The Experts below are selected from a list of 327 Experts worldwide ranked by ideXlab platform
Chun Wang - One of the best experts on this subject based on the ideXlab platform.
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facile synthesis and characterization of disulfide cross linked hyaluronic acid hydrogels for protein delivery and cell encapsulation
Biomacromolecules, 2011Co-Authors: Sun Young Choh, Daisy P Cross, Chun WangAbstract:Injectable hyaluronic acid (HA) hydrogels cross-linked via disulfide bond are synthesized using a thiol−disulfide exchange reaction. The production of small-molecule reaction product, pyridine-2-thione, allows the hydrogel formation process to be monitored quantitatively in real-time by UV spectroscopy. Rheological tests show that the hydrogels formed within minutes at 37 °C. Mechanical properties and equilibrium swelling degree of the hydrogels can be controlled by varying the ratio of HA pyridyl disulfide and macro-cross-linker PEG-dithiol. Degradation of the hydrogels was achieved both enzymatically and chemically by disulfide reduction with distinctly different kinetics and profiles. In the presence of hyaluronidase, hydrogel mass loss over time was linear and the degradation was faster at higher enzyme concentrations, suggesting surface-limited degradation. The kinetics of hydrogel erosion by glutathione was not linear, nor did the erosion rate correlate linearly with glutathione concentration, sugge...
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facile synthesis and characterization of disulfide cross linked hyaluronic acid hydrogels for protein delivery and cell encapsulation
Biomacromolecules, 2011Co-Authors: Sun Young Choh, Daisy P Cross, Chun WangAbstract:Injectable hyaluronic acid (HA) hydrogels cross-linked via disulfide bond are synthesized using a thiol-disulfide exchange reaction. The production of small-molecule reaction product, pyridine-2-thione, allows the hydrogel formation process to be monitored quantitatively in real-time by UV spectroscopy. Rheological tests show that the hydrogels formed within minutes at 37 °C. Mechanical properties and equilibrium swelling degree of the hydrogels can be controlled by varying the ratio of HA pyridyl disulfide and macro-cross-linker PEG-dithiol. Degradation of the hydrogels was achieved both enzymatically and chemically by disulfide reduction with distinctly different kinetics and profiles. In the presence of hyaluronidase, hydrogel mass loss over time was linear and the degradation was faster at higher enzyme concentrations, suggesting surface-limited degradation. The kinetics of hydrogel erosion by glutathione was not linear, nor did the erosion rate correlate linearly with glutathione concentration, suggesting a bulk erosion mechanism. A cysteine-containing chemokine, stromal cell-derived factor 1α, was successfully encapsulated in the hydrogel and released in vitro without chemical alteration. Several different cell types, including fibroblasts, endothelial cells, and mesenchymal stem cells, were successfully encapsulated in the hydrogels with high cell viability during and after the encapsulation process. Substantial cell viability in the hydrogels was maintained up to 7 days in culture despite the lack of adhesion between the HA matrix and the cells. The facile synthesis of disulfide-cross-linked, dual-responsive degradable HA hydrogels may enable further development of bioactive matrices potentially suitable for tissue engineering and drug delivery applications.
Leslie B Poole - One of the best experts on this subject based on the ideXlab platform.
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roles for the two cysteine residues of ahpc in catalysis of peroxide reduction by alkyl hydroperoxide reductase from salmonella typhimurium
Biochemistry, 1997Co-Authors: Holly R Ellis, Leslie B PooleAbstract:The catalytic properties of cysteine residues Cys46 and Cys165, which form intersubunit disulfide bonds in the peroxidatic AhpC protein of the alkyl hydroperoxide reductase (AhpR) system from Salmonella typhimurium, have been investigated. The AhpR system, composed of AhpC and a flavoprotein reductase, AhpF, catalyzes the pyridine nucleotide-dependent reduction of organic hydroperoxides and hydrogen peroxide. Amino acid sequence analysis of the disulfide-containing tryptic peptide demonstrated the presence of two identical disulfide bonds per dimer of oxidized AhpC located between Cys46 on one subunit and Cys165 on the other. Mutant AhpC proteins containing only one (C46S and C165S) or no (C46,165S) cysteine residues were purified and shown by circular dichroism studies to exhibit no major disruptions in secondary structure. In NADH-dependent peroxidase assays in the presence of AhpF, the C165S mutant was fully active in comparison with wild-type AhpC, while C46S and C46,165S displayed no peroxidatic activity. In addition, only C165S was oxidized by 1 equiv of hydrogen peroxide, giving a species that was stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF. Oxidized C165S also reacted rapidly with a stoichiometric amount of the thiol-containing reagent 2-nitro-5-thiobenzoic acid to generate a mixed disulfide, and was susceptible to inactivation by hydrogen peroxide, strongly supporting its identification as a cysteine sulfenic acid (Cys46-SOH). The lack of reactivity of the C46S mutant toward peroxides was not a result of inaccessibility of the remaining thiol as demonstrated by its modification with 5, 5'-dithiobis(2-nitrobenzoic acid), but could be due to the lack of a proximal active-site base which would support catalysis through proton donation to the poor RO- leaving group. Our results clearly identify Cys46 as the peroxidatic center of AhpC and Cys165 as an important residue for preserving the activity of wild-type AhpC by reacting with the nascent sulfenic acid of the oxidized protein (Cys46-SOH) to generate a stable disulfide bond, thus preventing further oxidation of Cys46-SOH by substrate.
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flavin dependent alkyl hydroperoxide reductase from salmonella typhimurium 2 cystine disulfides involved in catalysis of peroxide reduction
Biochemistry, 1996Co-Authors: Leslie B PooleAbstract:The two-component alkyl hydroperoxide reductase enzyme system from Salmonella typhimurium catalyzes the pyridine nucleotide-dependent reduction of alkyl hydroperoxide and hydrogen peroxide substrates. This system is composed of a flavoenzyme, AhpF, which is related to the disulfide-reducing enzyme thioredoxin reductase, and a smaller protein, AhpC, which lacks a chromophoric cofactor. We have demonstrated that NADH-linked reduction of AhpF under anaerobic conditions converts two cystine disulfide centers to their dithiol forms. The AhpC cystine disulfide center, shown to exist as an intersubunit disulfide bond, is stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF and can be reoxidized by ethyl hydroperoxide. Disulfide bridges within oxidized AhpF form between Cys129 and Cys132 and between Cys345 and Cys348; the two C-terminal half-cystine residues, Cys476 and Cys489, exist as free thiol groups in oxidized AhpF and play no role in catalysis. Removal of the N-terminal 202-amino acid segment containing the Cys129-Cys132 disulfide center obliterates the ability of AhpF to transfer electrons to 5,5'-dthiobis(2-nitrobenzoic acid) (DTNB) and AhpC. NADH added anaerobically to AhpF causes spectral changes consistent with preferential reduction of both disulfides relative to flavin reduction; the reduction potentials of the disulfide centers are thus appropriately poised for electron transfer from NADH and flavin to disulfide-containing substrates (AhpC or DTNB), and ultimately to peroxides from AhpC. Blue, neutral flavin semiquinone is also generated in high yields during reductive titrations (91% yield during dithionite titrations), although the relatively slow formation of this species indicates its catalytic incompetence. A long wavelength absorbance band beyond 900 nm attributable to an FADH2-->NAD+ charge transfer interaction is generated during NADH, but not dithionite, titrations and may be indicative of a species directly involved in the catalytic cycle. A catalytic mechanism including the transient formation of cysteine sulfenic acid within AhpC is proposed.
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flavin dependent alkyl hydroperoxide reductase from salmonella typhimurium 1 purification and enzymatic activities of overexpressed ahpf and ahpc proteins
Biochemistry, 1996Co-Authors: Leslie B Poole, Holly R EllisAbstract:The two components, AhpF and AhpC, of the Salmonella typhimurium alkyl hydroperoxide reductase enzyme system have been overexpressed and purified from Escherichia coli for investigations of their catalytic properties. Recombinant proteins were isolated in high yield (25−33 mg per liter of bacterial culture) and were shown to impart a high degree of protection against killing by cumene hydroperoxide to the host E. coli cells. We have developed quantitative enzymatic assays for AhpF alone and for the combined AhpF/AhpC system which have allowed us to address such issues as substrate specificity and inhibition by thiol reagents for each protein. All assays gave identical results whether overexpressed S. typhimurium proteins from E. coli or proteins isolated directly from S. typhimurium were used. Anaerobic hydroperoxide reductase assays have demonstrated that cumene hydroperoxide, ethyl hydroperoxide, and hydrogen peroxide can all be reduced by the combined enzyme system. AhpF possesses multiple pyridine nuc...
Michael Zharnikov - One of the best experts on this subject based on the ideXlab platform.
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self assembly of a pyridine terminated thiol monolayer on au 111
Langmuir, 2009Co-Authors: Christophe Silien, Manfred Buck, Dorothee Lahaye, Neil R Champness, Tobias Weidner, G Goretzki, Michael ZharnikovAbstract:Self-assembled monolayers (SAMs) of 3-(4-pyridine-4-yl-phenyl)-propane-1-thiol (PyP3) on Au(111)/mica have been studied by scanning tunneling microscopy (STM), polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), high-resolution X-ray photoemission spectroscopy (HRXPS), and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. The quality of the SAM is found to be strongly dependent on the solvent. Substantial gold corrosion is observed if pure ethanol is used. In contrast, highly ordered and densely packed SAMs are formed from acetonitrile or a KOH/ethanol mixture. The structure is described by a 2√3 × √3 unit cell with the aromatic moiety oriented nearly perpendicular to the surface. The PyP3 films form with the pyridine moiety deprotonated. Variation of pH allows reversible protonation without measurable damage of the SAM.
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self assembled monolayers of a bis pyrazol 1 yl pyridine substituted thiol on au 111
Langmuir, 2008Co-Authors: Cai Shen, Nirmalya Ballav, Manfred Buck, M Haryono, Andreas Grohmann, Michael ZharnikovAbstract:Self-assembled monolayers (SAMs) of a bis(pyrazol-1-yl)pyridine-substituted thiol (bpp-SH) on Au (111)/mica were studied with scanning tunneling microscopy (STM), X-ray photoelectron spectroscopy (XPS), and near-edge X-ray absorption fine structure spectroscopy (NEXAFS). Using substrates precoated with perylene-3,4,9,10-tetracarboxylic acid dianhydride (PTCDA), preparation at elevated temperatures yields highly ordered layers whose structure is described by a rectangular (5 × √3) unit cell containing one molecule. The bis(pyrazol-1-yl)pyridine (bpp) units exhibit π-stacking along the ⟨112⟩ direction, and they are tilted significantly. We conclude the three imine nitrogen atoms in the bpp headgroup adopt a trans,trans arrangement.
Sun Young Choh - One of the best experts on this subject based on the ideXlab platform.
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facile synthesis and characterization of disulfide cross linked hyaluronic acid hydrogels for protein delivery and cell encapsulation
Biomacromolecules, 2011Co-Authors: Sun Young Choh, Daisy P Cross, Chun WangAbstract:Injectable hyaluronic acid (HA) hydrogels cross-linked via disulfide bond are synthesized using a thiol−disulfide exchange reaction. The production of small-molecule reaction product, pyridine-2-thione, allows the hydrogel formation process to be monitored quantitatively in real-time by UV spectroscopy. Rheological tests show that the hydrogels formed within minutes at 37 °C. Mechanical properties and equilibrium swelling degree of the hydrogels can be controlled by varying the ratio of HA pyridyl disulfide and macro-cross-linker PEG-dithiol. Degradation of the hydrogels was achieved both enzymatically and chemically by disulfide reduction with distinctly different kinetics and profiles. In the presence of hyaluronidase, hydrogel mass loss over time was linear and the degradation was faster at higher enzyme concentrations, suggesting surface-limited degradation. The kinetics of hydrogel erosion by glutathione was not linear, nor did the erosion rate correlate linearly with glutathione concentration, sugge...
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facile synthesis and characterization of disulfide cross linked hyaluronic acid hydrogels for protein delivery and cell encapsulation
Biomacromolecules, 2011Co-Authors: Sun Young Choh, Daisy P Cross, Chun WangAbstract:Injectable hyaluronic acid (HA) hydrogels cross-linked via disulfide bond are synthesized using a thiol-disulfide exchange reaction. The production of small-molecule reaction product, pyridine-2-thione, allows the hydrogel formation process to be monitored quantitatively in real-time by UV spectroscopy. Rheological tests show that the hydrogels formed within minutes at 37 °C. Mechanical properties and equilibrium swelling degree of the hydrogels can be controlled by varying the ratio of HA pyridyl disulfide and macro-cross-linker PEG-dithiol. Degradation of the hydrogels was achieved both enzymatically and chemically by disulfide reduction with distinctly different kinetics and profiles. In the presence of hyaluronidase, hydrogel mass loss over time was linear and the degradation was faster at higher enzyme concentrations, suggesting surface-limited degradation. The kinetics of hydrogel erosion by glutathione was not linear, nor did the erosion rate correlate linearly with glutathione concentration, suggesting a bulk erosion mechanism. A cysteine-containing chemokine, stromal cell-derived factor 1α, was successfully encapsulated in the hydrogel and released in vitro without chemical alteration. Several different cell types, including fibroblasts, endothelial cells, and mesenchymal stem cells, were successfully encapsulated in the hydrogels with high cell viability during and after the encapsulation process. Substantial cell viability in the hydrogels was maintained up to 7 days in culture despite the lack of adhesion between the HA matrix and the cells. The facile synthesis of disulfide-cross-linked, dual-responsive degradable HA hydrogels may enable further development of bioactive matrices potentially suitable for tissue engineering and drug delivery applications.
Holly R Ellis - One of the best experts on this subject based on the ideXlab platform.
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roles for the two cysteine residues of ahpc in catalysis of peroxide reduction by alkyl hydroperoxide reductase from salmonella typhimurium
Biochemistry, 1997Co-Authors: Holly R Ellis, Leslie B PooleAbstract:The catalytic properties of cysteine residues Cys46 and Cys165, which form intersubunit disulfide bonds in the peroxidatic AhpC protein of the alkyl hydroperoxide reductase (AhpR) system from Salmonella typhimurium, have been investigated. The AhpR system, composed of AhpC and a flavoprotein reductase, AhpF, catalyzes the pyridine nucleotide-dependent reduction of organic hydroperoxides and hydrogen peroxide. Amino acid sequence analysis of the disulfide-containing tryptic peptide demonstrated the presence of two identical disulfide bonds per dimer of oxidized AhpC located between Cys46 on one subunit and Cys165 on the other. Mutant AhpC proteins containing only one (C46S and C165S) or no (C46,165S) cysteine residues were purified and shown by circular dichroism studies to exhibit no major disruptions in secondary structure. In NADH-dependent peroxidase assays in the presence of AhpF, the C165S mutant was fully active in comparison with wild-type AhpC, while C46S and C46,165S displayed no peroxidatic activity. In addition, only C165S was oxidized by 1 equiv of hydrogen peroxide, giving a species that was stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF. Oxidized C165S also reacted rapidly with a stoichiometric amount of the thiol-containing reagent 2-nitro-5-thiobenzoic acid to generate a mixed disulfide, and was susceptible to inactivation by hydrogen peroxide, strongly supporting its identification as a cysteine sulfenic acid (Cys46-SOH). The lack of reactivity of the C46S mutant toward peroxides was not a result of inaccessibility of the remaining thiol as demonstrated by its modification with 5, 5'-dithiobis(2-nitrobenzoic acid), but could be due to the lack of a proximal active-site base which would support catalysis through proton donation to the poor RO- leaving group. Our results clearly identify Cys46 as the peroxidatic center of AhpC and Cys165 as an important residue for preserving the activity of wild-type AhpC by reacting with the nascent sulfenic acid of the oxidized protein (Cys46-SOH) to generate a stable disulfide bond, thus preventing further oxidation of Cys46-SOH by substrate.
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flavin dependent alkyl hydroperoxide reductase from salmonella typhimurium 1 purification and enzymatic activities of overexpressed ahpf and ahpc proteins
Biochemistry, 1996Co-Authors: Leslie B Poole, Holly R EllisAbstract:The two components, AhpF and AhpC, of the Salmonella typhimurium alkyl hydroperoxide reductase enzyme system have been overexpressed and purified from Escherichia coli for investigations of their catalytic properties. Recombinant proteins were isolated in high yield (25−33 mg per liter of bacterial culture) and were shown to impart a high degree of protection against killing by cumene hydroperoxide to the host E. coli cells. We have developed quantitative enzymatic assays for AhpF alone and for the combined AhpF/AhpC system which have allowed us to address such issues as substrate specificity and inhibition by thiol reagents for each protein. All assays gave identical results whether overexpressed S. typhimurium proteins from E. coli or proteins isolated directly from S. typhimurium were used. Anaerobic hydroperoxide reductase assays have demonstrated that cumene hydroperoxide, ethyl hydroperoxide, and hydrogen peroxide can all be reduced by the combined enzyme system. AhpF possesses multiple pyridine nuc...
