -OH, respectively.
Quantitation of K + ion leakage
As shown in Figure 4, addition of long-chain fatty alcohols to bacterial suspensions led to the immediate leakage of K + ions from the cells.We investigated initial rates of leakage of K + ions and amounts of K + ions leaked from bacterial cells (Figure 5).In the presence of C 12 -OH and C 11 -OH, both values were larger than in the presence of other long-chain fatty alcohols.The long-chain fatty alcohols used in this study each possess a long aliphatic carbon chain and one hydroxyl group and they differ in terms of the length of the aliphatic carbon chain.The activity against S. aureus was most evident when the carbon chain length ranged from 10 to 13.When there were 10 or 11 aliphatic carbon atoms in the carbon chains, bactericidal activity was both evident and potent (Table 1).The bactericidal activity results were supported by the time-kill assay results (Figure 3).K + ion leakage assays indicated that C 10 -OH and C 11 -OH induced the leakage of K + ions more rapidly than the other tested alcohols.We reported previously that the initial rate of K + ion leakage reflects the damage to cell membranes when a reagent that affects cell membranes is added to a bacterial suspension [19].than those induced by TTO.The total amount of K + ions that leaks from bacterial cells reflects the antibacterial activity that is mediated by cell membrane damage.
Our results indicate that the antibacterial activity of C 10 -OH and C 11 -OH were mediated by damage to cell membranes that allowed leakage of K + ions, with subsequent reactions that induced the further leakage.MIC and MBC measurements (Table 1) confirmed that C 12 -OH and C 13 -OH had the most effective bacteriostatic activity among the long-chain fatty alcohols tested.K + ion leakage induced by C 12 -OH and C 13 -OH was less conspicuous than that observed in the presence of C 10 -OH and C 11 -OH and similar to that observed in the presence of long-chain fatty alcohols that did not have effective antibacterial activity.Thus, the expression of bacteriostatic or bactericidal activity was dependent on the length of the aliphatic carbon chain.We are now studying the nature of the antibacterial effects of C 12 -OH and C 13 -OH.
Some authors have reported that the antibacterial activity of hydrophobic compounds depended on their water/octanol partition coefficients [17,18].The water/octanol partition coefficients of the long-chain fatty alcohols used in this study are listed in Table 1.If activity were dependent on the partition coefficient, the activity would be expected to increase with elongation of the aliphatic carbon chain.Our results do not support such a conclusion.There is a defined range of chain lengths for induction of effective inhibition of the growth of S. aureus.The expression of antibacterial activity was determined by the length of the aliphatic carbon chain rather than by the water/octanol partition coefficient.
MIC and MBC measurements confirmed that C 12 -OH and C 13 -OH had the most effective bacteriostatic activity among the long-chain fatty alcohols tested.Leakage of K + ions in response to these alcohols was smaller than those in response to C 10 -OH and C 11 -OH and similar to that obtained with long-chain fatty alcohols that did not exhibit effective antibacterial activity.Therefore, it appears that the length of the aliphatic carbon chain might determine the nature of the antibacterial activity.
Compounds with alkyl chains are attracting attention since they have some novel attributes [22][23][24][25], for example, the ability to promote antibacterial activity and to resensitize methicillin-susceptible and -resistant S. aureus to antibiotics.Their properties are probably related to their alkyl groups since the effective length of the carbon chain in each alkyl group is in harmony with our results.Although there have been many studies of the antibacterial activities of fatty alcohols, some variations in activity have been missed, perhaps because of the use of high concentrations, on the order of mg mL -1 , in determinations of MICs.A detailed reevaluation is clearly needed of the antibacterial activities of hydrophobic compounds at appropriate concentrations.
General
All long-chain fatty alcohols used in this study were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).Staphylococcus aureus FDA209P was used as the standard strain [1].
Broth dilution method
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each alcohol were determined by the micro broth dilution method.The MIC was determined by the NCCLS method [26].The compound to be tested was added to aliquots of Mueller Hinton broth (100 µL, Difco, Detroit, MI, USA).An aliquot of an overnight culture of S. aureus (ca. 1 x 10 5 cfu mL -1 ) was added to each sample.Each culture was incubated in air without shaking at 37°C for 24 h.
Figure 4 .
Figure 4. Changes in the concentration of K + ions in a suspension of S. aureus FDA209P in response to 1-decanol.The arrow indicates the addition of 1-decanol.The results are typical of three assays that gave similar results.See text for full details.
Table 1 .
Antimicrobial effects of 1-alkanols on the growth of S. aureus and n-octanol/water partition coefficients. | v3-fos-license |
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} | pes2o/s2orc | Purification and properties of a bacteriophage-induced endo-N-acetylneuraminidase specific for poly-alpha-2,8-sialosyl carbohydrate units.
The soluble form of a bacteriophage-induced endo-N-acetylneuraminidase (Endo-N) specific for hydrolyzing oligo- or poly-alpha-2,8-linked sialosyl units in sources as disparate as bacterial and neural membrane glycoconjugates was purified approximately 10,000-fold and characterized. The enzyme appears homogenous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a subunit Mr 105,000. This corresponds to one of the higher Mr phage proteins which comprises 7.5% (by weight) of the total phage protein. The holoenzyme is active at neutral pH and has a Mr by gel filtration of 328,000, suggesting that the active enzyme is a trimer. Endo-N requires a minimum of 5 sialyl residues (DP5, where DP represents degree of polymerization) for activity. The limit digest products from the alpha-2,8-linked polysialic acid capsule of Escherichia coli K1 are DP4 with some DP3 and DP1,2. DP2-4 do not appear to inhibit depolymerization of polysialic acid. Endo-N digestion of the polysialosyl moiety on neural cell adhesion molecules yields sialyl oligomers with DP3 and DP4. The presence of a terminal sialitol changes both the distribution of limit digestion products and the apparent minimum substrate size. Higher Mr alpha-2,8-linked sialyl polymers (approximately DP200) are better substrates (Km 50-70 microM) than sialyl oligomers of approximately DP10-20 (Km 1.2 mM). Endo-N activity is inhibited by DNA and several other poly-anions tested. An examination of the distribution of intermediate products shows that Endo-N binds and cleaves at random sites on the polysialosyl chains, in contrast to initiating cleavage at one end and depolymerizing processively. Endo-N can serve as a specific molecular probe to detect and selectively modify poly-alpha-2,8-sialosyl carbohydrate units which have been implicated in bacterial meningitis and neural cell adhesion.
types has attracted attention because of its association with meningitis in human and animal neonates (6)(7)(8)(9). A structurally identical polysialic acid capsule is present in Neisseria meningitidis serogroup B (9). Although the wide spread occurrence and biological significance of sialic acid has long been recognized (10, l l ) , the nonbacterial occurrences of polysialic acid was unknown until recently. This was due in part because of the lability of poly-a-2,8-sialosyl linkages to pH 5 or to heating (100 "C/5 min) at neutral pH (12) and to the difficulties in readily distinguishing between mono-and polysialic acid. Recently, prokaryotic derived probes have been developed that permit the simple detection of poly-a-2,8-sialosyl carbohydrate units in embryonic neural cell adhesion molecules (N-CAM)' (13). N-CAMS function in neural cell-cell interaction and neural development (14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Evidence that the polysialosyl epitope of N-CAM has an effect on N-CAM-mediated adhesion between living cells and that the amount of this carbohydrate is important for normal development of neural tissue has also been obtained (24). Further studies on the role of polysialic acid in these and other processes and on the molecular characteristics of polysialic acid biosynthesis will be facilitated by the use of specific, well-characterized reagents that permit detection and selective modification of the polysialosyl moiety. Several endoand exoglycosidases have proven to be highly useful reagents for structural analysis of glycoconjugates (25-28). One such enzyme is an endo-N-acetylneuraminidase (Endo-N) associted with bacteriophages that specifically recognize the polysialic acid capsule of E. coli K1 as a receptor (13). Initial studies utilizing Endo-N and some of its properties have been reported (13,24,29,30). Whereas the enzyme used appeared to be free of proteolytic and exoneuraminidase activity, further experiments to detect polysialosyl units possibly associated with neurological disorders and to elucidate the biological effects of desialylated N-CAM on neural development will be more easily interpreted by using an enzyme that has been purified to homogeneity and is well characterized. Two reports
3553
on some of the properties of a bacteriophage-bound endoneuraminidase have appeared (31,32); however, a purified, soluble endoneuraminidase may have a distinct advantage, particularly for in vivo microinjection experiments to study the functional significance of polysialosyl units on N-CAM. The purification of a soluble endoneuraminidase and its specificity for various substrates containing sialic acid were recently reported (33), but no information was presented as to the size of the limit digestion products or the minimal substrate size.
This report describes the purification and properties of the soluble form of a bacteriophage-induced endo-N-acetylneuraminidase that is specific for degrading at neutral pH oligo-or poly-a-2,8-sialic acid units in prokaryotic and eurkaryotic glycoconjugates. Availability of the purified enzyme provides a valuable molecular probe to detect, modify selectively, and study the structure, synthesis, and function of poly-a-2,8-sialosyl carbohydrate units. These results are discussed in relation to the properties of the previously described forms of endoneuraminidases (31)(32)(33) and the minimum sialyl oligomer size required to interact with the substrate-binding site of the enzyme.
Characterization of Bacteriophage KIF Proteins
The CsC1-purified phage particles isolated as described under "Experimental Procedures" were analyzed by SDS-PAGE to characterize the molecular weights and relative abundance of the phage-associated proteins ( Table I). The K1F phage banded at a density between 1.3 and 1.6 g/cm3 in the CsCl gradient, indicating that it was similar to the endoneuraminidase-containing phages studied by Kwiatkowski et al. (31) and Finne and Makela (32). These phage particles were reported to have a bouyant density of 1.47 g/cm3, but in neither case were the proteins characterized. The K1F phage isolated here appeared homogenous in that only a single, high molecular weight DNA band was seen upon agarose electrophoresis after phenol/chloroform extraction and ethanol precipitation. As shown in Table I, the molecular weights of the major K1F-associated proteins were similar to other capsulespecific bacteriophages previously studied. The three capsular depolymerases previously characterized were reported to be spike elements of the phage that existed as a high molecular weight complex of 313,000 ( E . coli K l l ) , 245,000 ( E . coli 429), and 379,000 (Klebsiella aerogenes). The holoenzymes appeared to consist of non-identical subunits, as shown in Table I. In contrast, Endo-N is an oligomer ( M , 328,000) composed of three identical subunits of M , 105,000 each. The purpose in purifying the phage and characterizing its proteins was 3fold. First, determination of the protein components of the phage served as a determinant of purity of the soluble depolymerase since Endo-N should consist of one or more of these proteins. Second, a comparison of the relative substrate specificities and other enzymological properties of the phageassociated and soluble Endo-N depolymerase might reveal differences due to immobilization of the enzyme in the phage particle. Third, at least in some cases, it might prove more facile to purify the phage-bound enzyme to homogeneity since the enzyme is naturally enriched in the purified phage preparation, i.e. purification of the phage particle could serve as an "affinity" purification step.
Treatment of purified phage K1F with either 4 M guanidinium C1 or 0.1% sodium dodecyl sulfate destroyed Endo-N activity. However, two other procedures: either mild acid treatment, previously shown to be effective in solubilizing depolymerase-containing phages 429 (42) and K11 (43), or treatment with 8 M urea, previously used to solubilize the K . aerogenes capsular polysaccharide depolymerase (45), were effective in quantitatively solubilizing Endo-N from K1F phage particles. However, substantial losses of activity accompanied further attempts at purification, possibly due to varying degrees of reaggregation of the phage proteins during subsequent purification procedures. Therefore, the soluble form of the enzyme in K1F phage lysates of E. coli K1 was purified to homogeneity, as described below.
Purification of Soluble Endo-N from KIF Phage Lysates
The purification procedure described under "Experimental Procedures" and summarized in Table I1 allowed the purification to apparent homogeneity of about 4.4 mg of Endo-N in 3 days. Overnight incubation of purified Endo-N with a number of high and low molecular weight proteins and glycoproteins prior to SDS-PAGE showed no contaminating protease activity. The enzyme was purified greater than 10,000-fold, based on the lysate activity, which, however, might be underestimated. The 2-fold increase in total activity, and the apparent high degree of -fold purification are probably due to the removal of an inhibitor(s) of Endo-N at the final purification step. Various polyanions, including DNA, chondroitin sulfate, and poly-y-D-glutamic acid, inhibit Endo-N, presumably through ionic interactions since Endo-N is bound to heparin-agarose (results not shown). At least part of the inhibition during purification appears to be due to the presence of DNA. The purified glycohydrolase (20 pg ml-') was inhibited >90% by E. coli DNA (0.75 mg ml-') and treatment of fractions from initial stages of the purification with DNase (-1 mg ml-') resulted in a subsequent %fold increase in Endo-N activity. Thus, it is likely that it is the removal of DNA from the phage lysate by the hydroxylapatite column that is responsible for the increase in total activity and is therefore the most important purification step. The -fold purification shown in Table I1 may be an overestimation due to the unknown amount of inhibition of Endo-N activity in the lysate. However, we believe these results are the most accurate presentation of the data in the absence of detailed characterization of inhibitory factors that might be present in the phage lysate.
The ammonium sulfate pellet ( Table 11, Step 2) was reextracted with buffer to enhance recovery of Endo-N activity entrained in the mass of insoluble debris. The main purpose of the high speed centrifugation (Step 3) was to recover K1F phage, which is why both the total activity and the specific activity decreased at this stage. This step can be eliminated For acid treatment of K1F phage particles, the pH of a purified K1F phage suspension was lowered to 3.5 by addition of an equal volume of 0.4 M glycine HCl (pH 3.5). An immediate precipitate formed. After 10 min at pH 3.5, the pH was readjusted to pH 7.4 by the addition of 1.8 M Tris (pH 8.8), and the high molecular weight material was removed by ultracentrifugation (120,000 x g, 1 h). For solubilization with urea, the phage suspension was made 8 M in urea, incubated at 37 "C for 30 min, and then subjected to ultracentrifugation as above. if only the soluble Endo-N activity is desired. Heating the high speed supernatant at 60°C for 25 min (Step 4) and refractionation with 40% ammonium sulfate (Step 5) resulted in a substantial loss of protein and an approximately 7-fold purification. The most significant purification step was obtained by chromatography of the active fractions from the hydrophobic interaction chromatography (Step 6) on a double column of hydroxylapatite connected in series with DEAE-Trisacryl (Step 7). A single, symmetrical peak of Endo-N activity and protein was eluted at about 0.15 M NaC1.4 When this material was analyzed by SDS-PAGE (Fig. 2, lane 9), it was found to consist of one major protein band of greater than 99% homogeneity that co-migrated with the M , 105,000 protein of the K1 phage (Fig. 2, lane IO). The a-2,gd 0 Endo-N activity was measured by product formation, as described under ''Experimental Procedures," which yields initial reaction rates with all the substrates used. Thus, V, , is expressed as micromoles of NeuNAc-reducing equivalents formed min" mg of protein".
tracking dye had reached the bottom of the tube, the gel was removed and cut into 2-mm slices. The slices were eluted for 2 h with 50 mM Tris buffer (pH 7.5) and then assayed for Endo-N activity. The only gel slice that showed Endo-N activity contained only the M , 105,000 protein, as shown by subsequent SDS-PAGE. Thus, the purification procedure reported here allows the facile purification to homogeneity of sufficient quantities of Endo-N for further enzymological studies and to specifically detect, modify selectively, and study the function of poly-a-2,8-sialosyl units in a variety of biological systems. In the only previous report of the purification of a soluble endoneuraminidase (33), the enzyme was reported to consist of two subunits ( M , 74,000 and 38,500). This difference in size and number of subunits may be due to different phages being used. That enzyme had been purified 238-fold and had a specific activity of 0.95 (pmol of sialic acid released per min/mg of protein). In contrast, the Endo-N reported here was purified -10,000-fold and is markedly more efficient (-4,200 pmol min-l mg-'). It is thus possible that an inhibitor was present and that one of the two reported subunits could be a contaminant or a proteolytic cleavage product.
Properties of Endo-N-acetylneuraminidase
Kinetic Constants-Relatively little is known about the kinetic constants and enzyme mechanisms of endoglycohydrolases, in particular, endo-N-acetylneuraminidases. Here the apparent K,,, and VmaX for both the phage-bound and soluble form of Endo-N were determined for several oligoand polysialosyl substrates, as summarized in Table 111. No significant difference in apparent K,,, values was found between the K1F phage-bound and soluble form of the enzyme for either the low or high molecular weight a-2,8-linked substrates or the a-2,8-a-2,9-mixed linkage polymer (Table 111). The high molecular weight a-2,8-linked sialyl polymer (-DP150-200) appeared to be a substantially better substrate (apparent Km-50-70 p~) than the shorter (DP10-20) sialyl oligomers (apparent Km-1.2-1.6 mM). The large difference in K,,, values found here for Endo-N action on long polymers and short oligomers of sialic acid suggests that a different enzyme mechanism might be involved in processing these two different substrates! However, since the total number of A recent study (48) on the catalytic mechanism of polyphosphate glucokinase found that the apparent K,,, for polyphosphates was markedly different for chain lengths of 32 or -724. An examination of the variation of K,,, with chain length of polyphosphates demonstrated a distinct inflection at a chain length of 100, which appeared to coincide with a change in the mechanism of enzyme action from processive at longer chain lengths to nonprocessive at shorter chain lengths.
potential cleavage sites is about the same at these concentrations of oligo-and polysialic acid, this suggests that the apparent affinity of the enzyme for these two substrates is a function of the concentration of potential cleavage sites and hence is approximately the same. Possible mechanisms of Endo-N cleavage are discussed in more detail below. Unexpected was the observation that the alternating a-2,8-a-2,9ketosidically linked capsular sialyl polymer from E. coli N67 appeared to be a better substrate (apparent K,,, 6.6 PM) than the a-2,8-linked polymer from E. coli K1 ( K , 51 PM). Only the a-2,8 linkages in this polymer, however, were hydrolyzed by Endo-N (see below). The K,,, for polysialic acid of the free form of endoneuraminidase reported by Tomlinson and Taylor (33) was 7.4 mM or 145-fold higher than what was determined here.
Products of Endo-N Limit Digestion-The products of prolonged digestion (limit digestion) of various oligomers and polymers of sialic acid were examined. As shown in Fig. 3a, the primary digestion product of U-14C-labeled, high molecular weight poly-a-2,8-a-2,9-sialic acid was DP8 with a smaller amount of DP4,6,10,12,14. Since Endo-N does not hydrolyze a-2,g-linked polysialic acid (results not shown), this pattern was due to the specific cleavage of the a-2,8 linkages within the mixed linkage polymer, producing only oligomers containing an even number of sialyl units. This is in contrast to the spectrum of sialyl oligomers with an even and odd number of units obtained by the partial and random hydrolysis of polya-2,8-linked sialic acid (see below; Fig. 5 and 7). in the reducing termini after reduction with NaB3H4 (c) were subjected to overnight digestion by Endo-N, and the products were identified by HPLC as described under "Experimental Procedures." but in this case, the mixed linkage polymer appeared to be a poor substrate, and the digestion products were reported to be DP3,4,6,7 and higher residues, indicating that some cleavage of a,2-9 linkages may have occurred (49). As stated above, the primary hydrolysis product of the poly-a-2,8-a-2,9 capsule by Endo-N was DP8. In contrast, the primary digestion product of U-14C-labeled, high molecular weight poly-a-2,8sialic acid was DP4 with a smaller amount of DP3 and DP1,2 (Fig. 3b). These results further confirm that Endo-N was specific for the poly-a-2,8 linkage in the poly-a-2,8-~~-2,9mixed linkage polymer. The molar distribution of products shown in Fig. 3b is consistent with what can be calculated for the random cleavage of a polymer with a minimum substrate size of DP5. Unexpectedly, the major digestion product from colominic acid (DP10-20) that had been reduced with NaB3H4 was DP3 and not DP4 (Fig. 3c), although smaller amounts of DP4 and DP1,2 were present. These results show that sialyl oligomers terminating in sialitol perturbed Endo-N catalysis and yielded different reaction products than oligomers terminating in sialic acid. As described below, reduction also changes the minimum substrate size from DP5 to DP6. No significant difference was seen between K1F phage-bound and -soluble Endo-N with respect to the products of limit digestion (results not shown).
Minimum Substrate Size-The products of limit digestion of poly-a-2,8-sialic acid suggested that the smallest substrate cleaved by Endo-N was DP5, yielding primarily the tetramer, DP4 (Fig. 3b). However, some DP5 remained in the limit digest of colominic acid that had been reduced with NaB3H4, suggesting that the minimum substrate size for a sialyl oligomer terminating in sialitol was DP6. DP5 is not seen in Fig. 3c because it was present in much smaller amounts than the major limit digestion product (DP3) derived from reduced colominic acid. In the example shown, DP5 contained 2370 cpm. T o determine directly the minimum number of sialyl units required to serve as a substrate, U-14C-labeled sialyl oligomers and sialyl oligomers terminating in 3H-labeled sialitol (labeled by reduction with NaB3H4) were purified as described under "Experimental Procedures." Each oligomer of defined length was then incubated overnight at 37 "C with an excess of Endo-N, and the reaction products were analyzed by HPLC. Minus enzyme controls showed no degradation. Under these conditions, reduced DP5-03H ((NeuNAc),-NeuNAc-03H) (Fig. 4a) was not hydrolyzed by Endo-N, whereas reduced DP6-03H and D7-03H were (Fig. 4, b and c), again giving DP3-03H as the major digestion product. However, when nonreduced U-"C-labeled oligomers were used, different results were obtained. As shown in Fig. 4d, DP4 was not cleaved by Endo-N, but DP5 was, yielding DP4 and DP1 (Fig. 4e). Whereas the K , for DP5 was not determined, the results shown in Fig. 4e suggest that it is a relatively poor substrate since complete hydrolysis did not occur, even after an overnight incubation. Cleavage of DPlO (Fig. 4f) again gave close to the expected molar distribution of products with some DP5 remaining unhydrolyzed. Analysis of the digestion products of DP6-8 gave similar results (not shown). We conclude from these results that the minimum substrate size for Endo-N is DP5. DP5 and higher oligomers were cleaved by Endo-N to give DP4 and sialic acid as major products (Fig. 4e). In contrast, reduced DP5 (DP5-03H) was not cleaved by Endo-N (Fig. 4a), but DP6-03H and higher reduced oligomers were hydrolyzed to DP3-03H (Fig. 4, b and c). Thus, the presence of a terminal sialitol changed the minimum substrate size from DP5 to DP6 and also appeared to alter the Endo-N cleavage pattern. Confirmation of this conclusion was provided as shown in Fig. 4 (g-i). In these experiments, 14C-labeled DP5, which served as a substrate (Fig. 4e), was reduced with NaBH, to form [14C]DP5-OH and tested for its ability to serve as a substrate for Endo-N. As shown in Fig. 4g, this reduced oligomer no longer served as a substrate. Reduced DP6 ( [14C]DP6-OH) and DP7 ( [14C]DP7-OH) both served as substrates (Fig. 4, h and i ) . Interestingly, the proportion of product found as DP3 with both [14C]DP6-OH and [14C]DP7-OH was again markedly increased over the nonreduced oligomers. Therefore, these results demonstrate that a terminal sialitol residue not only changes the minimum substrate size but also changes the cleavage pattern from that expected for completely random, as seen with nonreduced substrate, to one that is biased toward producing DP3-OH. In addition, an examination of the digestion products of (NeuNAc),-NeuNAc-03H of DP5-16 shows that this bias was not restricted to oligomers that were close to the minimum substrate size (Table IV). Indeed, the relative proportion of observed products appears to be independent of oligomer length, up to at least DP16, with the exception of DP8 where relatively lower amounts of DP3 and higher amounts of DP4 were observed. This latter result suggests that oligomers containing -eight sialyl units may have a different conformation in solution.
The results of Endo-N digestion of different oligomers of polysialic acid clearly show that the miminum requirement for cleavage is c~-2,8-(NeuNAc)~. This is in contrast to previously described phage-bound endoneuraminidases that were reported to require a minimum of eight cu-2,8-linked sialic acid units (31,32). In the present study, comparison of reduced and nonreduced oligomers of sialic acid has demonstrated that reduction of the reducing terminus to sialitol changes the apparent substrate specificity of Endo-N. In a previous study (32), reduced oligomers of a-B&linked sialic acid were used to define the substrate specificity of a bacteriophage (PK1A)-bound endoneuraminidase. The possible influence of reduction of the substrates on enzyme specificity and activity was not addressed. The previous study by Finne and Makela (32) reported that their enzyme required a minimum of 8 sialyl residues and that cleavage produced a DP3, derived from the nonreducing terminus, and a DP5, derived from the reducing end. It was therefore concluded that digestion of brain polysialosyl glycopeptides by endoneuraminidase would leave 5 sialyl residues attached to the glycopeptide moiety (32). However, this conclusion may have to be reconsidered in light of the results presented here.
Kinetics of Endo-N Digestion of /U-'4CJPoly-~-2,8-sialic Acid-Examination of the products produced by the action of a glycohydrolase as a function of time can yield information about the mode of enzymatic action. At low Endo-N to substrate ( [U-14C]poly-cu-2,8-sialic acid, DP150-200) ratios, an apparent lag in the formation of limit digest products (DP2-4) was seen when the reaction products were examined by paper chromatography and radiochromatographic scanning (results not shown). Under these conditions, the increase in number of free reducing termini, measured by thiobarbituric acid, was linear, suggesting that the initial products of Endo-N action were intermediate size oligomers (10 < DP > 100) that were not resolved by paper chromatography. This would imply that the mechanism of Endo-N hydrolysis of polysialic acid was random since a processive mechanism (repetitive hydrolysis from one end) would produce limit digest products during the initial phase of the reaction. To examine the mechanism of Endo-N catalysis, a detailed kinetic analysis of the products of Endo-N hydrolysis of [U-'4C]poly-cu-2,8-sialic acid was carried out (Fig. 5). When Endo-N digestion of substrate was 75% complete (4 min, Fig. 5a), only oligomers of DP3-8 were present. This suggested either that intermediate size oligomers were preferred substrates or that Endo-N catalysis might be processive with intermediate length oligomers (10 < DP > 50). An examination of the relative rates of disappearance of DP5-8 (Fig. 5, 6-e) supports the earlier conclusion that the smaller oligomers were poorer substrates. These results also confirm the conclusion that DP5 was the minimum substrate size for Endo-N since the major product of limit digestion was DP4 (Fig. 5, d and e).
Partial Digestion Products of Different Length Oligomers of (NeuNAc) ,-NeuNAc-03H-To differentiate whether the products of partial digestion of [U-'4C]polysialic acid noted above were due to a processive mechanism or to preferential hydrolysis of intermediate oligomers, the products of partial Endo-N digestion of DP7-03H to DP14-03H were examined (Fig. 6). DP7-03H and DPS-03H were degraded directly to DP3-03H without the formation of intermediate species.
However, the formation of intermediates became more apparent with the longer chain oligomers (DP10-03H to DP14-03H). This shows that for the intermediate size oligomers, the mechanism of Endo-N hydrolysis is random and not processive. Moreover, for the longer oligomers (DP10-03H to DP14-03H), cleavage is primarily on the distal (nonreducing end) of the terminal sialitol, in contrast to the preferential cleavage on the proximal (reducing end) of the terminal sialitol observed with DP7-03H to DP9-03H. This is additional evidence consistent with the conclusion that conformational differences may exist between sialyl oligomers when their size approaches a DP9, 10. The presence of a terminal sialitol influences Endo-N action even with intermediate length oligomers where presumably binding and hydrolysis near the reducing terminus is hindered due to the presence of sialitol. However, the apparent random mechanism of Endo-N hydrolysis under these conditions is not due to the terminal sialitol since partial Endo-N digestion of nonreduced ["C] DP12 also formed intermediate sized oligomers (Fig. 7). Model of Endo-N Catalysis-Several models have been proposed to account for the binding and subsequent hydrolysis of oligomeric substrates by depolymerases (50)(51)(52)(53)(54)(55)(56)(57). In these models, the active site of the enzyme is postulated to consist of a series of subsites, each of which is capable of binding a single carbohydrate moiety. These models can be used to predict time-dependent changes in the distribution of products, for example, amylase action (58, 59), when the subsite binding affinities have been determined by other methods or the subsite binding affinities can be determined by measuring product distribution as a function of time (60,61). Whereas a detailed, quantitative treatment of Endo-N action in this manner is beyond the scope of this study, a schematic model of Endo-N and substrate interaction (Fig. 8) is instructive and allows some qualitative assessments to be made.
In the case of the nonreduced substrate (Fig. Sa), interaction of sialic acid residues with enzyme-binding subsites is approximately equipotential, although occupancy of subsite 1 is preferred to subsite 2 since cleavage of [l4CJDP5 gives primarily DP4 and sialic acid (Fig. 4e). Additional, long range interactions are probably also important since sialic acid oligomers of intermediate length appear to be better substrates than shorter oligomers (Table 111). In the case of oligomers up to DP9 with a terminal sialitol (Fig. 86), the presence of the alcohol causes preferential binding of the terminal sialitol at subsite 7, favoring cleavage predominantly at (NeuNAc),-(NeuNAc),-NeuNAc-OH. Since DP6-OH is cleaved, this could indicate that occupancy of subsite 2, al-
Conclusions
The purification procedure reported here provides sufficient quantities of highly purified (homogenous) soluble Endo-N, free of proteases and exoneuraminidase activities, suitable for future studies on the biosynthesis and biological function of poly-a-2,8-sialosyl-containing glyco-conjugates. The following considerations are relevant to the future use of Endo-N as a probe for polysialic acid. Since Endo-N catalysis is random and long polymers are substantially better substrates than short ones, high enzyme concentrations and long incubation periods may be necessary to achieve complete digestion of oligomers that are only slightly larger than the minimum substrate size (DP5). Since the minimum substrate size is DP5, the release of sialic acid from a homooligomer by Endo-N would indicate the presence of an oligomer of >DP4. However, the lack of release of sialic acid does not necessarily indicate a polymer of <DP5 since Endo-N could be sterically hindered due to an attached reducing end. Indeed, here it was found that merely the presence of a terminal sialitol changes both the apparent minimum substrate size and the cleavage pattern obtained. Experiments using Endo-N to study the biosynthesis of polysialosyl units in neural cell adhesion molecules and to probe the function of polysialic acid in a variety of biological systems are currently in progress in a number of laboratories. | v3-fos-license |
2017-09-09T01:42:46.347Z | 2011-04-12T00:00:00.000 | 20034388 | {
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} | pes2o/s2orc | Influence of a polyclonal antibody preparation on the in situ degradability of three energy sources
________________________________________________________________________________ Abstract The objective of this study was to evaluate the effect of a polyclonal antibody preparation (PAP) against specific ruminal bacteria on the in situ degradability of dry-grounded maize grain (DMG), high moisture maize silage (HMMS) starch and citrus pulp (CiPu) pectin. Nine ruminally cannulated cows were used in a 3 x 3 Latin square design, replicated three times in a factorial arrangement of treatments of two rumen modifiers represented by monensin and PAP plus a control group, and the three energy sources (DMG, HMMS and CiPu). Each period had 21 days, where 16 were used for adaptation to treatment and five for data collection. The group treated with PAP showed an effect on the soluble fraction (“a”) of DMG starch, decreasing it by respectively 45.3% and 45.4% compared to the CON and MON groups. No effect of PAP was observed for any in situ degradability parameters of starch from HMMS or pectin of CiPu. It was concluded that the polyclonal antibody preparation had limited effect on the in situ degradability of the tested energy sources. ________________________________________________________________________________
Introduction
The use of ionophores for ruminal fermentation modulation has been employed with great success for better utilization of diets.However, the possible health effects of the use of these additives are a cause for concern and new methods of ruminal fermentation manipulation are beginning to be tested.The European Community, a major importer of meat from Brazil, by Regulation (EC) 1831/2003(Europe, 2003)), banned the use of antibiotics and coccidiostats as feed additives for cattle.This regulation reinforces the need of new feed additive development.The objective of this study was to evaluate the in situ degradation of the dry matter and starch from dry-grounded maize grain (DMG), high moisture maize silage (HMMS) and pectin from citrus pulp (CiPu), as influenced by a polyclonal antibody preparation against specific rumen bacteria in cows fed a high concentrate diet.The bacteria species are Streptococcus bovis, Fusobacterium necrophorum, Clostridium aminophilum, Peptostreptococcus anacrobius and Clostridium sticklandii.
Materials and Methods
The trial was conducted at the College of Veterinary Medicine and Animal Science at the University of São Paulo (USP), Brazil.Nine ruminally cannulated Holstein x Zebu non-pregnant dry cows (690 44 kg BW) were used in 3 x 3 Latin square experimental design with three periods of 21 d each.Treatments were arranged as a 3 x 3 factorial arrangement of two all-feed additives monensin ([MON] or polyclonal antibody preparation [PAP]), plus a control group and three energy sources in the diet (dry-grounded maize grain [DMG], high moisture maize silage [HMMS] and citrus pulp [CiPu]).Cows were housed in a tie-stall barn equipped with individual feed bunks, rubber-matted floors and automatic water fountains common to two animals.There were fans in the ceiling in order to relieve the high temperatures during the day.Body weight was measured at the beginning of period one (d 1) and at the end of each of the three periods (d 21) at the same time each day.
Diets were fed as total mixed rations (TMR) with a ratio of concentrate to forage of 70 : 30 (DM basis, Table 1).Diets were offered twice daily at 08:00 and 16:00 for ad libitum consumption (minimum of 10% feed refusal).The forage source was fresh sugarcane chopped to a theoretical average particle size of 1.18 cm; measurement taken by the Penn State Particle Size Separator (Lammers et al., 1996).MON and PAP were offered directly through the rumen cannula twice daily, just before the meals.MON (Rumensin, Elanco Animal Health, Indianapolis, I.N., USA) at 300 mg/animal/day was administered in absorbent tissue paper and PAP (CAMAS Inc., Le Centre, MN) at 10 mL/animal/day using a 10 mL syringe.The latter product contained antibodies against Streptococcus bovis, Fusobacterium necrophorum and some strains of proteolytic bacteria (Peptostreptococcus sp., Clostridium aminophilum and Clostridium sticklandii).
Each period had 21 days, where 16 days were used for adaptation to treatments and five days for data collection.The in situ degradability of DM and starch or pectin from the energy sources was measured by the nylon bag technique (Mehrez & Ørskov, 1977).Dry matter was determined according to AOAC (1990).Starch concentration was determined by the method described by Pereira & Rossi (1995) after extraction of soluble carbohydrates (Hendrix, 1993).Pectin was determined by the method described by Van Soest et al. (1991).
For degradation, parameters were estimated using the model proposed by Ørskov & McDonald (1979): p = a + b (1-e -ct ), where p is the degradation at each time; "a" is the soluble fraction; "b", the potentially degradable fraction of the insoluble fraction that is degraded at a rate "c"; "c" is the rate of degradation of fraction "b"; and "t" is the incubation period in hours.The parameters "a", "b" and "c" from exponential equation were used to calculate the potential degradability (Pd = a + b), which represents the quantity of feed that can be solubilized or degraded in the rumen if time is not a limiting factor.The effective ruminal degradability (Ed) was calculated according to the mathematical model proposed by Ørskov where K is the passage rate of solids from the rumen, accepted here as either 0.02, 0.05 or 0.08 %/h.Degradability data were calculated by the difference in weight of nylon bags before and after rumen incubation and adjusted according to the equation of Ørskov & McDonald (1979).Results were analyzed by the Statistical Analysis System software (SAS, 2001).Firstly, the residue normality was verified by the Shapiro-Wilk test (PROC UNIVARIATE).Data (dependent variable) that did not meet this assumption were submitted to logarithmic transformation [Log (X+1)] or square root adjustment [RQ (X+1/2)].Original or transformed data, when this last procedure was necessary, were submitted to analysis of variance by PROC GLM (General Linear Models) procedure.The model accounted for the effect of feed additive, energy source, the interaction of feed additive x energy source, period and animal.The effects of the main factors (feed additive and energy source) were separated by Duncan test.Effects were declared significant at P 0.05.
Results and Discussion
The results of the influence of polyclonal antibody on the in situ degradability parameters of starch from DMG and HMMS and pectin from CiPu are presented on Tables 2, 3 and 4, respectively.
The group treated with PAP showed an effect (P = 0.037) in the soluble fraction "a" of DMG starch, decreasing it by 45.3% and 45.4% compared to the CON and MON groups, respectively (Table 2).
It was observed that the treatment with MON decreased the value of the potentially degradable fraction "b" of DM of HMMS by 16.1% compared to the CON group, but there was no difference when PAP was administered (Table 3).No effect of rumen modifier was observed for any of the in situ degradability parameters of starch of HMMS.In general, irrespective of treatments, in situ degradability values of HMMS were higher than the ones described by Jobim et al. (1999).These authors mentioned that high moisture maize silages had higher soluble fractions "a" in comparison to whole plant or ear of maize silages (grain +
Table 2
Effect of rumen modifier on the in situ degradability of dry matter and starch of dry-grounded maize grain 1Rumen modifier: CON -control; MON -monensin; PAP -polyclonal antibody preparation. 2.e.m. -standard error of the mean; 3 Prob.-statisticalprobabilities for rumen modifier effect; 4 Ed -effective degradability; 5 Pd -potential degradability.The South African Journal of Animal Science is available online at http://www.sasas.co.za/sajas.asp
Table 3
Effect of rumen modifier on the in situ degradability of dry matter and starch of high moisture maize grain a,b,c Means without common superscripts differ, P 0.05 (Duncan). 1Rumen modifier: CON -control; MON -monensin; PAP -polyclonal antibody preparation. 2s.e.m. -standard error of the mean; 3 Prob.-statistical probabilities for rumen modifier effect; 4 Ed -effective degradability; 5 Pd -potential degradability. | v3-fos-license |
2017-05-03T00:32:21.438Z | 2011-05-30T00:00:00.000 | 96077953 | {
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} | pes2o/s2orc | Photothermal Reactions of Nitrosobenzene and Halonitrosobenzenes in Solid-state
Photothermal reactions of the dimers of nitrosobenzene, m-chloronitrosobenzene, and pchloronitrosobenzene were studied in solid-state by IR spectroscopy at low temperatures and by X-ray powder diffraction. It was found for the first time that photothermal cycle (photolytic dissociation followed by thermal dimerization) could successively be performed also with Z-configured dimeric nitrosobenzene. Halonitrosobenzenes dimers with E-configuration afforded different photo behavior depending on the position of halogen atom on the benzene ring: while m-halonitrosobenzenes do not dissociate under UV irradiation, p-chloronitrosobenzene, as well as previously studied p-bromonitrosobenzene photolyses very efficiently with recovering of the original crystal phase. Kinetics of thermal dimerization was measured in solid-state, and it was found that the reaction phase transformation occurs as a two-dimensional growth through the crystal. (doi: 10.5562/cca1714)
INTRODUCTION
While nitrosobenzenes appear in solution as equilibrium of two forms, monomers and dimers, in solid-state most of them crystallize predominantly in the form of dimers (azodioxides). 1 In our previous work we have found that under cryogenic conditions in solid state, such azodioxides undergo photodissociation to corresponding nitrosobenzene monomers, which, in turn, redimerize quickly by warming above some critical temperature. 2Since these processes include breaking and rebinding the azodioxide nitrogen-nitrogen bonds, the system represents a sort of molecular OFF-ON switch, (Scheme 1).
The reaction mechanism of such a solid-state photodissociation of p-bromonitrosobenzene dimer was studied in detail by in situ X-ray diffraction analysis of the single-crystal-to-single-crystal transformation. 3Fast thermal redimerization above 170 K was explained as a consequence of a very close contact of neighboring Natoms in crystals of in situ prepared monomers.This non-covalent distance is even for 23 % shorter than the sum of the van der Waals radii.
For possible application of such a molecular OFF-ON switch, the system must satisfy the condition that the dissociation-dimerization process is operative not only under cryogenic conditions, but also at room tem-perature.In this line it becomes necessary to search for more promising systems, which could afford such photothermal effect.
In this work we extend the investigation of reactivity, kinetics, as well as topochemistry of nitrosobenzene derivatives to Z-nitrosobenzene dimer 1, and mand p-chloronitrosobenzene, 2 and 3 (Scheme 1).Structure 1 is interesting because of its Z-stereochemistry.Namely, all the nitrosobenzene dimers previously studied by solid-state photochemistry were E-stereomers.Investigation of 2 and 3 is expected to provide new information about the effect of molecular packing on the photodissociation in the crystal lattice.Position of the chlorine atom on the benzene ring could have critical influence on the reaction path.
EXPERIMENTAL
Nitrosobenzene dimers were prepared by standard methods. 4hotolysis.A high-pressure Hg lamp 250 W was used for photolysis.FT-IR kinetics.The solid-state dimerization rate was measured by following the temporal change in transmittance of the ONNO asymmetric stretching signal at 1258 cm −1 and the NO stretching signal of monomer at 1494 cm −1 .Bruker Equinox FT-IR spectrometer with 1 cm −1 resolution was used for this measurement.The sample was prepared as a standard KBr pellet cooled by Leybold-Heraeus ROK 10 -300 cyclic helium cryostat.Powder diffractogram was taken on Philips PW 1840 diffractometer, Bragg-Brentan geometry, 2θ = 5-35°.
RESULTS AND DISCUSSION
Nitrosobenzene dimer, 1.After photolysis of crystals of dimer 1 in KBr pellet at 23 K by a high-pressure Hg lamp, the signals at 1398 cm −1 and 1412 cm −1 assigned to symmetric and asymmetric stretching vibrations of the Z-ONNO group strongly decreased in their intensity, and the new signals at 1503 cm −1 , originated from N=O stretching vibration of the nitroso monomer, appeared in the spectrum (Figure 1b).
By warming the sample to room temperature, the signals of the starting dimer were recovered, while the signal of the monomer disappeared (Figure 1c).Because the process can be visualized as changing the color from yellowish (dimer) to blue (monomer), the reaction is also an example of photochromic/thermochromic effect.The observed temperature of this thermal transition from monomer to dimer was between 265 K and 275 K.The photolysis of 1 has been repeated at different temperatures, and it was found that the highest temperature at which this compound undergoes photochromic dissociation is 245 K.This temperature is much higher than in the case of previously observed photoreaction of p-bromonitrosobenzene (170 K). 2 The transformation cycle that includes photodissociation followed by thermal redimerization was repeated five times.Relative intensity of the 1503 cm −1 band assigned to the monomer NO stretching vibration was more or less recovered after an each cycle (Figure 2).Evidently, this OFF-ON switching system has a high efficiency.
Chloronitrosobenzene dimers, 2 and 3. Irradiation of m-chloronitrosobenzene dimer 2 cooled in KBr pellet to 20 K did not afford any change in the IR spectrum.No dissociation was observed at any temperature between 15 K and the room temperature.The same experiment was made with m-bromo derivative of dimer 2 which also did not afford photodissociation.In contrast, dimer 3, in which the halogen atom is in para position, readily undergoes photodissociation at 25 K.The effect was characterized by a disappearance of the IR signal at 1258 cm −1 assigned to asymmetric stretching vibration of the E-ONNO group, 5 and the appearance of the signal characteristic for monomer NO stretching vibration at 1494 cm −1 (Figure 3).The thermal redimerization started at the temperature as high as 295 K.Moreover, at this temperature we have succeeded to measure kinetics of dimerization by following the intensity of the disappearing NO stretching band at 1494 cm −1 .The obtained kinetic curve is a sigmoid that is typical for solid-state reactions and phase transformations (Figure 4). 6From the analysis of the reaction by using the standard Avrami-Erofeev model, the reaction in the crystal can be described as a two-dimensional growth of the product phase. 6Namely, linear plot of logarithms of the extent of the reaction, (α) versus logarithm of time (Sharp-Hancock plot) gives the coefficient m = 2.58 characteristic for the two-dimensional process (Figure 5).Phase reversibility of the photothermal cycle.The powder X-ray diffractograms were recorded for polycrystalline samples before photolysis, and at the end of the cycle, i.e. after thermal redimerization.As it can be seen in Figure 6, the diffractograms do not differ, and it follows that the compound after photodissociation followed by redimerization recovers its origins crystal phase.
This finding supports the previous conclusions about the topochemical reversibility of such cycle, obtained from observations of the single-crystal-to-singlecrystal transformations.The hindered solid-state photodissociation of meta halogen derivatives (2 and 4) can be explained by formation of interhalogen bonds 7 (Cl---Cl, or Br---Br) in their crystal lattice (Figure 7).The Br…Br contacts are identified by typical interatomic distance of 3.76 Å.If such bonds are in meta-position, they can hinder the necessary partial rotation of benzene rings, which always accompanies dissociation.Oppositely, benzene rings have freedom to rotate partially and to enable dissociation if interhalogen bonds are formed between halogens in para-position (Figure 7).Note, that 2 and 4 are isostructural because they have analogous unit cell, which for 2 have parameters a (Å) 12.302(2), b (Å) 3.268(1), c (Å) 13.261(4), beta (°) 107.53( 6)).
CONCLUSIONS
Since in previous investigations only trans nitroso dimers were successfully photodissociated in the solidstate, here we found for the first time that the photothermal cycle (photolytic dissociation followed by thermal dimerization) could successively be performed also with Z-configured nitrosobenzene dimers and that the process occurs at much higher temperature than in previously studied E-derivatives.
In the second part of this work we studied the solid-state photothermal transformations of dimeric halonitrosobenzenes with E-configuration in more details.They afforded different photo behavior depending on the position of halogen atom on the benzene ring: while m-halonitrosobenzene dimers do not dissociate under UV irradiation, dimeric p-chloronitrosobenzene, as well as previously studied p-bromonitrosobenzene photolyse very efficiently with recovering of the original crystal phase.Nonreactivity of meta dimers is explained by hindering the necessary rotation of benzene rings caused by interhalogen bonds formed in the crystal lattice.From the kinetics of thermal dimerization measured in solid-state, which afforded typical sigmoid curve, it was found that the process progress as a two-dimensional growth of the product phase.
Figure 1 .
Figure 1.FT-IR spectrum of 1 in KBr pellet (a) at 23 K, (b) after 30 min of photolysis at 23 K by a high pressure Hg lamp, (c) after warming to 300 K.The signal characteristic for ON-NO stretching vibration of dimer is labeled with o, while the signal characteristic for NO stretching vibration of monomer is labeled with *.
Figure 2 .
Figure 2. Cyclic change in the intensity of the band assigned to monomer NO stretching vibration (1503 cm −1 ) of 1 after successive photodissociations (blue line) and thermal dimerizations (red line).
Figure 3 .
Figure 3. FT-IR spectrum of 3 in KBr pellet (a) at 20 K, (b) after 50 min of photolysis at 20 K by a high pressure Hg lamp, (c) after warming to 300 K. o labels dimer, * labels monomer.
Figure 4 .
Figure 4. Kinetic curve of solid-state dimerization of 3 in KBr pellet at 295 K.
Figure 5 .
Figure 5. Linear Sharp-Hancock plot obtained from kinetics data for solid-state dimerization of 3. | v3-fos-license |
2018-04-03T05:57:23.525Z | 2017-10-23T00:00:00.000 | 35006283 | {
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} | pes2o/s2orc | Heavy metal and nutrient uptake in plants colonizing post-flotation copper tailings
Copper ore mining and processing release hazardous post-flotation wastes that are difficult for remediation. The studied tailings were extremely rich in Cu (1800 mg kg−1) and contaminated with Co and Mn, and contained very little available forms of P, Fe, and Zn. The plants growing in tailings were distinctly enriched in Cu, Cd, Co, Ni, and Pb, and the concentration of copper achived the critical toxicity level in shoots of Cerastium arvense and Polygonum aviculare. The redundancy analysis demonstrated significant relationship between the concentration of available forms of studied elements in substrate and the chemical composition of plant shoots. Results of the principal component analysis enabled to distinguish groups of plants which significantly differed in the pattern of element accumulation. The grass species Agrostis stolonifera and Calamagrostis epigejos growing in the tailings accumulated significantly lower amounts of Cu, but they also had the lowest levels of P, Fe, and Zn in comparison to dicotyledonous. A. stolonifera occurred to be the most suitable species for phytostabilization of the tailings with regard to its low shoot Cu content and more efficient acquisition of limiting nutrients in relation to C. epigejos. The amendments improving texture, phosphorus fertilization, and the introduction of native leguminous species were recommended for application in the phytoremediation process of the tailings.
Introduction
Mining and processing of copper ores extensively alter the environment, and the main difficulty is a huge amount of flotation tailings which comprise even 96% of the mass of run-of-mine ores with worldwide production estimated to be two billion tons in 2011 (Gordon 2002;Onuaguluchi and Eren 2012). These tailings pose an enormous problem in EU countries, particularly in Poland where the copper production accounted over 421,000 t, which was accompanied by tailing deposition which amounted to 8.5 million tons in 2014 (Brown et al. 2016). The tailings consist mainly of very fine grinding rocks, and, as a result of the beneficiation process, they contain high concentrations of copper and other trace elements (Łuszczkiewicz 2000;BREF 2009;Wang et al. 2014). These kinds of wastes are usually deposited without any treatment in extensive ponds which pose opened sources of pollutants for neighboring ecosystems. The migration of heavy metals leads to the contamination of air, water, soil, and sediments, which negatively affects all organisms, including humans (Baycu et al. 2015;Venkateswarlu et al. 2016).
The correct presentation of Table 4 is shown in this paper.
The original version of this article was revised.
Responsible editor: Elena Maestri
Among the technologies that may be employed in remediation of metal-contaminated lands and especially in the case of large sites, phytoremediation constitutes the most useful and cost-effective way that can be applied with minimum environmental impact (Khan et al. 2004). In the phytoextraction technique, certain plants are used (e.g., hyperaccumulators of trace elements) that have the extraordinary ability to take up and accumulate contaminants in their aboveground parts. BPhytomining,^however, has certain limitations and induces ecological risk because of introducing potentially toxic metals into the food chain or the improper disposal of contaminated biomass (Mench et al. 2010;Venkateswarlu et al. 2016). Reduction of metal mobility and bioavailability can be achieved due to the phytostabilization technique which uses suitable plant species and associated microbes for revegetation of contaminated sites. This enables the stabilization of the site area and pollution control, visual improvement, and removal of threats for herbivores and human beings (Wong 2003;Sheoran et al. 2010).
Establishing vegetation on abandoned metalliferous wastes is usually difficult because these artificial habitats create unfavorable conditions for plant growth. They are characterized by poor physical properties of substrate with unsuitable air-water conditions, high content of toxic metals, salinity, low fertility, and microbial activity (Forsberg 2008;Neuschütz 2009;Sheoran et al. 2010;Rybak et al. 2014). As a result, these wastelands are often devoid of any vegetation or have only sparse plant cover and are unable to create sustaining and healthy ecosystems. Especially, soils with high or contrasting metal contents impose strong selective pressure on colonizing plants which can persist in such habitats due to a wide range of adaptations (Baker 1981;Kinzel and Lechner 1992;Broadley et al. 2003). The lack of nutrients or their low availability is a similarly important selective factor, which hardly constrains vegetation development (Kazakou et al. 2008;Sheoran et al. 2010;Turnau et al. 2010). However, the problem of metal dispersion and nutrient deficiency can be solved by using clean topsoil to cover the waste substrate, but usually it must be transported from distant areas, which is costly, especially in the case of large wastelands. In this connection, revegetation of metalliferous wastes should be carried out with attentively selected plants that must be metal resistant and adapted to nutrient-deficient soils and can improve soil biological activity, grow quickly, and form dense canopies and root systems. These plants should be also adapted to local environmental conditions and be of native origin (Mench et al. 2010;Sheoran et al. 2010), so species spontaneously colonizing particular sites should be primarily considered and implemented for phytoremediation practices. Our main aims in the present study were (1) to evaluate heavy metal and nutrient contents in shoots of plants that colonize post-flotation copper tailings and juxtapose these data with chemical composition of the examined species growing on an unpolluted site, (2) to reveal the interactions between plant chemical composition and the specific properties of the copper tailings, and (3) to select plants and formulate general recommendations for the phytoremediation of these wastes. The tested hypothesis was whether the plant species that grow spontaneously on the copper tailings may be used as suitable organisms for their phytoremediation.
Study sites
The studies were carried out in a BWartowice 3^tailings pond area (51°12′ 38.34″ N 15°40′ 53.58″ E) which is located in Warta Bolesławiecka district, Lower Silesia province, SW Poland (Fig. 1). This pond covers about 232 ha and had been in mining activity for storing tailings from the copper ore flotation process until 1989. The main materials forming the tailings are silica (quartz), carbonate minerals (dolomite, calcite), and clay minerals, as well as to a lesser extent shale copper ores, marl, shale, and anhydrite.
Despite of natural succession lasting over 20 years, the pond's surface remains almost bare with very poor vegetation. There are only infrequent and widely scattered stands, of a surface area of approx. 2 m 2 , of stunted specimens of Pinus sylvestris L. and Populus tremula L. as well as a few herbaceous species with dominance of Agrostis stolonifera L. Only the places that are overburden and with a little share of the copper tailings have greater and more diverse plant cover. A few attempts at the reclamation of the tailings were made in the past, but they were costly and ineffective, resulting in very sparse plant cover.
In comparison to the tailings pond, an urban wasteland with grassland and ruderal vegetation was chosen as a reference site which inhabited these plant species that lived in the pond. It was located in a relatively clean area nearby the Ślęza River in Wrocław city, SW Poland ( Fig. 1).
Sampling and chemical analysis
Because of the extremely poor vegetation that covers the pond, only five herbaceous plant species could be examined in this study, such as Agrostis stolonifera L. and Calamagrostis epigejos (L.) Roth (both from the Poaceae family), Cerastium arvense L. (Caryophyllaceae), Polygonum aviculare L. (Polygonaceae), and Tussilago farfara L. (Asteraceae). Samples of these species and substrates were collected from the tailings pond and the reference site, and they consisted of aboveground plant parts and copper tailings or reference soil taken from the plant rooting zone (10-20 cm depth). In the tailings pond, each species with substrate was collected randomly from three to five sampling sites. There were ten sampling sites in total with different compositions of collected species. At the reference site, the samples were taken randomly from four quadrates of a surface area 25 m 2 with similar plant composition.
An analysis of granulometric composition of the copper tailings was carried out with use of the aerometric method. Organic carbon was determined by a CHNS analyzer from CE Instruments (Skjemstad and Baldlock 2008) after removal of carbonates; pH was measured in distilled water in the 1:2 (weight/volume) ratio (Hendershot et al. 2008).
Total contents of 13 elements (Ca, Cd, Co, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb, and Zn) in the copper tailings were determined by ICP-OES after microwave digestion in concentrated nitric acid with hydrogen peroxide oxidation (1.0 g of substrate treated with 10 mL of concentrated HNO 3 and 2 mL H 2 O 2 , Merck Darmstadt). Appropriate Sigma-Aldrich ICP standards were used for working standards after matching a matrix.
Available forms of some elements (Ca, Co, Cu, Fe, K, Mg, Mn, P, and Zn) in air-dried and sieved (Ø 2-mm sieve) samples of the tailings and the soil were determined after extraction in 0.2 mol L − 1 CH 3 COOH + 0.25 mol L − 1 NH 4 Cl, 0.005 mol L −1 + C 8 H 8 O 7 (citric acid) and 0.05 mol L −1 HCl pH = 1.3 by Yanai et al.'s (2000) method. The elements Mg, Fe, Mn, Zn, Cu, and Co were analyzed by atomic absorption spectrometry with a Varian SpectrAA 220 FS apparatus using an air-acetylene oxidizing flame, after optimization at specific wavelengths. Potassium and calcium were determined by atomic emission spectrometry with use of the air-acetylene flame for K detection, and the oxidizing nitrous oxideacetylene flame for Ca. The caesium-lanthanum buffer (by Schinkel, Merck Darmstadt) was added as a releasing agent for K analysis. Colorimetric determination of phosphorus was done by (molybdenum blue) Murphy and Riley's (1962) method with a Thermo Scientific Evolution 600 UV-Vis spectrophotometer at 715-nm wavelength.
Plant samples were dried and ground with a stainless steel cutter mill equipped with a 2-mm sieve. Next, 2.5 g of airdried material was weighted into quartz crucibles and ashed in an oven, with a program ensuring slow increase of temperature to 500°C, maintaining this temperature over 6 h, and then slowly cooling down. The ashed samples were dissolved in Fig. 1 Location of the study sites and the view of BWartowice 3^tailings pond with extremely poor vegetation 6 mol dm −3 HCl (Sillanpää 1982), evaporated, and then transferred with 0.2 mol dm −3 HCl into a 50-mL volumetric flask. Element contents in plants were determined in conditions adequate for each element, as described above.
Data analysis
Significance of differences between study sites in terms of element contents in substrates as well as plant species was evaluated with use of Student's t test after verification of a normal distribution by Shapiro-Wilk's W test and data Box-Cox transformation. The calculations were carried out with Statistica software version 10 (StatSoft 2011).
To reduce the amount of variables and for data exploration purposes, principal component analysis (PCA) was performed to analyze the variance in element contents in plants and redundancy analysis (RDA) was used to relate variation in the chemical composition of plants to the element contents in examined substrates. These linear methods of ordination were chosen after preliminary application of detrended correspondence analysis (DCA) of the plant data, which yielded axes of short gradients, i.e., below 0.4 SD (Lepš and Šmilauer 2003). In the PCA, a set of 13 elements and 37 samples of plant species from the copper tailings and the reference soil was analyzed. Student's t test was used to assess the significance of differences between groups of the plant samples distinguished in the PCA ordination. The RDA was based on a 9 × 37 element-by-sample matrix, and the Monte Carlo permutation test (499 permutations under a reduced model) was performed to evaluate the statistical significance of constrained axes as well as of each descriptive variable in the model of regression of forward selection. Computations and ordination plots in PCA and RDA were made using CANOCO 4.55 for Windows software (ter Braak and Šmilauer 2002).
Substrate properties
The granulometric composition of the copper tailings (Table 1) was characterized by the majority of a fraction with grains of a diameter lower than 0.05 mm. This fact indicates the unsuitable air-water conditions with the oxygen deficiency that occurred in wet seasons, which is particularly unfavorable for the plant root growth. Additionally, other authors (Spiak et al. 2012) reported that the tailings had an adverse water capacity because of too low content of the productive and easily accessible water (related to the matrix potential from 2.2 to 3.7 pF), which especially occurred in the top layer. Under such circumstances, the seedling mortality can be very high, which restricts plant settlement in a colonization process of the bare pond surface.
The copper tailings had low content of organic carbon (< 0.3%) which mainly derived from black shale and lignite that are present in copper ore deposits, and from flotation agents. Due to the high content of carbonate minerals, they are characterized by an alkaline reaction with the pH H2O value which ranged between 8.0 and 8.5. The pH value of the reference soil was in a range 5.8-6.2.
The total element contents found in the copper tailings are presented in Table 2. The mean phosphorus and potassium contents in the tailings were higher than the averages recorded for relatively poor Polish soils as well as European mean values, which results from the ore beneficiation in a flotation process. The total contents of Ca (16.3%) and Mg (2.4%) were very high in comparison to mean soil levels given by Lis et al.
(2012) (0.47 and 0.10%, respectively) and Salminen et al. (2005) (2.53 and 1.04%, respectively). These occurrences are due to the fact that the tailings developed as assorted material from dolomite rocks. Among microelements, the total Cu content was extremely high (1800 mg kg −1 ) which is almost 13 times higher than the maximum allowable content of this element in European agricultural soils, as reported by Kabata-Pendias and Pendias (2001) (Cu < 140 mg kg −1 ) following EU legislation (CD 1986). This is despite the fact that the concentration of Cu decreases from about 2% in the mined ore to 0.18% in the deposited tailings. The total content of Co and Mn was very high too (20 and 1900 mg kg −1 , respectively), and it exceeded the mean values of these elements for Polish as well as European soils, according to Lis et al. and Mn were also in accordance with the lower values of the allowable content of these elements in European agricultural soils, as given by Kabata-Pendias and Pendias (2001) (20 and 1500 mg kg −1 , respectively); thus, the copper tailings can be considered as Co-and Mn-contaminated. The total Fe and Ni contents were a few times as high as Polish soil average values (6700 and 6.0 mg kg −1 , respectively) (Lis et al. 2012), but they were not enriched in relation to European means. The total Pb level was comparable to the means determined for Polish and European soils. The obtained values of total Cd and Zn were lower than the means reported by Lis et al. (2012) (0.8 and 88 mg kg −1 , respectively). The total Na content was much lower compared to the European mean. The chemical composition of the copper tailings and the reference soil differed significantly (t test, P < 0.05) in terms of content of the available forms of all analyzed elements ( Table 3). The tailings were extremely rich in available Cu which was over 40 times as high as that of the soil. Levels of available Ca and Mg were very high too, and the calcium content was above seven times and magnesium content three times as high as that of the soil. Similarly, the content of available K, Fe, Co, and Mn was also considerably higher in the tailings. On the other hand, the copper tailings have several times lower content of the available P and a lower level of the available Zn, compared to the reference soil.
Element content in plants
A comparison of chemical composition of plant shoots collected from the copper tailings and the reference soil revealed the occurrence of many considerable differences (Table 4). The plants from copper tailings, except for T. farfara, had higher Ca content. The highest mean calcium value was found in C. arvense (29 g kg −1 ), and this was about seven times as high as that from the soil. As opposed to the other species, T. farfara accumulated calcium at a very high level (21 g kg −1 ), which was the same in both study sites. The shoot Mg content was considerably higher in the plants from tailings, and P. aviculare, C. arvense, and T. farfara accumulated magnesium above the requirement for optimal plant growth, according to Marschner (2012) (1.5-3.5 g kg −1 ). Among the species growing in tailings, grasses, i.e., A. stolonifera and C. epigejos, were characterized by the lowest Ca and Mg levels. The plants from copper tailings, relative to those from the soil, accumulated phosphorus at the same level as in the case of dicotyledonous, or had a significantly lower P content (t test, P < 0.05) than in both grass species with the lowest result noted in C. epigejos. The K accumulation pattern differed widely among the studied sites and the species. The plants from tailings had much higher Na content with the highest result (78.7 mg kg −1 ) found in P. aviculare which accumulated 17 times as much sodium as that from the soil.
The shoot Cu content was significantly higher (t test, P < 0.05) in all plants from tailings compared to those growing in the reference soil. The highest amounts were found in C. arvense and P. aviculare (47.0 and 37.8 mg kg −1 , respectively). These values were nearly eight and ten times as high as those of the soil environment, and they were within the range of critical toxicity Cu level for plants, according to Kabata-Pendias and Pendias (2001) (20-100 mg kg −1 ) as well as the European Union legislation (EU 2008). The experimental critical toxic values for cultivating plants were estimated to be 8-40 mg Cu kg −1 (Davis and Beckett 1978;Macnicol and Beckett 1985), but we cannot state unambiguously if the toxic effect occurs in the species mentioned above because relevant data are rather scarce.
All species from tailings accumulated cadmium and cobalt above normal values for plants (0.2 and 1.0 mg kg −1 , respectively) but below the toxic levels, as given by Kabata-Pendias and Pendias (2001). The highest Cd amounts were found in shoots of C. epigejos, C. arvense, and P. aviculare (0.8-0.6 mg kg −1 , respectively). The species from tailings were distinctly enriched in Ni and Pb whose contents approached the maximum of the normal ranges (5 and 10 mg kg −1 , respectively; Kabata-Pendias and Pendias 2001). The Mn content was within the normal values for plants in all studied species. As opposed to the others, T. farfara did not accumulate higher amounts of Cd, Ni, Mn, and Pb growing in the tailings environment.
The shoot Fe content was much lower in all species growing in tailings compared to those from the soil, and statistically significant differences (t test, P < 0.05) were found in most cases. The iron levels for the examined grasses were below the range of critical deficiency concentration (50-150 mg Fe kg −1 , according to Marschner The results are presented as mean with standard deviation (SD) and median; t test probability level (P) for comparison of means of both substrates, significant differences in bold text a Determined according to the Yanai et al. 2000 The results are presented as values of mean ± standard deviation (SD) and Median, t test probability level (P) for comparison of means of both study sites, significant differences are italicized Environ Sci Pollut Res (2018) 25:824-835 (2012)), and the lowest result was found in C. epigejos. The shoot Zn accumulation showed low variability among study sites and plant species. The lowest Zn level obtained in C. epigejos (17.1 mg kg −1 ) was within a range of the critical deficiency concentration given by Marschner (2012) (5-20 mg kg −1 ). In PCA, the highest eigenvalues were achieved for the first two principal components (0.603 and 0.133, respectively) which explained 73.6% in total of the variability in shoot element contents. The first principal component was strongly determined by Cu, Mg, Pb, Na, Ca, and Co with the linear correlation coefficient r ranging from 0.96 (Cu) to 0.85 (Co), respectively; the influence of Ni was much weaker (r = 0.56). The second component mainly represented the gradient of Fe, P, and Zn (r = 0.91 to 0.62, respectively). Values of the variables which determine the first and second principal components are linearly uncorrelated.
The PCA ordination (Fig. 2) indicated a few groups of plant samples distinguished by the gradient of the first and second factors. The plant samples from the reference soil, except for T. farfara, formed one group where the first axis returned negative scores (the lowest tissue content of Cu, Mg, Pb, Na, Ca, Co, and Ni) and the second axis returned high positive or close to zero scores (the highest and near to medium Fe, P, and Zn contents). The plant samples from tailings were divided into two clearly separated groups of various species. The first one consisted of C. arvense and P. aviculare which had the highest positive scores of the first axis (the highest or high Cu, Mg, Pb, Na, Ca, Co, and Ni contents) and positive or close to zero scores of the second axis (high and medium levels of Fe, P, and Zn). The second one included A. stolonifera and C. epigejos which differed from the other samples from tailings by the lower scores of the first axis and had the lowest scores of the second axis. These two grasses accumulated significantly lower amounts of most elements considerable in this PCA model i.e., Cu, Mg, Na, Ca, Co, Fe, P, and Zn, in relation to C. arvense and P. aviculare growing in tailings (t test, P < 0.0001) as well as T. farfara but in this case without Zn (t test, P < 0.0001 to < 0.02, respectively). These species also differed significantly in levels of Fe, P, Cu, Ca, Ni Na, and Zn (t test, P < 0.00001 to < 0.04, respectively) compared to the group of samples from the reference soil. The samples of T. farfara, both from the soil and the copper tailings, were placed in a central part of the diagram and were not sharply separated from each other or from the other samples. T. farfara from soil differed significantly from the other species growing in the same environment by higher contents of Ca, Co, Pb, and Mg (t test, P < 0.0001-0.04), whereas T. farfara from tailings contained considerably lower amounts of Mn, Zn, Cu, Mg, and Pb than C. arvense and P. aviculare from the same study site (t test, P < 0.0001-0.031, respectively).
Relation of element content in plants to the concentration in substrates
Redundancy analysis (RDA) demonstrated a significant relationship between element contents in plants and concentrations of the available forms of elements in substrates. In the resulting RDA model, nine tested explanatory variables, i.e., the concentration of available P, K, Ca, Mg, Fe, Cu, Co, Mn, and Zn in substrates, explained 43.5% of the total variability in the element contents in plant samples. The first canonical axis accounted for 33.5% of the variation and was highly statistically significant (Monte Carlo permutation test, P = 0.002), whereas the second axis was not significant in this model. The forward stepwise selection of variables revealed that each of the subset of the explanatory variables, i.e., the concentration of Mn, Ca, Zn, Mg, Cu, and P, was statistically significant (Monte Carlo permutation test, P = 0.002) when tested independently and explained the variation at the comparable level, Lambda 1 = 0.31 (Mn) to 0.27 (P). In addition, all these six descriptors were highly cross-correlated (all correlation coefficients > 0.83), but the values of Mn, Ca, Mg, and Cu were mutually negatively related to Zn and P. The explanatory effect of Co concentration in substrates was also significant but lower than others (Lambda 1 Co = 0.17, P = 0.002). The effect of K and Fe levels in substrates appeared to be insignificant. The RDA diagram (Fig. 3) presents the ordination of the nutrient contents in plants along gradients of the element concentrations in substrates. The location and length of vectors in the ordination space indicate the relations and their strength. It was noticeable that the high levels of Mn, Ca, Mg, Cu, and Co that occurred in the copper tailings were mutually significantly correlated with the high amounts of Cu, Ni, Na, Pb, Ca, Mg, Co, and Mn in plants (the negative part of the first axis). On the other side, the higher levels of available P and Zn, characteristic of the reference soil, were mutually significantly related with the higher Fe and P contents in plants. Additionally, there was no significant correlation between the content of Fe and Zn in the studied substrates and plants, and all associations of Cd and K contents in plants with each other variable were insignificant.
Discussion
The obtained results revealed that plant species growing in the tailings and the reference soil varied significantly in shoot element contents and that plant accumulation patterns depended on the site conditions as well as systematic position of the plant, and the latter occurred distinctly in the specific copper tailings environment. It was also found that the contents of the available Ca, Mg, P, Cu, Co, and Mn in the studied substrates were significantly and positively correlated with the content of these elements in the plant shoots while correlations for the available Fe and Zn were insignificant.
Under the same site conditions, the shoot calcium content differs considerably between plant species and higher phylogenetic units (Broadley et al. 2003), and monocotyledons, including the Poaceae family, have generally lower Ca and Mg shoot contents than dicotyledons (Thompson et al. 1997). This corresponds to our findings of the significantly lower levels of these elements in A. stolonifera and C. epigejos from the tailings environment that is highly enriched in these elements. The ability to accumulate Ca in the shoots is also determined by plant physiotype. Among the oxalate plants, members of the Caryophyllaceae and Polygonaceae family increase the shoot Ca content proportionally to increasing Ca supply (Kinzel and Lechner 1992). A similar phenomenon was also observed in C. arvense and P. aviculare in our study. Furthermore, C. arvense can detoxify the Mg surplus in sparingly soluble oxalate salt (Kinzel and Weber 1982;Popp 1983). It was also demonstrated that T. farfara, as a calcium accumulator, took up Ca in spite of its low concentration in nutrient solution and the uptake and growth of this species were stimulated by higher Ca ion supply (Kinzel and Lechner 1992). These results can explain the high Ca accumulation levels found in this species both in the reference soil and in the copper tailings.
Both P. aviculare and C. arvense growing in the copper tailings were characterized by the highest shoot Cu content as well as comparatively high levels of all studied trace elements, which implies their high metal accumulation capacity. Similarly, other authors also found high levels of Zn, Cd, Cu, and Pb in shoots of P. aviculare growing on Zn tailings (Carrillo-Gonzales and Gonzales-Chavez 2006) and in an urban environment (Polechońska et al. 2013). Additionally, high efficiency of the shoot Na accumulation confirms the salt tolerance of this species demonstrated in the other study (Arnaud and Vincent 1988). C. arvense and other members of Caryophyllaceae are important components of serpentine vegetation, and they exhibit adaptation features to colonize these specific substrata (Kasowska and Koszelnik-Leszek 2014) Fig. 3 Redundancy analysis (RDA) ordination plot-the effect of Fe, Ca, Co, Cu, K, Mg, Mn, P, and Zn concentration in the copper tailings and the reference soil on the content of some elements in plants (according to PCA). The less significant variables (< 0.5 correlation coefficient with axes) are not presented. Black and red vectors indicate the explanatory and the response variables, respectively together with tolerance mechanisms of high metal levels (Kazakou et al. 2008). It was found that this species accumulated shoot Ni at relatively moderate levels (Lombini et al. 1998).
The studied grass species from the copper tailings, besides the lower shoot Ca and Mg levels, accumulated significantly lower amounts of other elements as Cu, Co, and Na than the examined species of dicotyledons. Generally, grasses are regarded as metal excluders, as defined by Baker (1981). A. stolonifera can evolve tolerant populations that have a greater capacity to accumulate copper in the roots and restrict transport of this element to the shoots, probably due to the existence of an efficient copper-complexing mechanism (Wu et al. 1975). This species may also evolve magnesium and salt tolerance, and the last with Na exclusion mechanism from roots and shoots (Wu 1981). Likewise, Fitzgerald et al. (2003) obtained lower Cu content in shoots of A. stolonifera compared to the roots as well as the shoots of dicotyledons. Similarly, C. epigejos growing on ash deposits accumulated much less Cu in the shoots than in the roots (Mitrović et al. 2008), and the shoot Cu level was comparable to that obtained in our study. Lehmann and Rebele (2004) stated that the copper tolerance of the C. epigejos population from a copper smelter was at a comparable level to that of copper-tolerant A. stolonifera.
Accumulation of cobalt was below the critical toxicity level in all studied species despite of its high content in the copper tailings, which implies reduced bioavailability of this element in this substrate. The solubility and toxicity of Co to plants decrease with an increase in the exchangeable Ca content in the soil solution (Li et al. 2009). This probably occurs in the tailings, because of their very high total and available Ca contents. The uptake and distribution of Co in plants is speciesdependent, and this element is accumulated mainly in the roots (Palit et al. 1994;Page and Feller 2005). However, some studies reported that higher amounts of cobalt were retained in the root systems of monocotyledons while its transport from the roots to the shoots in dicotyledons was highly effective (Bakkaus et al. 2005;Collins et al. 2010).
Low amounts of phosphorus were detected in shoots of the species from copper tailings despite of its high total content in this substrate. The low bioavailability of this element can be attributed to high pH, and the abundance of calciummagnesium carbonates occurred in the tailings. The excess of carbonates intensifies P sorption on their surface, and the high content of Ca and Mg increases phosphorus precipitation in the soil solution as Ca-Mg phosphates (Lindsay 1979;Sharpley et al. 1989) which are poorly soluble at high pH [(Ca 3 (PO 4 ) 2 Ksp = 1.4 × 10 29 , Mg 3 (PO 4 ) 2 Ksp = 6.3 × 10 26 ] (Corbridge 2013). It is known that plant species and their genotypes can develop a wide range of adaptive mechanisms involved in phosphorus acquisition (Ramaekers et al. 2010). However, especially at low P availability, enhanced P uptake could be achieved by releasing protons, carboxylates, and other exudates into the rhizosphere . For instance, in alkaline and calcareous soils, oxalic acid compared to other organic anions was considered to be the most effective in mobilizing unavailable phosphorus (Ström et al. 2005;Wang and Chen 2015). It can be suggested that the very low shoot P content obtained in A. stolonifera and C. epigejos in the copper tailings is indicative of the little effective Pmobilizing strategy used by these grasses under calcareous conditions. Additionally, diminishing in P uptake effectiveness in the tailings habitat could also be caused by lack of arbuscular mycorrhiza (AM), because this symbiosis commonly occurs in both these species and was found in their roots in the reference soil (unpublished data). In the copper tailings, typically mycorrhizal species A. stolonifera, C. epigejos, and T. farfara realized P uptake only by their root mechanisms, which resulted in P deficiency in the case of the grasses.
The alkaline soil environment strongly modifies the Fe uptake (Schenkeveld et al. 2014b), and the increase in pH by one unit diminishes Fe solubility 1000-fold, which can induce Fe deficiency aggravated by the presence of free carbonates (Coulombe et al. 1984;Inskeep and Bloom 1984). As a result, much lower shoot Fe contents were found in all species growing in the copper tailings compared to the reference soil, with the critical deficiency concentration occurring in both grasses. In neutral and calcareous soils with Fe deficiency, grasses excrete phytosiderophores (PS) for iron acquisition that in turn form soluble Fe complexes easily taken up (Römheld and Marschner 1986;Reichman and Parker 2005). PS can also mobilize other trace elements, particularly Cu, Zn, Ni, Co, Cd, and Mn (Romheld 1991;Schenkeveld et al. 2014a, b). This can increase their bioavailability but also may result in competition between Fe(III) and other metals for binding to PS complexes. Especially, the mobilization of Cu can diminish solubilized Fe(III) content (Reichman and Parker 2005), which might aggravate the iron deficiency that occurred in both grass species in tailings conditions. Inputs of PS into the rhizosphere strongly depend on the Fe status of the plant, the species, and the genotype (Bashir et al. 2006;Pereira et al. 2014); the diurnal PS release cycle; and other factors (Reichman and Parker 2005). Thus, the mechanisms involved in strategy II of Fe uptake and interactions with other elements could result in the lowest iron content found in shoots of A. stolonifera and C. epigejos as well as in differences in levels of accumulation of other metals that were present in these both grass species in tailings conditions.
Reaction of the soil solution also affects manganese availability but to a lesser extent than in the case of iron. According to Sanders (1983), decreasing the acidity from pH 5.2 to 7.3 reduces Mn solubility over 200-fold. Then also the high content of both total and available Ca and Mg forms acts antagonistically to Mn uptake (Maas et al. 1969) and the adsorption of Mn on carbonate surfaces diminishes the amount of soluble manganese (McBride 1979). However, besides decreased manganese bioavailability, the Mn content in the plants from copper tailings was higher in the same species compared to the reference soil, but this differentiation of Mn levels was without significant relation to the groups distinguished by PCA, and high Mn levels were found in C. arvense, P. aviculare, and C. epigejos. According to Lambers et al. (2015), plants that release carboxylates in their phosphorus-acquisition strategy tend to have high leaf manganese concentrations because of possibility of Mn mobilization even under its low availability. This association between considerable leaf Mn levels and carboxylate release in P-impoverished habitats was suggested for plants without arbuscular mycorrhiza even at lack of a significant correlation between shoot Mn and P concentrations . Similarly, the absence of this correlation could also be observed in RDA in our study. In this connection, the high shoot Mn level found together with the lowest P value in C. epigejos from the copper tailings implies that this species has particular difficulties in P uptake in the tailings environment.
The copper tailings had low zinc content. Additionally, the uptake of this nutrient is also limited at high pH and carbonate content and clay minerals can strongly absorb this element (Alloway 2009;Lambers et al. 2008). The lowest zinc level found in C. epigejos from tailings was comparable to the result obtained on alkaline ash deposits (Mitrović et al. 2008). It was also noted that this species growing on Zn-Pb wastes had the lowest shoot Zn content compared to other species (Wójcik et al. 2014). Exudation of phytosiderophores in grasses or carboxylates can mobilize Zn in alkaline conditions . However, Marschner (2012) also lists other solutes excreted by dicotyledonous at Zn deficiency. In our study, the shoot P, Fe, and Zn contents were significantly correlated with a single principal component which distinguished A. stolonifera and C. epigejos growing in tailings from other plants by the lowest content of these elements. This implies a similar mode of acquisition mechanisms of these nutrients in these species which proved very little effective in the copper tailings conditions.
The investigation contributes towards a better understanding of the mechanisms used by plants to respond to specific properties of tailings from the copper mining industry in an effort to develop an efficient strategy for their remediation. The most suitable plant species for this process proved to be A. stolonifera due to its demonstrated low shoot heavy metal content, which is important in the context of entering these elements into the food chain. In contrast to C. epigejos, creeping bent grass also acquired limiting nutrients P, Fe, and Zn more efficiently, which can result in a competitive advantage of this species in a plant community on the tailings. Considering also our earlier results (Spiak et al. 2012), in the phytostabilization process we propose to apply any kind of amendment (e.g., sand) to improve the tailings structure and the air-water relations at least in the surface layer, and to sow legumes from Trifolium and Medicago genera using seeds inoculated with Rhizobium bacteria. As hosts for rhizobia and arbuscular mycorrhizal fungi, these plants can fix nitrogen and uptake phosphorus more effectively, which enhances both their growth and that of neighboring plants. It seems that legumes could effectively colonize the tailings because high Ca and Co contents enhance their nodule formation (Smit et al. 1989;Arrigioni et al. 2013;Kliewer and Evans 1963). Before establishing AM, phosphorus fertilization should be used to stimulate plant growth and symbiotic associations. Following the vegetation development, processes of acidification usually intensify. This can trigger releasing higher amounts of copper and other potentially toxic metals into the tailings environment. Thus, monitoring of the site properties, the plant responses, and the presence and activity of microorganisms will be required for the evaluation of effectiveness of the remediation.
Conclusions
The tailings from the copper mining industry had a lot of unfavorable features for vegetation development, especially the adverse air-water conditions, the low availability of nutrients such as P, Fe, and Zn, and a very high content of Cu, Co, and Mn. The chemical composition of plants colonizing the tailings reflected the concentration of the available forms of Ca, Mg, P, Cu, Co, and Mn, but the species differed distinctly in their response to the tailings properties. Polygonum aviculare and Cerastium arvense had high shoot accumulation capacity of all studied elements with the highest Cu contents lying within the range of critical toxicity for plants, whereas the grass species had lower levels of majority of elements, especially Cu, P, Fe, and Zn. Agrostis stolonifera proved to be the most suitable species for phytostabilization of the tailings with regard to its low shoot heavy metal content and, as opposed to Calamagrostis epigejos, more efficient acquisition of the limiting nutrients. The remediation process of the tailings, in view of potentially high costs, should at least involve the application of amendments improving their texture, phosphorus fertilization, and the introduction of native leguminous species.
Open Access This article is distributed under the terms of the Creative Comm ons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. | v3-fos-license |
2016-03-22T00:56:01.885Z | 2015-10-15T00:00:00.000 | 1197556 | {
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"year": 2015
} | pes2o/s2orc | Two-Dimensional N-Glycan Distribution Mapping of Hepatocellular Carcinoma Tissues by MALDI-Imaging Mass Spectrometry
A new mass spectrometry imaging approach to simultaneously map the two-dimensional distribution of N-glycans in tissues has been recently developed. The method uses Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS) to spatially profile the location and distribution of multiple N-linked glycan species released by peptide N-glycosidase F in frozen or formalin-fixed tissues. Multiple formalin-fixed human hepatocellular carcinoma tissues were evaluated with this method, resulting in a panel of over 30 N-glycans detected. An ethylation reaction of extracted N-glycans released from adjacent slides was done to stabilize sialic acid containing glycans, and these structures were compared to N-glycans detected directly from tissue profiling. In addition, the distribution of singly fucosylated N-glycans detected in tumor tissue microarray cores were compared to the histochemistry staining pattern of a core fucose binding lectin. As this MALDI-IMS workflow has the potential to be applied to any formalin-fixed tissue block or tissue microarray, the advantages and limitations of the technique in context with other glycomic methods are also summarized.
Introduction
Hepatocellular carcinoma (HCC), a subtype of liver cancer that accounts for approximately two-thirds of all liver cancers, is among the most common and aggressive malignancies worldwide [1,2]. Early detection disease biomarkers are highly sought after and offer the potential to drastically improve patient outcome. Both proteomic and glycomic approaches have led to significant advances in HCC biomarker research [2]. Glycoproteins such as peroxiredoxin 3 [3,4], osteopontin [5][6][7], and alpha fetoprotein (AFP) [8] have been identified as potential HCC biomarkers. However, detection of these proteins alone display limited sensitivity in the background of liver cirrhosis or benign diseases. It has been observed that Į-1,6 core fucosylated AFP offers improved specificity for HCC than AFP alone [8][9][10]. Currently, core fucosylated AFP assessed through the AFP-L3 test, is the only test approved by the United States Food and Drug Administration for the detection of hepatocellular carcinoma. Elevated levels of core fucosylation are not specific for AFP, but are also observed on transferrin, alpha-1-antitrypsin, GP73 and other proteins [11][12][13][14][15]. Total serum glycomic studies have also identified increases in core Į-1,6 linked and Į-1,3 linked outer-arm fucosylation, N-glycan branching, and sialylation in HCC samples [16][17][18][19].
We have recently developed a matrix-assisted laser desorption-ionization imaging mass spectrometry (MALDI-IMS) approach for profiling N-glycans directly in frozen and formalin-fixed paraffin-embedded (FFPE) tissues [20,21]. The method relies on the spraying of a molecular coating of peptide N-glycosidase F (PNGaseF) to release N-glycans directly on tissues mounted on glass slides prior to matrix application. Relative to other classes of biomolecules targeted by MALDI-IMS, the method offers ready identification of the individual glycan species and creation of structural reference databases [21]. In this report, the utility of the method is demonstrated for profiling multiple N-glycans using different FFPE tissue slices of HCC. Current data analysis approaches are also summarized, as well as use of a novel derivatization method to stabilize sialic acids in a linkage-dependent/differentiating manner, and therefore better characterize larger, sialic acid containing N-glycans [22]. As the methodology is still evolving, areas to improve glycan detection and structural characterization by MALDI-IMS are discussed, as well as the context of how other glycan analysis approaches complement the method. As nearly all current cancer biomarkers are glycoproteins or carbohydrate antigens [23,24], a global analysis of N-glycans from HCC tissue sections using MALDI-IMS could extend and complement the continued development of N-glycan biomarkers associated with HCC.
FFPE Tissues
All but one tissue sections used in this study were de-identified and obtained commercially from Biochain (Newark, CA, USA). These include a slide with two FFPE tissue sections from a 60 year old female with a poorly differentiated hepatocellular carcinoma; patient matched tumor and normal FFPE tissue sections from a 53 year old male with hepatocellular carcinoma, and a patient matched renal cell carcinoma tumor and lymph node tissue with renal cell carcinoma metastasis from a 28 year old female. A subset of a commercially available renal tissue microarray (TMA) from Biochain was also analyzed, where two cores from each patient (oriented vertically) were present. One de-identified pancreas tissue was obtained by MUSC and was determined to be not human research classifications by the respective Institutional Review Boards at MUSC.
Washes for Deparaffinization and Rehydration
Slide preparation proceeded as described in our previous paper [21]. Briefly, FFPE tissue sections not acquired precut from Biochain, were sectioned at 5 ȝm and mounted on positively charged slides compatible with the Bruker slide adaptor. Standard ITO coated slides used for most MALDI imaging studies do not need to be used with this FTICR-MALDI configuration. All slides were heated at 60 °C for 1 h to ensure tissue adhesion to the slide. After cooling, the slide was deparaffinized by washing with xylene and rehydrated in a series of ethanol and water washes. Citraconic anhydride (Thermo) was used as the antigen retrieval buffer and the retrieval process took place over 25 min in a vegetable steamer. After allowing the buffer to cool, the buffer was gradually exchanged to 100% water. The slide was then desiccated to dryness prior to enzymatic digestion.
N-Glycan MALDI-IMS
An ImagePrep spray station (Bruker Daltonics, Billerica, MA, USA) was used to coat the slide with a 0.2 mL aqueous solution of PNGaseF (20 μg total/slide) as previously described [21]. As negative control, adjacent control tissue slices were shielded from PNGaseF application by covering the tissue section with a glass slide. Digestion occurred in a humidified chamber at 37 °C for 2 h. Slides were desiccated prior to Į-cyano-4-hydroxycinnamic acid matrix application (0.021 g CHCA in 3 mL 50% acetonitrile/50% water and 12 μL 25% TFA) using the ImagePrep sprayer. Released glycan ions were detected using a Solarix dual source 7T FTICR mass spectrometer (Bruker Daltonics) (m/z 690-5000) with a SmartBeam II laser operating at 1000 Hz, a laser spot size of 25 ȝm. Following MS analysis, data was loaded into FlexImaging Software focusing on the range m/z = 1000-4000 and reduced to 0.95 ICR Reduction Noise Threshold. FlexImaging 4.0 (Bruker Daltonics) was used to generate images of differentially expressed glycans. Observed glycans were searched against the glycan database generated using GlycoWorkbench [25]. Presented glycan structures were generated in GlycoWorkbench and represent putative structures determined by combinations of accurate m/z and off-slide derivatization experiments. CASI/CID was done as previously described [20,21].
Ethyl Esterification
N-glycans were extracted from slides as described previously and dried by vacuum centrifugation [20]. The ethyl esterification protocol, including the modification and enrichment, was adapted from Reiding et al. [22]. Briefly, 2 μL water and 40 μL 0.25 M HOBt/EDC were added to dried glycans followed by incubation at 37 °C for 1 h. 40 μL acetonitrile was added and the mixture was placed at í20 °C for 20 min. Glycans were enriched using cotton-HILIC tips according to Selman et al. [26]. Briefly, cotton wool composed of 100% cotton (Assured, Rio Rancho, NM, USA) was inserted in 20 μL tips and equilibrated with 10 μL water three times followed by 10 μL 85% ACN three times. Samples were loaded and unbound material removed by washing three times with 10 μL 85% acetonitrile with and without 1% TFA, respectively. Tip-bound glycans were eluted in 10 μL water. Enriched and modified glycans were spotted on an Anchorchip MALDI plate (Bruker Daltonics) with 2,5-dihydroxybenzoic acid (DHB) at a concentration of 5 mg/mL in 50% ACN/50% water/1 mM NaOH. Ethanol was then used for recrystallization.
Lectin Histochemistry
An HCC TMA slide (Catalog No.: Z7020059, Lot No.: B506168) was purchased from Biochain, Inc. This array consisted of 16 cases of liver cancer, each in duplicate, with corresponding uninvolved tissue from the same patient acting as controls. All the tissues were from surgical resection. Patients had a mean age of 47.56 years (range of 33-68 years) with a 8:1 ratio of males to females. Tissue slides were deparaffinized by using PROTOCOL SafeClear II clearing agent (Fisher Scientific), followed by rehydration though a series of graded ethanol. Lectin histochemistry was performed at room temperature, unless otherwise indicated. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide, followed by heat-mediated antigen retrieval using Universal Antigen Retrieval Reagent (R & D Systems, Minneapolis, MN, USA). Tissues were fixed with 4% formaldehyde solution followed by permeabilization with 0.5% IGEPAL. Prior to staining, the TMA slides were blocked using serum-free Dako Protein Block (Carpinteria, CA, USA). Next, the detection of core fucosylation was conducted using biotinylated recombinant N224Q rAAL lectin [27], diluted in Dako High Background Reducing Diluent solution to make the working final concentration of 500 ng/mL. Bound, biotinylated lectin was detected using streptavidin horseradish peroxidase (Vector Laboratory, Burlingame, CA, USA), and color was developed using 3,3'-diaminobenzidine (DAB) Chromogen from Dako (Carpinteria, CA, USA). Slides were visualized using an E200 Binocular Compound Biological Microscope from Nikon Instrument (Melville, NY, USA) and an IX71 Inverted Microscope from Olympus Imaging America, Inc. (Center Valley, PA, USA).
Influence of Histopathology on MALDI-IMS of N-Glycans
The ability to profile and determine the two-dimensional localization and histopathology of multiple N-glycans in tissues has been developed using a MALDI-IMS approach [20,21]. The goal of the studies were to present new data for analysis of N-glycan distribution in different FFPE hepatocellular carcinoma tissues, in order to highlight the capabilities and limitations of the method, how it compares with other glycan analysis approaches, and identify areas of improvement. Initial MALDI-IMS of a commercially available FFPE HCC tissue of complex histopathology demonstrated the specific release of N-glycans following PNGaseF digestion and the ability of released glycans to distinguish tissue subtypes and pathologies. Two serial sections were prepared identically with the exception that one tissue received PNGaseF while the other was shielded from enzyme application to serve as a negative control. Average spectra of each tissue region revealed a robust signal increase following PNGaseF digestion ( Figure 1a) compared to the control tissue ( Figure 1b). Putative glycan structures were annotated using GlycoWorkbench [25] based on mass accuracy and previous studies [20,21], but no information is available regarding specific anomeric linkages. All glycan structures reported herein are the [M + Na] + adducts unless otherwise noted, and a representative list of detected N-glycan compositions are in Table 1. Three representative glycans, Hex5dHex1HexNAc4NeuAc1 (m/z 2100.759, blue), Hex5HexNAc4 (m/z 1663.582, red) and Hex9HexNAc2 (m/z 1905.612, green) were selected and shown in an overlay image ( Figure 1c). The three glycans map to the tissue histopathology marked on the H & E stain (Figure 1d), withthree evident tissue morphologies; necrosis (outlined in red), HCC tissue (outlined in green) and fibroconnective tissue (outlined in blue). The three N-glycans in Figure 1c were selected to illustrate the regiospecific distribution of them in relation to the different histopathologies. Images of other N-glycans localized to the three regions are shown in Supplemental Figure 1. Interestingly, high mannose glycans were elevated in the tumor tissue, while fucosylated or sialylated/fucosylated complex diantennary glycans were elevated in the fibroconnective tissue.
While the initial experiment demonstrated the ability of N-glycan localization to define pathology in HCC tissue blocks, the ability to distinguish tumor vs. normal tissue is of more clinical significance. Analysis of a patient matched tumor and normal FFPE tissue sample revealed overall glycan heterogeneity between the two tissues. While Hex8HexNAc2 (m/z 1743.565, red) is present in both the normal and tumor sections, Hex7HexNAc6 (m/z 2393.854, green) is largely absent in the normal tissue (Figure 2a,b). This observation is evident in the image overlay, where the normal tissue image is red in color due to the presence of Hex8HexNAc2 and the absence of Hex7HexNAc6, while the tumor tissue is yellow due to the presence of both Hex7HexNAc6 and Hex8HexNAc2 (Figure 2c,d). This finding is consistent with our previous studies made in the analysis of an HCC TMA. Interestingly, while Hex8HexNAc2 is present in both the matched tumor and normal tissues and was elevated in the normal HCC tissue (Figure 2a), it is elevated in the tumor tissue compared to necrotic and fibroconnective tissue regions (Supplemental Figure S1). This trend emphasizes the importance of coupling histological analysis with the MALDI-IMS technique. Table 1. Comparative Analysis of N-glycans from MALDI-IMS and Ethyl Esterification. N-glycans from an HCC tumor tissue were extracted as described for Figure 4. Theoretical glycan m/z values were generated using GlycoWorkbench and are displayed as the [M + Na] + value. The m/z values of the N-glycans from MALDI-IMS and following the ethyl esterification protocol are also provided as the [M + Na] + value, unless otherwise stated. N-glycans bearing a single sialic acid residue are a lactonized or b ethylated. In the case where one glycan has two sialic acid residues, the glycan has c one lactonized and one ethylated sialic acid or d two ethylated sialic acids.
Similarities of Glycan Distribution across Tissue Types
The distribution of N-glycans in different fibroconnective tissues from other sources besides HCC tissues was further assessed. Lymph tissue with a clear cell renal cell carcinoma (ccRCC) metastasis (Figure 3a), pancreatic cancer and adjacent normal tissue (Figure 3b), and the hepatocellular carcinoma tissue presented earlier (Figure 3c), all with regions of fibrous and/or fibroconnective tissue, were analyzed by MALDI-IMS for N-glycans. The regions of fibroconnective tissue (outlined in black) and fibrous tumor tissue (outlined in red) are defined on the H & E image for each tissue (Figure 3). In each tissue, three diantennary N-glycans were commonly detected at higher levels in the fibrous/fibroconnective tissue compared to adjacent regions: Hex5HexNAc4 (m/z 1663.582), Hex5dHex1HexNAc4 (m/z 1809.661), and Hex5dHex1HexNAc4NeuAc1 (m/z 2122.720; [M í H + 2Na] + ). In all tissues, the Hex5dHex1HexNAc4 NeuAc1 glycan had the highest specificity for fibrous tissue regions. The Hex5HexNAc4 was detected at greater signal intensity in more regions of the tissues, and we have also noticed that this glycan is likely a marker of tissue regions where blood is present, illustrated in Figure 3b. These same glycans have also been detected primarily in stroma regions of prostate cancer tissues [28]. We hypothesize this shared glycan structural motif across tissues reflects the glycoproteins carrying them, most likely collagen and collagen-binding proteins. Glycoproteomic experiments are ongoing to assess this.
Validation of N-Glycans Structures by off-Slide Glycomic Analysis
MALDI ionization of glycans often results in the loss of labile sialic acids from glycans. We performed a recently introduced glycan modification approach, termed ethyl esterification [22], to assess whether the loss of sialic acids is occurring in MALDI-IMS experiments. This procedure results in lactonization of Į-2,3 linked sialic acids and ethyl esterification of Į-2,6 linked sialic acids, resulting in a 46.04 Da mass shift [22]. Following ethyl esterification of glycans, glycans were enriched using cotton-HILIC tips. Two HCC slides from adjacent slices were prepared for on-slide PNGaseF digestion. One slide was processed as described for MALDI-IMS analysis. For the other slide, glycans were retrieved from the tissue, subjected to the ethyl esterification protocol, and spotted on an anchorchip MALDI plate. An annotated spectrum is provided (Figure 4). A list of 33 representative N-glycans present in the MALDI-IMS and/or ethyl esterification experiments, as well as the glycan's theoretical m/z value is provided in Table 1. The results demonstrate a close overlap of observed m/z values for both approaches with theoretical m/z values obtained from GlycoWorkbench. Additionally, a majority of N-glycans were present in both the MALDI-IMS and off-tissue ethyl esterification experiments. However, when comparing the ethyl esterification average spectrum (Figure 4) with the MALDI-IMS average spectrum (Figure 1a), it is evident that the peak intensities of complex and sialylated N-glycans are more predominant in the ethyl esterification spectrum compared to the MALDI-IMS experiment: an example being Hex5HexNAc4NeuAc1 which has the highest signal intensity in the ethyl esterification spectrum (m/z 1936.692 and m/z 1982.733), but is only detected as minor peaks in the imaging average spectrum (m/z 1954.589 and m/z 1976.707). This result may be attributed to a reduction in signal intensity of sialylated N-glycans in MALDI-IMS due to a loss of sialic acid upon in-source decay, reflected in the comparatively higher level of detection of Hex5HexNAc4 (m/z 1663.582) in the MALDI-IMS average spectrum. However, as this comparison involves a purified glycan preparation vs. the analysis of glycans directly from the more complex tissue without any purification, the differences between the two may represent other conditions besides in-source decay.
Figure 4. Off-Tissue Ethyl Esterification of HCC Glycans Correlates with MALDI-IMS
Results. N-glycans extracted from HCC tissue sections were modified by ethyl esterification followed by cotton HILIC tip enrichment [22,26]. Enriched glycans were spotted on a MALDI plate and analyzed in positive ion mode by MALDI-FTICR. The core fucosylation in the indicated structures has yet to be confirmed by other methods. , and matched non-tumor adjacent (one core) tissues was profiled by MALDI-IMS [21], or stained by lectin histochemistry with a recombinant core fucose binding lectin, N224Q-rAAL [27]. Four representative TMA core sections were selected for comparison. Lectin histochemistry indicated tumor cores with low (top panel, (a,b)) or high (bottom panel, (c,d)) staining. Additional staining images are provided in Supplemental Figure S2. The image for each lectin stained core was captured individually, and images in the panel are from a 4× magnification by light microscopy. The individual core images were re-grouped in the panel to match the MALDI-IMS image orientation. Shown in the MALDI images are four monofucosylated glycans: 1. Hex5dHex1HexNAc4 (m/z = 1809.661); 2. Hex5dHex1HexNAc4 NeuAc1 (m/z = 2122.720 + 2Na); 3. Hex6dHex1HexNAc5 (m/z = 2174.772); 4. Hex7dHex1 HexNAc6 (m/z = 2539.904).
Detection of Core Fucosylation in HCC Tissue Microarrays
In addition to the dissociation of labile residues, another historical structural limitation of MALDI analysis is differentiating glycans with a core fucose vs. those with an outer arm fucose. To explore solutions for this limitation, the localization of glycans from a MALDI-IMS experiment were compared to a histochemistry staining with a core fucose binding lectin. We have recently reported MALDI-IMS profiling can be applied to TMA, and demonstrated this feature on a small HCC TMA of 48 tissue cores [22]. A recently described recombinant lectin [27], N224Q-rAAL, that is specific to binding of core fucosylated glycans was used to stain a serial section of the HCC TMA. This lectin has been used to detect increased core fucosylation of N-glycans associated with HCC [27]. Staining intensities of high vs. low core fucose expressing tissues were determined and compared to the corresponding MALDI glycan images of compositions with single fucoses. As shown in Figure 5 for a subset of tissue cores with low and high core fucose staining, there was a consistent detection of four singly fucosylated glycans by MALDI imaging in tumor cores. These glycans display a higher intensity in tissue cores that exhibit high levels of lectin staining (Figure 5c,d). Conversely, another tissue core set with low N224Q-rAAL staining had significantly lower signal intensities of the same four glycans (Figure 5a). In contrast, a different low N224Q-rAAL staining core set had high amounts of signal intensities for the mono-fucosylated glycans, suggesting that the fucose position in these glycans could be an outer arm modification (Figure 5b). Higher resolution light field microscope images of the lectin staining of these tissue cores are shown in Supplemental Figure S2. This example illustrates the potential utility of combining class specific lectin staining of tissues with the MALDI-IMS detection of individual glycans, an approach particularly amenable to fucose and sialic acid containing complex N-glycans.
Multiple approaches will be needed to clarify the positions of the fucoses in these glycans, and will involve tandem mass spectrometry characterization. The FTICR-MALDI used in these studies is capable of selective ion capturing, termed CASI, and when coupled with collision-induced dissociation (CID), can determine glycan composition as previously reported [21]. However, these were proof-of-concept examples. To examine whether core fucosylation could be determined by this process, further analysis was done from single fucosylated glycans captured by CASI/CID directly from tissue. As shown in the spectra in Figure 6, CID of two singly fucosylated glycans of Hex5dHex1HexNAc4 (m/z = 1809.656; Figure 6a) and Hex4dHex1HexNAc4 (m/z = 1647.598; Figure 6b) resulted in loss of the terminal N-acetylglucosamine and fucose. The CID of a non-fucosylated Hex5HexNAc4 (m/z = 1663.581) is shown in comparison (Figure 6c). The peak isolated by CASI is shown in the top panel for each glycan. This general approach should be applicable to assist in distinguishing core fucosylation of simpler N-glycans detected by MALDI-IMS.
Discussion
Profiling of N-glycans in fresh/frozen tissues, FFPE tissue blocks and TMAs is a new application of MALDI-IMS methodology and new approach in glycobiology that has the potential to identify systemic disease markers and elucidate disease etiology. This methodology is particularly relevant for cancer tissues, as most known cancer biomarkers are glycoproteins or carbohydrate antigens [23,24], including AFP for HCC [2]. The MALDI-IMS glycan profiling data presented herein, although from only a limited number of HCC tissues, illustrates this potential. Compared to other biomolecules targeted by MALDI-IMS like lipids, metabolites and proteins/peptides, N-glycans offer a particular advantage in that there are relatively fewer total glycan ion signals that are detected in a given tissue. Assigning the underlying composition for each glycan is rather straight-forward on the basis of accurate masses, albeit anomeric linkages are not differentiated. In addition to mass accuracy, glycan compositions can be validated by a variety of approaches, such as CID and derivatization. Structural standards for many of the simpler glycans are available, several glycan reference databases are available, and CID of glycans directly from tissue slides is possible using the MALDI-FTICR instrument [21].
Knowing the identity of the glycan offers many other opportunities for application to cell biology and genomic studies, particularly as these structures are synthesized by specific glycosyltransferases, and modified by specific glycosidases. This facilitates comparative analysis of genomic data for these enzymes from the same tissues, and several studies have been reported attempting to develop predictive algorithms for MALDI-detected glycans and gene transcripts [29][30][31]. Knowing the glycan composition also allows inference of different cell biology aspects related to biosynthesis, vesicular trafficking or degradation processes involving N-glycans. We have found across multiple tumor types, and illustrated herein, that identifying a specific tumor glycan profile in tissues is fairly straightforward [21,28]. Herein, this principle has been further expanded as consistent glycan signatures for fibroconnective tissue have been observed across cancer types (Figure 3). We are further testing whether detecting changes in glycan profiles can be used as surrogate indicators of changes in glycoprotein expression associated with cancer development and progression, as well as responses to treatment. Current efforts are also directed at using the N-glycan profiles as a tissue map to identify disease-specific regions of interest for subsequent glycopeptide analysis strategies [32,33].
There still remain many challenges associated with improving the approach, some of which were highlighted in the results. Detection of high mass N-glycans by MALDI mass spectrometry, particularly those glycans containing multiple sialic acids, has always been a challenge as previously described [34][35][36][37]. We are addressing these challenges in three ways, at the instrumentation level, with on-slide analysis approaches, and with off-slide analysis approaches. The source configuration of the Solarix 7T FTICR instrument used for these experiments is already inherently amenable to better detection of sialic acid-containing glycans. Compared to most MALDI-TOF instruments (i.e., non-FTICR), less vacuum and a cooling gas following laser desorption/ionization is present. This combination has been reported to aid in the retention of sialic acids on glycoconjugates [38,39]. The ability to selectively capture specific glycan ions directly from tissue for CID is also an advantage of the Solarix instrument [21]. Comparison of N-glycan profiles of the same tissue slides in different MALDI-TOF instruments is ongoing, with emphasis on how detection of sialic acid containing glycans differ across instrument configurations. In this context, different MALDI matrix formulations are also being tested and compared. Combining these approaches with glycan class-specific lectin staining is another complementary approach, highlighted in Figure 5. These are all cumulatively being assessed in the optimization for detection of higher mass glycan species.
Currently, based on mass accuracy of standards and derivatization experiments [20,21], we show that while sialylated N-glycans are detected in MALDI-IMS (Figure 1a), they appear to be of lower abundance than may be naturally occurring (Figure 4). Attempts to globally modify N-glycan reducing ends (i.e., 3-aminoquinoline, [40]) or specifically modifying labile sialic acid residues (i.e., p-toluidine or ethyl esterification) represent potential solutions, as previously reported [22,41,42]. We believe that some modification of the above off-slide derivation approaches used for N-glycans could be adapted to an on-slide MALDI-IMS tissue profiling workflow. Additionally, performing MALDI-IMS of N-glycans in negative ion mode could be a reasonable approach for partially retaining sialic acids of N-glycans, although much optimization remains to be done. For both fucose and sialic acid residue determination, the use of linkage specific fucosidases and sialidases are additional options. Applying sialidase in combination with PNGaseF followed by MALDI-IMS glycan imaging has been demonstrated for mouse brain tissues [20].
Given the ability to use FFPE tissues as the primary source for N-glycan imaging and their general availability, there is a potential to leverage this approach to discover glycan biomarkers of HCC and other cancer types. To facilitate biomarker discovery, the described techniques must be used with large numbers of tissue samples on sample slides or in tissue microarray format, highlighting the need for an accurate method to directly compare between samples/experiments. In the examples provided for HCC, N-glycan signatures were capable of distinguishing tumor from non-tumor tissue regions. To determine the significance of these glycans, a similar study involving an increased number of tissue cores is required. Ongoing research is directed at determining best practices for data normalization and incorporating internal standards into experimental workflows, and are issues common to all MALDI-IMS experiments [43]. Only after large enough data sets are generated will we be able to determine the best approach to analyze these larger sample sets. Use of TMAs for glycan MALDI-IMS can certainly facilitate the increase in sample numbers [44]. Use of MALDI-IMS alone may not be enough, as it is the type of method that can be complemented with existing and emerging quantitative mass spectrometry approaches [45], as well as lectin staining of tissues directly or use of lectin arrays [46]. A recent report using mouse kidney tissues has linked MALDI-IMS of N-glycans with LC-MS/MS characterization of the glycans [47]. This combination of MALDI, LC-MS/MS and array approaches will likely yield a more complete analysis option of FFPE tumor tissues.
Conclusions
The profiling and tissue mapping of N-glycans offers many new avenues of glycobiology research that can be initiated by, and complemented with, MALDI-IMS strategies. Challenges and limitations of the approach are evident, but we believe most can be addressed moving forward as more samples are analyzed, new derivation and matrix strategies tested, and through long-term, continued improvement in instrumentation platforms. | v3-fos-license |
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