Superhydrophobic and Slippery Lubricant-Infused Flexible Transparent Nanocellulose Films by Photoinduced Thiol-Ene Functionalization.

PubMed

Guo, Jiaqi; Fang, Wenwen; Welle, Alexander; Feng, Wenqian; Filpponen, Ilari; Rojas, Orlando J; Levkin, Pavel A

2016-12-14

Films comprising nanofibrillated cellulose (NFC) are suitable substrates for flexible devices in analytical, sensor, diagnostic, and display technologies. However, some major challenges in such developments include their high moisture sensitivity and the complexity of current methods available for functionalization and patterning. In this work, we present a facile process for tailoring the surface wettability and functionality of NFC films by a fast and versatile approach. First, the NFC films were coated with a layer of reactive nanoporous silicone nanofilament by polycondensation of trichlorovinylsilane (TCVS). The TCVS afforded reactive vinyl groups, thereby enabling simple UV-induced functionalization of NFC films with various thiol-containing molecules via the photo "click" thiol-ene reaction. Modification with perfluoroalkyl thiols resulted in robust superhydrophobic surfaces, which could then be further transformed into transparent slippery lubricant-infused NFC films that displayed repellency against both aqueous and organic liquids with surface tensions as low as 18 mN·m -1 . Finally, transparent and flexible NFC films incorporated hydrophilic micropatterns by modification with OH, NH 2 , or COOH surface groups, enabling space-resolved superhydrophobic-hydrophilic domains. Flexibility, transparency, patternability, and perfect superhydrophobicity of the produced nanocellulose substrates warrants their application in biosensing, display protection, and biomedical and diagnostics devices.

A novel strategy for global analysis of the dynamic thiol redox proteome.

PubMed

Martínez-Acedo, Pablo; Núñez, Estefanía; Gómez, Francisco J Sánchez; Moreno, Margoth; Ramos, Elena; Izquierdo-Álvarez, Alicia; Miró-Casas, Elisabet; Mesa, Raquel; Rodriguez, Patricia; Martínez-Ruiz, Antonio; Dorado, David Garcia; Lamas, Santiago; Vázquez, Jesús

2012-09-01

Nitroxidative stress in cells occurs mainly through the action of reactive nitrogen and oxygen species (RNOS) on protein thiol groups. Reactive nitrogen and oxygen species-mediated protein modifications are associated with pathophysiological states, but can also convey physiological signals. Identification of Cys residues that are modified by oxidative stimuli still poses technical challenges and these changes have never been statistically analyzed from a proteome-wide perspective. Here we show that GELSILOX, a method that combines a robust proteomics protocol with a new computational approach that analyzes variance at the peptide level, allows a simultaneous analysis of dynamic alterations in the redox state of Cys sites and of protein abundance. GELSILOX permits the characterization of the major endothelial redox targets of hydrogen peroxide in endothelial cells and reveals that hypoxia induces a significant increase in the status of oxidized thiols. GELSILOX also detected thiols that are redox-modified by ischemia-reperfusion in heart mitochondria and demonstrated that these alterations are abolished in ischemia-preconditioned animals.

LC-MS-based characterization of the peptide reactivity of chemicals to improve the in vitro prediction of the skin sensitization potential.

PubMed

Natsch, Andreas; Gfeller, Hans

2008-12-01

A key step in the skin sensitization process is the formation of a covalent adduct between skin sensitizers and endogenous proteins and/or peptides in the skin. Based on this mechanistic understanding, there is a renewed interest in in vitro assays to determine the reactivity of chemicals toward peptides in order to predict their sensitization potential. A standardized peptide reactivity assay yielded a promising predictivity. This published assay is based on high-performance liquid chromatography with ultraviolet detection to quantify peptide depletion after incubation with test chemicals. We had observed that peptide depletion may be due to either adduct formation or peptide oxidation. Here we report a modified assay based on both liquid chromatography-mass spectrometry (LC-MS) analysis and detection of free thiol groups. This approach allows simultaneous determination of (1) peptide depletion, (2) peptide oxidation (dimerization), (3) adduct formation, and (4) thiol reactivity and thus generates a more detailed characterization of the reactivity of a molecule. Highly reactive molecules are further discriminated with a kinetic measure. The assay was validated on 80 chemicals. Peptide depletion could accurately be quantified both with LC-MS detection and depletion of thiol groups. The majority of the moderate/strong/extreme sensitizers formed detectable peptide adducts, but many sensitizers were also able to catalyze peptide oxidation. Whereas adduct formation was only observed for sensitizers, this oxidation reaction was also observed for two nonsensitizing fragrance aldehydes, indicating that peptide depletion might not always be regarded as sufficient evidence for rating a chemical as a sensitizer. Thus, this modified assay gives a more informed view of the peptide reactivity of chemicals to better predict their sensitization potential.

Jack bean urease: the effect of active-site binding inhibitors on the reactivity of enzyme thiol groups.

PubMed

Krajewska, Barbara; Zaborska, Wiesława

2007-10-01

In view of the complexity of the role of the active site flap cysteine in the urease catalysis, in this work we studied how the presence of typical active-site binding inhibitors of urease, phenylphosphorodiamidate (PPD), acetohydroxamic acid (AHA), boric acid and fluoride, affects the reactivity of enzyme thiol groups, the active site flap thiol in particular. For that the inhibitor-urease complexes were prepared with excess inhibitors and had their thiol groups titrated with DTNB. The effects observed were analyzed in terms of the structures of the inhibitor-urease complexes reported in the literature. We found that the effectiveness in preventing the active site cysteine from the modification by disulfides, varied among the inhibitors studied, even though they all bind to the active site. The variations were accounted for by different extents of geometrical distortion in the active site that the inhibitors introduced upon binding, leaving the flap either open in AHA-, boric acid- and fluoride-inhibited urease, like in the native enzyme or closed in PPD-inhibited urease. Among the inhibitors, only PPD was found to be able to thoroughly protect the flap cysteines from the further reaction with disulfides, this apparently resulting from the closed conformation of the flap. Accordingly, in practical terms PPD may be regarded as the most suitable inhibitor for active-site protection experiments in inhibition studies of urease.

Thiol-reactive amphiphilic block copolymer for coating gold nanoparticles with neutral and functionable surfaces

PubMed Central

Chen, Hongwei; Zou, Hao; Paholak, Hayley J.; Ito, Masayuki; Qian, Wei; Che, Yong; Sun, Duxin

2014-01-01

Nanoparticles designed for biomedical applications are often coated with polymers containing reactive functional groups, such as –COOH and –NH2, to conjugate targeting ligands or drugs. However, introducing highly charged surfaces promotes binding of the nanoparticles to biomolecules in biological systems through ionic interactions, causing the nanoparticles to aggregate in biological environments and consequently undergo strong non-specific binding to off-target cells and tissues. Developing a unique polymer with neutral surfaces that can be further functionalized directly would be critical to develop suitable nanomaterials for nanomedicine. Here, we report a thiol-reactive amphiphilic block copolymer poly(ethylene oxide)-block-poly(pyridyldisulfide ethylmeth acrylate) (PEO-b-PPDSM) for coating gold nanoparticles (AuNPs). The resultant polymer-coated AuNPs have almost neutral surfaces with slightly negative zeta potentials from -10 to 0 mV over a wide pH range from 2 to 12. Although the zeta potential is close to zero we show that the PEO-b-PPDSM copolymer-coated AuNPs have both good stability in various physiological conditions and reduced non-specific adsorption of proteins/biomolecules. Because of the multiple pyridyldisulfide groups on the PPDSM block, these individually dispersed nanocomplexes with an overall hydrodynamic size around 43.8 nm can be directly functionalized via disulfide-thiol exchange chemistry. PMID:24729795

Efficient Site-Specific Labeling of Proteins via Cysteines

PubMed Central

Kim, Younggyu; Ho, Sam O.; Gassman, Natalie R.; Korlann, You; Landorf, Elizabeth V.; Collart, Frank R.; Weiss, Shimon

2011-01-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70–90%, and specificities are better than ~95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis. PMID:18275130

Efficient site-specific labeling of proteins via cysteines.

PubMed

Kim, Younggyu; Ho, Sam O; Gassman, Natalie R; Korlann, You; Landorf, Elizabeth V; Collart, Frank R; Weiss, Shimon

2008-03-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

Analysis of function-related interactions of ATP, sodium and potassium ions with Na+- and K+-transporting ATPase studied with a thiol reagent as tool.

PubMed

Grosse, R; Eckert, K; Malur, J; Repke, K R

1978-01-01

The paper describes the interaction of ATP, Na+ and K+ with (NaK)-ATPase exploiting the inactivation by reaction with NBD-chloride as an analytical tool for the evaluation of enzyme ligandation with the various effectors. 1. The inactivation of (NaK)-ATPase by reaction with NBD-chloride showing under all conditions studied a pseudo first-order rate rests on the alkylation of thiol groups in or near catalytic centre. ATP bound to catalytic centre prevents from enzyme inactivation by NDD-chloride through protection of these thiol groups from alkylation. Na+ and K+ affect the reactivity of the thiol groups towards NBD-chloride either indirectly via influencing ATP binding or more directly via changing the conformation of catalytic centre. Proceeding from these interrelations, the interaction of the various effectors with the enzyme was analyzed. 2. The K'D-values of various nucleotides determined by our approach correspond to the values obtained by independent methods. As shown for the first time, two catalytic centres per enzyme molecule exist. They exhibit high or low affinity to both ATP and ADP apparently caused by anticooperative interaction of the half-units of the enzyme through intersubunit communication ("half-of-the-sites reactivity"). 3. In the absence of ATP, Na+ or K+ ligandation of (NaK)-ATPase produce opposite effects on the reactivity of the thiol groups of catalytic centres reflecting different changes of their conformation. This corresponds to the well-known antagonistic effect of Na+ and K+ on some partial reactions of (NaK)-ATPase. The Na+ and K+ concentrations required to change thiol reactivity are rather high, i.e. the ionophoric centres for both Na+ and K+ are not readily accessible for cation complexation in the absence of enzyme complexation with ATP. 4. Na+ being without effect on ATP binding to the enzyme also does not influence the inactivating reaction with NBD-chloride while K+ by decreasing ATP binding dramatically decreases the protective effect of ATP. The K+ affinity of the enzyme-ATP complex is by more than two orders of magnitude higher than that of free enzyme. Na+ ligandation of the K+-liganded enzyme-ATP complex reverses the effect of K+ ligandation and produces a protective effect which distinctly surpasses that of the complexation of free enzyme with ATP. Hence, the enzyme molecule carries simultaneously ionophoric centres for both Na+ and K+. 5. The findings that per enzyme molecule ionophoric centres for Na+ and K+, and two catalytic centres with anticooperative interaction coexist corroborate the corresponding basic predictions of the flip-flop concept of (NaK)-ATPase pump mechanism, and explain some peculiar kinetic features of transport and enzyme activities of (NaK)-ATPase.

Redox Reactivity of Cerium Oxide Nanoparticles Induces the Formation of Disulfide Bridges in Thiol-Containing Biomolecules.

PubMed

Rollin-Genetet, Françoise; Seidel, Caroline; Artells, Ester; Auffan, Mélanie; Thiéry, Alain; Vidaud, Claude

2015-12-21

The redox state of disulfide bonds is implicated in many redox control systems, such as the cysteine-cystine couple. Among proteins, ubiquitous cysteine-rich metallothioneins possess thiolate metal binding groups susceptible to metal exchange in detoxification processes. CeO2 NPs are commonly used in various industrial applications due to their redox properties. These redox properties that enable dual oxidation states (Ce(IV)/Ce(III)) to exist at their surface may act as oxidants for biomolecules. The interaction among metallothioneins, cysteine, and CeO2 NPs was investigated through various biophysical approaches to shed light on the potential effects of the Ce(4+)/Ce(3+) redox system on the thiol groups of these biomolecules. The possible reaction mechanisms include the formation of a disulfide bridge/Ce(III) complex resulting from the interaction between Ce(IV) and the thiol groups, leading to metal unloading from the MTs, depending on their metal content and cluster type. The formation of stable Ce(3+) disulfide complexes has been demonstrated via their fluorescence properties. This work provides the first evidence of thiol concentration-dependent catalytic oxidation mechanisms between pristine CeO2 NPs and thiol-containing biomolecules.

Methemoglobin formation from butylated hydroxyanisole and oxyhemoglobin. Comparison with butylated hydroxytoluene and p-hydroxyanisole.

PubMed

Stolze, K; Nohl, H

1992-01-01

The widely used food additives butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) react with oxyhemoglobin, thereby forming methemoglobin. The reaction rates were measured using visible spectroscopy, and second order rate constants were established for BHA and compared with p-hydroxyanisole. Using ESR we investigated the involvement of free radical reaction intermediates. The expected one-electron oxidation product of BHA and BHT, the phenoxyl radical, could only be detected with pure 3-t-butyl-4-hydroxyanisole and oxyhemoglobin. With the commercial mixture of 2- and 3-t-butyl-4-hydroxyanisole a very strong ESR signal of a secondary free radical species was observed, similar to the one observed earlier with p-hydroxyanisole and dependent on the presence of free thiol groups, so that we assumed the intermediate existence of a perferryl species, the MetHb-H2O2 adduct. In a second series of experiments we investigated the reactivity of this postulated intermediate with BHA and BHT, starting with a pure MetHb/H2O2-phenol mixture in a stopped-flow apparatus linked to the ESR spectrometer, detecting the expected phenoxyl radicals from BHA and p-hydroxyanisole. Due to the low solubility and decreased reactivity of BHT only traces of phenoxyl type radical were found together with a high concentration of unreacted perferryl species. The reactivity of BHA, BHT and p-hydroxyanisole with free thiol groups is demonstrated by an increased reaction rate in the presence of the thiol group blocking substance NEM.

Monohalogenated acetamide-induced cellular stress and genotoxicity are related to electrophilic softness and thiol/thiolate reactivity.

PubMed

Pals, Justin A; Wagner, Elizabeth D; Plewa, Michael J; Xia, Menghang; Attene-Ramos, Matias S

2017-08-01

Haloacetamides (HAMs) are cytotoxic, genotoxic, and mutagenic byproducts of drinking water disinfection. They are soft electrophilic compounds that form covalent bonds with the free thiol/thiolate in cysteine residues through an S N 2 reaction mechanism. Toxicity of the monohalogenated HAMs (iodoacetamide, IAM; bromoacetamide, BAM; or chloroacetamide, CAM) varied depending on the halogen substituent. The aim of this research was to investigate how the halogen atom affects the reactivity and toxicological properties of HAMs, measured as induction of oxidative/electrophilic stress response and genotoxicity. Additionally, we wanted to determine how well in silico estimates of electrophilic softness matched thiol/thiolate reactivity and in vitro toxicological endpoints. Each of the HAMs significantly induced nuclear Rad51 accumulation and ARE signaling activity compared to a negative control. The rank order of effect was IAM>BAM>CAM for Rad51, and BAM≈IAM>CAM for ARE. In general, electrophilic softness and in chemico thiol/thiolate reactivity provided a qualitative indicator of toxicity, as the softer electrophiles IAM and BAM were more thiol/thiolate reactive and were more toxic than CAM. Copyright © 2017. Published by Elsevier B.V.

Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions.

PubMed

Jackson, Paul A; Widen, John C; Harki, Daniel A; Brummond, Kay M

2017-02-09

Although Michael acceptors display a potent and broad spectrum of bioactivity, they have largely been ignored in drug discovery because of their presumed indiscriminate reactivity. As such, a dearth of information exists relevant to the thiol reactivity of natural products and their analogues possessing this moiety. In the midst of recently approved acrylamide-containing drugs, it is clear that a good understanding of the hetero-Michael addition reaction and the relative reactivities of biological thiols with Michael acceptors under physiological conditions is needed for the design and use of these compounds as biological tools and potential therapeutics. This Perspective provides information that will contribute to this understanding, such as kinetics of thiol addition reactions, bioactivities, as well as steric and electronic factors that influence the electrophilicity and reversibility of Michael acceptors. This Perspective is focused on α,β-unsaturated carbonyls given their preponderance in bioactive natural products.

High-throughput identification of promiscuous inhibitors from screening libraries with the use of a thiol-containing fluorescent probe.

PubMed

McCallum, Megan M; Nandhikonda, Premchendar; Temmer, Jonathan J; Eyermann, Charles; Simeonov, Anton; Jadhav, Ajit; Yasgar, Adam; Maloney, David; Arnold, Alexander Leggy

2013-07-01

Testing small molecules for their ability to modify cysteine residues of proteins in the early stages of drug discovery is expected to accelerate our ability to develop more selective drugs with lesser side effects. In addition, this approach also enables the rapid evaluation of the mode of binding of new drug candidates with respect to thiol reactivity and metabolism by glutathione. Herein, we describe the development of a fluorescence-based high-throughput assay that allows the identification of thiol-reactive compounds. A thiol-containing fluorescent probe, MSTI, was synthesized and used to evaluate small molecules from the Library of Pharmacologically Active Compounds (LOPAC) collection of bioactive molecules. LOPAC compounds that are known to react with sulfur nucleophiles were identified with this assay, for example, irreversible protease inhibitors, nitric oxide-releasing compounds, and proton-pump inhibitors. The results confirm that both electrophilic and redox reactive compounds can be quickly identified in a high-throughput manner, enabling the assessment of screening libraries with respect to thiol-reactive compounds.

Detection of intracellular glutathione using ThiolTracker violet stain and fluorescence microscopy.

PubMed

Mandavilli, Bhaskar S; Janes, Michael S

2010-07-01

Glutathione plays an important role in protecting mammalian cells from oxidative stress and cell death. Because reduced glutathione (GSH) represents the large majority of intracellular free thiols, cell-permeant, thiol-reactive fluorescent probes represent potentially useful indicators of intracellular GSH. The ThiolTracker Violet stain (a registered trademark of Invitrogen) is a bright fluorescent probe that is highly reactive to thiols and can be used as a convenient and effective indicator of intracellular GSH and general redox status by a variety of detection modalities. While this probe has been validated in flow cytometry and microplate fluorimetry assays, the following method will describe details on the use of the ThiolTracker Violet dye in traditional fluorescence microscopy, as well as high-content imaging and analysis.

Thiol-Based Redox Switches and Gene Regulation

PubMed Central

2011-01-01

Abstract Cysteine is notable among the universal, proteinogenic amino acids for its facile redox chemistry. Cysteine thiolates are readily modified by reactive oxygen species (ROS), reactive electrophilic species (RES), and reactive nitrogen species (RNS). Although thiol switches are commonly triggered by disulfide bond formation, they can also be controlled by S-thiolation, S-alkylation, or modification by RNS. Thiol-based switches are common in both prokaryotic and eukaryotic organisms and activate functions that detoxify reactive species and restore thiol homeostasis while repressing functions that would be deleterious if expressed under oxidizing conditions. Here, we provide an overview of the best-understood examples of thiol-based redox switches that affect gene expression. Intra- or intermolecular disulfide bond formation serves as a direct regulatory switch for several bacterial transcription factors (OxyR, OhrR/2-Cys, Spx, YodB, CrtJ, and CprK) and indirectly regulates others (the RsrA anti-σ factor and RegB sensory histidine kinase). In eukaryotes, thiol-based switches control the yeast Yap1p transcription factor, the Nrf2/Keap1 electrophile and oxidative stress response, and the Chlamydomonas NAB1 translational repressor. Collectively, these regulators reveal a remarkable range of chemical modifications exploited by Cys residues to effect changes in gene expression. Antioxid. Redox Signal. 14, 1049—1063. PMID:20626317

General Synthesis of Alkenyl Sulfides by Palladium-Catalyzed Thioetherification of Alkenyl Halides and Tosylates.

PubMed

Velasco, Noelia; Virumbrales, Cintia; Sanz, Roberto; Suárez-Pantiga, Samuel; Fernández-Rodríguez, Manuel A

2018-05-08

The cross-coupling reaction of alkenyl bromides with thiols catalyzed by palladium complexes derived from inexpensive dppf ligand is reported. These reactions occur under low catalyst loading and in high yields and display wide scope, including the coupling of bulky thiols and trisubstituted bromoolefins, and functional group tolerance. In addition, the thioetherification of less reactive chloroalkenes and, for the first time, alkenyl tosylates was accomplished using a catalyst generated from CyPF tBu alkylbisphosphine ligand.

Lignin-Based Materials Through Thiol-Maleimide "Click" Polymerization.

PubMed

Buono, Pietro; Duval, Antoine; Averous, Luc; Habibi, Youssef

2017-03-09

In the present report an environmentally friendly approach to transforming renewable feedstocks into value-added materials is proposed. This transformation pathway was conducted under green conditions, without the use of solvents or catalyst. First, controlled modification of lignin, a major biopolymer present in wood and plants, was achieved by esterification with 11-maleimidoundecylenic acid (11-MUA), a derivative from castor oil that contains maleimide groups, following its transformation into 11-maleimidoundecanoyl chloride (11-MUC). Different degrees of substitution were achieved by using various amounts of the 11-MUC, leading to an efficient conversion of lignin hydroxy groups, as demonstrated by 1 H and 31 P NMR analyses. These fully biobased maleimide-lignin derivatives were subjected to an extremely fast (ca. 1 min) thiol-ene "click" polymerization with thiol-containing linkers. Aliphatic and aromatic thiol linkers bearing two to four thiol groups were used to tune the reactivity and crosslink density. The properties of the resulting materials were evaluated by swelling tests and thermal and mechanical analyses, which showed that varying the degree of functionality of the linker and the linker structure allowed accurate tailoring of the thermal and mechanical properties of the final materials, thus providing interesting perspectives for lignin in functional aromatic polymers. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thiolated alkyl-modified carbomers: Novel excipients for mucoadhesive emulsions.

PubMed

Bonengel, Sonja; Hauptstein, Sabine; Leonaviciute, Gintare; Griessinger, Julia; Bernkop-Schnürch, Andreas

2015-07-30

The aim of this study was the design and evaluation of mucoadhesive emulsifying polymeric excipients. Three thiol bearing ligands with increasing pKa values of their sulfhydryl group, namely 4-aminothiophenol (pKa=6.86), l-cysteine (pKa=8.4) and d/l-homocysteine (pKa=10.0) were coupled to the polymeric backbone of alkyl-modified carbomer (PA1030). Resulting conjugates displayed 818.5μmol 4-aminothiophenol, 698.5μmol cysteine and 651.5μmol homocysteine per gram polymer and were evaluated regarding the reactivity of thiol groups, emulsifying and mucoadhesive properties. In general, the synthesized conjugates showed a pH dependent reactivity, whereby the fastest oxidation occurred in PA1030-cysteine, as almost no free thiol groups could be detected after 120min. Emulsification of medium chain triglycerides was feasible with all synthesized conjugates leading to oil-in-water-emulsions. Emulsions with PA1030-cysteine displayed the highest stability and the smallest droplet size among the tested formulations. Oxidation and consequently cross-linking of the thiomers prior to the emulsification process led to an overall decreased emulsion stability. Evaluating mucosal residence time of thiomer emulsions on porcine buccal mucosa, a 9.2-fold higher amount of formulation based on PA1030-cysteine remained on the mucosal tissue within 3h compared to the unmodified polymer. According to these results, the highest reactive ligand l-cysteine seems to be most promising in order to obtain thiolated polymers for the preparation of mucoadhesive o/w-emulsions. Copyright © 2015 Elsevier B.V. All rights reserved.

Reactivity measurement in estimation of benzoquinone and benzoquinone derivatives’ allergenicity

PubMed Central

Mbiya, Wilbes; Chipinda, Itai; Simoyi, Reuben H.; Siegel, Paul D.

2015-01-01

Benzoquinone (BQ) and benzoquinone derivatives (BQD) are used in the production of dyes and cosmetics. While BQ, an extreme skin sensitizer, is an electrophile known to covalently modify proteins via Michael Addition (MA) reaction whilst halogen substituted BQD undergo nucleophilic vinylic substitution (SNV) mechanism onto amine and thiol moieties on proteins, the allergenic effects of adding substituents on BQ have not been reported. The effects of inserting substituents on the BQ ring has not been studied in animal assays. However, mandated reduction/elimination of animals used in cosmetics testing in Europe has led to an increased need for alternatives for the prediction of skin sensitization potential. Electron withdrawing and electron donating substituents on BQ were assessed for effects on BQ reactivity toward nitrobenzene thiol (NBT). The NBT binding studies demonstrated that addition of EWG to BQ as exemplified by the chlorine substituted BQDs increased reactivity while addition of EDG as in the methyl substituted BQDs reduced reactivity. BQ and BQD skin allerginicity was evaluated in the murine local lymph node assay (LLNA). BQD with electron withdrawing groups had the highest chemical potency followed by unsubstituted BQ and the least potent were the BQD with electron donating groups. The BQD results demonstrate the impact of inductive effects on both BQ reactivity and allergenicity, and suggest the potential utility of chemical reactivity data for electrophilic allergen identification and potency ranking. PMID:26612505

Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions

PubMed Central

Jackson, Paul A.; Widen, John C.; Harki, Daniel A.; Brummond, Kay M.

2017-01-01

Although Michael acceptors display a potent and broad spectrum of bioactivity, they have largely been ignored in drug discovery because of their presumed indiscriminate reactivity. As such, a dearth of information exists relevant to the thiol reactivity of natural products and their analogs possessing this moiety. In the midst of recently approved acrylamide-containing drugs, it is clear that a good understanding of the hetero-Michael addition reaction and the relative reactivities of biological thiols with Michael acceptors under physiological conditions is needed for the design and use of these compounds as biological tools and potential therapeutics. This perspective provides information that will contribute to this understanding, such as kinetics of thiol addition reactions, bioactivities, as well as steric and electronic factors that influence the electrophilicity and reversibility of Michael acceptors. This perspective is focused on α,β-unsaturated carbonyls given their preponderance in bioactive natural products. PMID:27996267

Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

PubMed

Smith, M Ryan; Vayalil, Praveen K; Zhou, Fen; Benavides, Gloria A; Beggs, Reena R; Golzarian, Hafez; Nijampatnam, Bhavitavya; Oliver, Patsy G; Smith, Robin A J; Murphy, Michael P; Velu, Sadanandan E; Landar, Aimee

2016-08-01

Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study demonstrates for the first time that mitochondrial thiol modification inhibits metabolism via inhibition of both aconitase and GAC in a breast cancer cell model. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Mucoadhesive polymers: Synthesis and in vitro characterization of thiolated poly(vinyl alcohol).

PubMed

Suchaoin, Wongsakorn; Pereira de Sousa, Irene; Netsomboon, Kesinee; Rohrer, Julia; Hoffmann Abad, Patricia; Laffleur, Flavia; Matuszczak, Barbara; Bernkop-Schnürch, Andreas

2016-04-30

The aim of this study was to synthesize thiolated poly(vinyl alcohol) (PVA) and to evaluate its mucoadhesive properties. Thiourea and 3-mercaptopropionic acid were utilized in order to obtain thiolated PVAs, namely, TPVA1 and TPVA2, respectively. TPVA1 and TPVA2 displayed 130.44 ± 14.99 and 958.35 ± 155.27 μmol immobilized thiol groups per gram polymer, respectively, which were then evaluated regarding reactivity of thiol groups, swelling behavior and mucoadhesive properties. Both thiolated PVAs exhibited the highest reactivity at pH 8.0 whereas more than 95% of free thiol groups were preserved at pH 5.0. Thiolation of PVA decelerated water uptake and prolonged disintegration time of test discs compared to unmodified PVA. Contact time of TPVA1- and TPVA2-based test discs on porcine intestinal mucosa was 3.2- and 15.8-fold prolonged, respectively, in comparison to non-thiolated PVA as measured by rotating cylinder method. According to tensile studies on mucosa, the total work of adhesion (TWA) and the maximum detachment force (MDF) were increased when compared to PVA. Furthermore, thiolated PVAs preserved higher percentage of viable cells compared to unmodified PVA within 24h as evaluated by MTT assay. Accordingly, thiolated PVA represents a novel excipient that can likely improve the mucoadhesive properties of various pharmaceutical formulations. Copyright © 2016 Elsevier B.V. All rights reserved.

Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces.

PubMed

Neves-Petersen, Maria Teresa; Snabe, Torben; Klitgaard, Søren; Duroux, Meg; Petersen, Steffen B

2006-02-01

Photonic induced immobilization is a novel technology that results in spatially oriented and spatially localized covalent coupling of biomolecules onto thiol-reactive surfaces. Immobilization using this technology has been achieved for a wide selection of proteins, such as hydrolytic enzymes (lipases/esterases, lysozyme), proteases (human plasminogen), alkaline phosphatase, immunoglobulins' Fab fragment (e.g., antibody against PSA [prostate specific antigen]), Major Histocompability Complex class I protein, pepsin, and trypsin. The reaction mechanism behind the reported new technology involves "photonic activation of disulfide bridges," i.e., light-induced breakage of disulfide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol-reactive surfaces (see Fig. 1). Interestingly, the spatial proximity of aromatic residues and disulfide bridges in proteins has been preserved throughout molecular evolution. The new photonic-induced method for immobilization of proteins preserves the native structural and functional properties of the immobilized protein, avoiding the use of one or more chemical/thermal steps. This technology allows for the creation of spatially oriented as well as spatially defined multiprotein/DNA high-density sensor arrays with spot size of 1 microm or less, and has clear potential for biomedical, bioelectronic, nanotechnology, and therapeutic applications.

Hydrogen sulfide deactivates common nitrobenzofurazan-based fluorescent thiol labeling reagents.

PubMed

Montoya, Leticia A; Pluth, Michael D

2014-06-17

Sulfhydryl-containing compounds, including thiols and hydrogen sulfide (H2S), play important but differential roles in biological structure and function. One major challenge in separating the biological roles of thiols and H2S is developing tools to effectively separate the reactivity of these sulfhydryl-containing compounds. To address this challenge, we report the differential responses of common electrophilic fluorescent thiol labeling reagents, including nitrobenzofurazan-based scaffolds, maleimides, alkylating agents, and electrophilic aldehydes, toward cysteine and H2S. Although H2S reacted with all of the investigated scaffolds, the photophysical response to each scaffold was significantly different. Maleimide-based, alkylating, and aldehydic thiol labeling reagents provided a diminished fluorescence response when treated with H2S. By contrast, nitrobenzofurazan-based labeling reagents were deactivated by H2S addition. Furthermore, the addition of H2S to thiol-activated nitrobenzofurazan-based reagents reduced the fluorescence signal, thus establishing the incompatibility of nitrobenzofurazan-based thiol labeling reagents in the presence of H2S. Taken together, these studies highlight the differential reactivity of thiols and H2S toward common thiol-labeling reagents and suggest that sufficient care must be taken when labeling or measuring thiols in cellular environments that produce H2S due to the potential for both false-positive and eroded responses.

Synthesis and in vitro characterization of a novel S-protected thiolated alginate.

PubMed

Hauptstein, Sabine; Dezorzi, Stefanie; Prüfert, Felix; Matuszczak, Barbara; Bernkop-Schnürch, Andreas

2015-06-25

The object of this study was to synthesize and characterize a novel S-protected thiolated polymer with a high degree of modification. In this regard, an alginate-cysteine and an alginate-cysteine-2-mercaptonicotinic acid conjugate were synthesized. To achieve a high coupling rate of the thiol group bearing ligand cysteine to the polymer, the carbohydrate was activated by an oxidative ring opening with sodium periodate followed by a reductive amination to bind the primary amino group of cysteine to resulting reactive aldehyde groups. The obtained thiolated polymer displayed 1561±130μmol thiol groups per gram polymer. About one third of these thiol groups were S-protected by the implementation of a thiol bearing aromatic protection group via disulfide bond formation. Test tablets of both modified polymers showed improved stability against oxidation in aqueous environment compared to the unmodified alginate and exhibit higher water-uptake capacity. Rheological investigations revealed an increased viscosity of the S-protected thiolated polymer whereat the thiolated non S-protected polymer showed gelling properties after the addition of hydrogen peroxide. The mucoadhesive properties could be improved significantly for both derivatives and no alteration in biocompatibility tested on Caco-2 cell monolayer employing an MTT assay could be detected after modification. According to these results, both new derivatives seem promising for various applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Necrotic and apoptotic cell death induced by Captan on Saccharomyces cerevisiae.

PubMed

Scariot, Fernando J; Jahn, Luciane; Delamare, Ana Paula L; Echeverrigaray, Sergio

2017-08-01

Captan is one of the most widely used broad-spectrum fungicide applied to control several early and late diseases of grapes, apples, and other fruits and vegetables, and as other phthalimide fungicides is defined as a multisite compound with thiol-reactivity. Captan can affect non-target organisms as yeasts, modifying microbial populations and fermentation processes. In this study, we asked whether Captan thiol-reactivity and other mechanisms are involved in acute Captan-induced cell death on aerobic growing Saccharomyces cerevisiae. Thus for, we analyze cellular protein and non-protein thiols, cell membrane integrity, reactive oxygen species accumulation, phosphatidylserine externalization, and apoptotic mutants behavior. The results showed that when submitted to acute Captan treatment most cells lost their membrane integrity and died by necrosis due to Captan reaction with thiols. However, part of the cells, even maintaining their membrane integrity, lost their culture ability. These cells showed an apoptotic behavior that may be the result of non-protein thiol depletion and consequent increase of reactive oxygen species (ROS). ROS accumulation triggers a metacaspase-dependent apoptotic cascade, as shown by the higher viability of the yca1-deleted mutant. Together, necrosis and apoptosis are responsible for the high mortality detected after acute Captan treatment of aerobically growing cells of S. cerevisiae.

Furoxans (Oxadiazole-4 N-oxides) with Attenuated Reactivity are Neuroprotective, Cross the Blood Brain Barrier, and Improve Passive Avoidance Memory.

PubMed

Horton, Austin; Nash, Kevin; Tackie-Yarboi, Ethel; Kostrevski, Alexander; Novak, Adam; Raghavan, Aparna; Tulsulkar, Jatin; Alhadidi, Qasim; Wamer, Nathan; Langenderfer, Bryn; Royster, Kalee; Ducharme, Maxwell; Hagood, Katelyn; Post, Megan; Shah, Zahoor A; Schiefer, Isaac T

2018-05-07

Nitric oxide (NO) mimetics and other agents capable of enhancing NO/cGMP signaling have demonstrated efficacy as potential therapies for Alzheimer's disease. A group of thiol-dependent NO mimetics known as furoxans may be designed to exhibit attenuated reactivity to provide slow onset NO effects. The present study describes the design, synthesis, and evaluation of a furoxan library resulting in the identification of a prototype furoxan, 5a, which was profiled for use in the central nervous system. Furoxan 5a demonstrated negligible reactivity toward generic cellular thiols under physiological conditions. Nonetheless, cGMP-dependent neuroprotection was observed, and 5a (20 mg/kg) reversed cholinergic memory deficits in a mouse model of passive avoidance fear memory. Importantly, 5a can be prepared as a pharmaceutically acceptable salt and is observed in the brain 12 h after oral administration, suggesting potential for daily dosing and excellent metabolic stability. Continued investigation into furoxans as attenuated NO mimetics for the CNS is warranted.

Mass Spectrometry in Studies of Protein Thiol Chemistry and Signaling: Opportunities and Caveats

PubMed Central

Devarie Baez, Nelmi O.; Reisz, Julie A.; Furdui, Cristina M.

2014-01-01

Mass spectrometry (MS) has become a powerful and widely utilized tool in the investigation of protein thiol chemistry, biochemistry, and biology. Very early biochemical studies of metabolic enzymes have brought to light the broad spectrum of reactivity profiles that distinguish cysteine thiols with functions in catalysis and protein stability from other cysteine residues in proteins. The development of MS methods for the analysis of proteins using electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) coupled with the emergence of high-resolution mass analyzers have been instrumental in advancing studies of thiol modifications, both in single proteins and within the cellular context. This article reviews MS instrumentation and methods of analysis employed in investigations of thiols and their reactivity toward a range of small biomolecules. A selected number of studies are detailed to highlight the advantages brought about by the MS technologies along with the caveats associated with these analyses. PMID:25261734

Thiol trapping and metabolic redistribution of sulfur metabolites enable cells to overcome cysteine overload

PubMed Central

Deshpande, Anup Arunrao; Bhatia, Muskan; Laxman, Sunil; Bachhawat, Anand Kumar

2017-01-01

Cysteine is an essential requirement in living organisms. However, due to its reactive thiol side chain, elevated levels of intracellular cysteine can be toxic and therefore need to be rapidly eliminated from the cellular milieu. In mammals and many other organisms, excess cysteine is believed to be primarily eliminated by the cysteine dioxygenase dependent oxidative degradation of cysteine, followed by the removal of the oxidative products. However, other mechanisms of tackling excess cysteine are also likely to exist, but have not thus far been explored. In this study, we use Saccharomyces cerevisiae, which naturally lacks a cysteine dioxygenase, to investigate mechanisms for tackling cysteine overload. Overexpressing the high affinity cysteine transporter, YCT1, enabled yeast cells to rapidly accumulate high levels of intracellular cysteine. Using targeted metabolite analysis, we observe that cysteine is initially rapidly interconverted to non-reactive cystine in vivo. A time course revealed that cells systematically convert excess cysteine to inert thiol forms; initially to cystine, and subsequently to cystathionine, S-Adenosyl-L-homocysteine (SAH) and S-Adenosyl L-methionine (SAM), in addition to eventually accumulating glutathione (GSH) and polyamines. Microarray based gene expression studies revealed the upregulation of arginine/ornithine biosynthesis a few hours after the cysteine overload, and suggest that the non-toxic, non-reactive thiol based metabolic products are eventually utilized for amino acid and polyamine biogenesis, thereby enabling cell growth. Thus, cells can handle potentially toxic amounts of cysteine by a combination of thiol trapping, metabolic redistribution to non-reactive thiols and subsequent consumption for anabolism. PMID:28435838

Study of Highly Selective and Efficient Thiol Derivatization using Selenium Reagents by Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Kehua; Zhang, Yun W.; Tang, Bo

2010-08-15

Biological thiols are critical physiological components and their detection often involves derivatization. This paper reports a systemic mass spectrometry (MS) investigation of the cleavage of Se-N bond by thiol to form a new Se-S bond, the new selenium chemistry for thiol labeling. Our data shows that the reaction is highly selective, rapid, reversible and efficient. For instance, among twenty amino acids, only cysteine was found to be reactive with Se-N containing reagents and the reaction takes place in seconds. By adding dithiothreitol (DTT), the newly formed Se-S bond of peptides/proteins can be reduced back to free thiol. The high selectivitymore » and excellent reversibility of the reaction provide potential of using this chemistry for selective identification of thiol compounds or enriching and purifying thiol peptides/proteins. In addition, the derivatized thiol peptides have interesting dissociation behavior, which is tunable using different selenium reagents. For example, by introducing an adjacent nucleophilic group into the selenium reagent in the case of using ebselen, the reaction product of ebselen with glutathione (GSH) is easy to lose the selenium tag upon collision-induced dissociation (CID), which is useful to "fish out" those peptides containing free cysteine residues by precursor ion scan. By contrast, the selenium tag of N-(phenylseleno) phthalimide reagent can be stable and survive in CID process, which would be of value in pinpointing thiol location using a top-down proteomic approach. Also, the high conversion yield of the reaction allows the counting of total number of thiol in proteins. We believe that ebselen or N-(phenylseleno) phthalimide as tagging thiol-protein reagents will have important applications in both qualitative and quantitative analysis of different thiol-proteins derived from living cells by MS method.« less

Bifunctional Diaminoterephthalate Fluorescent Dye as Probe for Cross-Linking Proteins.

PubMed

Wallisch, Melanie; Sulmann, Stefan; Koch, Karl-Wilhelm; Christoffers, Jens

2017-05-11

Diaminoterephthalates are fluorescent dyes and define scaffolds, which can be orthogonally functionalized at their two carboxylate residues with functional residues bearing task specific reactive groups. The synthesis of monofunctionalized dyes with thiol groups for surface binding, an azide for click chemistry, and a biotinoylated congener for streptavidin binding is reported. Two bifunctionalized dyes were prepared: One with an azide for click chemistry and a biotin for streptavidin binding, the other with a maleimide for reaction with thiol and a cyclooctyne moiety for ligation with copper-free click chemistry. In general, the compounds are red to orange, fluorescent materials with an absorption at about 450 nm and an emission at 560 nm with quantum yields between 2-41 %. Of particular interest is the maleimide-functionalized compound, which shows low fluorescence quantum yield (2 %) by itself. After addition of a thiol, the fluorescence is "turned on"; quantum yield 41 %. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis of Single-walled Carbon Nanotubes Coated with Thiol-reactive Gel via Emulsion Polymerization.

PubMed

Nagai, Yukiko; Tsutsumi, Yusuke; Nakashima, Naotoshi; Fujigaya, Tsuyohiko

2018-06-15

Single-walled carbon nanotubes (SWNTs) have unique near-infrared absorption and photoemission properties that are attractive for in vivo biological applications such as photothermal cancer treatment and bioimaging. Therefore, a smart functionalization strategy for SWNTs to create biocompatible surfaces and introduce various ligands to target active cancer cells without losing the unique optical properties of the SWNTs is strongly desired. This paper reports the de-sign and synthesis of a SWNT/gel hybrid containing maleimide groups, which react with various thiol compounds through Michael addition reactions. In this hybrid, the method called carbon nanotube micelle polymerization was used to non-covalently modify the surface of SWNTs with a cross-linked polymer gel layer. This method can form an extremely stable gel layer on SWNTs; such stability is essential for in vivo biological applications. The monomer used to form the gel layer contained a maleimide group, which was protected with furan in endo-form. The resulting hybrid was treated in water to induce deprotection via retro Diels-Alder reaction and then functionalized with thiol com-pounds through Michael addition. The functionalization of the hybrid was explored using a thiol-containing fluores-cent dye as a model thiol and the formation of the SWNT-dye conjugate was confirmed by energy transfer from the dye to SWNTs. Our strategy offers a promising SWNT-based platform for biological functionalization for cancer targeting, imaging, and treatment.

Mitochondrial respiratory chain complexes as sources and targets of thiol-based redox-regulation.

PubMed

Dröse, Stefan; Brandt, Ulrich; Wittig, Ilka

2014-08-01

The respiratory chain of the inner mitochondrial membrane is a unique assembly of protein complexes that transfers the electrons of reducing equivalents extracted from foodstuff to molecular oxygen to generate a proton-motive force as the primary energy source for cellular ATP-synthesis. Recent evidence indicates that redox reactions are also involved in regulating mitochondrial function via redox-modification of specific cysteine-thiol groups in subunits of respiratory chain complexes. Vice versa the generation of reactive oxygen species (ROS) by respiratory chain complexes may have an impact on the mitochondrial redox balance through reversible and irreversible thiol-modification of specific target proteins involved in redox signaling, but also pathophysiological processes. Recent evidence indicates that thiol-based redox regulation of the respiratory chain activity and especially S-nitrosylation of complex I could be a strategy to prevent elevated ROS production, oxidative damage and tissue necrosis during ischemia-reperfusion injury. This review focuses on the thiol-based redox processes involving the respiratory chain as a source as well as a target, including a general overview on mitochondria as highly compartmentalized redox organelles and on methods to investigate the redox state of mitochondrial proteins. This article is part of a Special Issue entitled: Thiol-Based Redox Processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Detoxification of toxins by bacillithiol in Staphylococcus aureus

PubMed Central

Newton, Gerald L.; Fahey, Robert C.

2012-01-01

Bacillithiol (BSH), an α-anomeric glycoside of l-cysteinyl-d-glucosaminyl-l-malate, is a major low-molecular-mass thiol found in bacteria such as Bacillus sp., Staphylococcus aureus and Deinococcus radiodurans. Like other low-molecular-mass thiols such as glutathione and mycothiol, BSH is likely to be involved in protection against environmental toxins including thiol-reactive antibiotics. We report here a BSH-dependent detoxification mechanism in S. aureus. When S. aureus Newman strain was treated with monobromobimane and monochlorobimane, the cellular BSH was converted to the fluorescent S-conjugate BS-bimane. A bacillithiol conjugate amidase activity acted upon the BS-bimane to produce Cys-bimane, which was then acetylated by an N-acetyltransferase to generate N-acetyl-Cys-bimane, a mercapturic acid. An S. aureus mutant lacking BSH did not produce mercapturic acid when treated with monobromobimane and monochlorobimane, confirming the involvement of bacillithiol. Furthermore, treatment of S. aureus Newman with rifamycin, the parent compound of the first-line anti-tuberculosis drug, rifampicin, indicated that this thiol-reactive antibiotic is also detoxified in a BSH-dependent manner, since mercapturic acids of rifamycin were observed in the culture medium. These data indicate that toxins and thiol-reactive antibiotics are detoxified to less potent mercapturic acids in a BSH-dependent manner and then exported out of the cell in S. aureus. PMID:22262099

Thiol-Reactive Star Polymers Display Enhanced Association with Distinct Human Blood Components.

PubMed

Glass, Joshua J; Li, Yang; De Rose, Robert; Johnston, Angus P R; Czuba, Ewa I; Khor, Song Yang; Quinn, John F; Whittaker, Michael R; Davis, Thomas P; Kent, Stephen J

2017-04-12

Directing nanoparticles to specific cell types using nonantibody-based methods is of increasing interest. Thiol-reactive nanoparticles can enhance the efficiency of cargo delivery into specific cells through interactions with cell-surface proteins. However, studies to date using this technique have been largely limited to immortalized cell lines or rodents, and the utility of this technology on primary human cells is unknown. Herein, we used RAFT polymerization to prepare pyridyl disulfide (PDS)-functionalized star polymers with a methoxy-poly(ethylene glycol) brush corona and a fluorescently labeled cross-linked core using an arm-first method. PDS star polymers were examined for their interaction with primary human blood components: six separate white blood cell subsets, as well as red blood cells and platelets. Compared with control star polymers, thiol-reactive nanoparticles displayed enhanced association with white blood cells at 37 °C, particularly the phagocytic monocyte, granulocyte, and dendritic cell subsets. Platelets associated with more PDS than control nanoparticles at both 37 °C and on ice, but they were not activated in the duration examined. Association with red blood cells was minor but still enhanced with PDS nanoparticles. Thiol-reactive nanoparticles represent a useful strategy to target primary human immune cell subsets for improved nanoparticle delivery.

The Chemical Basis of Thiol Addition to Nitro-conjugated Linoleic Acid, a Protective Cell-signaling Lipid*♦

PubMed Central

Turell, Lucía; Vitturi, Darío A.; Coitiño, E. Laura; Lebrato, Lourdes; Möller, Matías N.; Sagasti, Camila; Salvatore, Sonia R.; Woodcock, Steven R.; Alvarez, Beatriz; Schopfer, Francisco J.

2017-01-01

Nitroalkene fatty acids are formed in vivo and exert protective and anti-inflammatory effects via reversible Michael addition to thiol-containing proteins in key signaling pathways. Nitro-conjugated linoleic acid (NO2-CLA) is preferentially formed, constitutes the most abundant nitrated fatty acid in humans, and contains two carbons that could potentially react with thiols, modulating signaling actions and levels. In this work, we examined the reactions of NO2-CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine, and β-mercaptoethanol) and human serum albumin. Reactions followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2-CLA located on the β- and δ-carbons with respect to the nitro group. The differential reactivity was confirmed by computational modeling of the electronic structure. The rates (kon and koff) and equilibrium constants for both reactions were determined for different thiols. LC-UV-Visible and LC-MS analyses showed that the fast reaction corresponds to β-adduct formation (the kinetic product), while the slow reaction corresponds to the formation of the δ-adduct (the thermodynamic product). The pH dependence of the rate constants, the correlation between intrinsic reactivity and thiol pKa, and the absence of deuterium solvent kinetic isotope effects suggested stepwise mechanisms with thiolate attack on NO2-CLA as rate-controlling step. Computational modeling supported the mechanism and revealed additional features of the transition states, anionic intermediates, and final neutral products. Importantly, the detection of cysteine-δ-adducts in human urine provided evidence for the biological relevance of this reaction. Finally, human serum albumin was found to bind NO2-CLA both non-covalently and to form covalent adducts at Cys-34, suggesting potential modes for systemic distribution. These results provide new insights into the chemical basis of NO2-CLA signaling actions. PMID:27923813

Carvedilol: relation between antioxidant activity and inhibition of the mitochondrial permeability transition.

PubMed

Oliveira, Paulo J; Esteves, Telma; Rolo, Anabela P; Monteiro, Pedro; Gonçalves, Lino; Palmeira, Carlos M; Moreno, António J

2003-01-01

The mitochondrial permeability transition (MPT) is an event related to severe oxidative stress (for example, during myocardial ischemia and reperfusion) and excessive mitochondrial calcium accumulation, also being implicated in cell death. In this study, we compared the effect of carvedilol on the cardiac MPT induced by calcium and phosphate (Ca/Pi) and calcium/carboxyatractyloside (Ca/Catr). Oxidative stress plays a major role in MPT induction by Ca/Pi, leading to the oxidation of protein thiol groups, in contrast with Ca/Catr, where such oxidation is secondary to MPT induction and is not caused by oxidative stress. Mitochondria were isolated from rat hearts and parameters related to MPT induction were evaluated (n = 5 for each inducer): mitochondrial swelling and oxidation of protein thiol groups (both measured by spectrophotometry). Using Ca/Pi, carvedilol protected mitochondria from MPT induction, particularly in its high conductance form. Its effect was demonstrated by analyzing the decrease in mitochondrial swelling amplitude. Simultaneously, we observed inhibition of protein thiol group oxidation (p < 0.001). By contrast, carvedilol did not show any protective effect with Ca/Catr. Carvedilol was only effective against the MPT when the oxidation of protein thiol groups was the cause and not the consequence of the MPT phenomenon. The results clearly show that during myocardial aggressions (ischemia and reperfusion, for example), the protective effect of carvedilol is primarily due to an antioxidant mechanism, inhibiting the production and effects of reactive oxygen species.

Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jong Seo; Dai, Ziyu; Aryal, Uma K.

2013-06-17

Proteolytic processing is a ubiquitous, irreversible posttranslational modification that plays an important role in cellular regulation in all living organisms. Herein we report a resin-assisted positive selection method for specifically enriching protein N-terminal peptides to facilitate the characterization of proteolytic processing events by liquid chromatography-tandem mass spectrometry. In this approach, proteins are initially reduced and alkylated and their lysine residues are converted to homoarginines. Then, protein N-termini are selectively converted to reactive thiol groups. We demonstrate that these sequential reactions were achieved with nearly quantitative efficiencies. Thiol-containing N-terminal peptides are then captured (>98% efficiency) by a thiol-affinity resin, a significantmore » improvement over the traditional avidin/biotin enrichment. Application to cell lysates of Aspergillus niger, a filamentous fungus of interest for biomass degradation, enabled the identification of 1672 unique protein N-termini and proteolytic cleavage sites from 690 unique proteins.« less

Development of Selective Colorimetric Probes for Hydrogen Sulfide Based on Nucleophilic Aromatic Substitution

PubMed Central

Montoya, Leticia A.; Pearce, Taylor F.; Hansen, Ryan J.; Zakharov, Lev N.; Pluth, Michael D.

2013-01-01

Hydrogen sulfide is an important biological signalling molecule and an important environmental target for detection. A major challenge in developing H2S detection methods is separating the often similar reactivity of thiols and other nucleophiles from H2S. To address this need, the nucleophilic aromatic substitution (SNAr) reaction of H2S with electron-poor aromatic electrophiles was developed as a strategy to separate H2S and thiol reactivity. Treatment of aqueous solutions of nitrobenzofurazan (7-nitro-1,2,3-benzoxadiazole, NBD) thioethers with H2S resulted in thiol extrusion and formation of nitrobenzofurazan thiol (λmax = 534 nm). This reactivity allows for unwanted thioether products to be converted to the desired nitrobenzofurazan thiol upon reaction with H2S. The scope of the reaction was investigated using a Hammett linear free energy relationship study, and the determined ρ = +0.34 is consistent with the proposed SN2Ar reaction mechanism. The efficacy of the developed probes was demonstrated in buffer and in serum with associated sub-micromolar detection limits as low as 190 nM (buffer) and 380 nM (serum). Furthermore, the sigmoidal response of nitrobenzofurazan electrophiles with H2S can be fit to accurately quantify H2S. The developed detection strategy offers a manifold for H2S detection that we foresee being applied in various future applications. PMID:23735055

Development of selective colorimetric probes for hydrogen sulfide based on nucleophilic aromatic substitution.

PubMed

Montoya, Leticia A; Pearce, Taylor F; Hansen, Ryan J; Zakharov, Lev N; Pluth, Michael D

2013-07-05

Hydrogen sulfide is an important biological signaling molecule and an important environmental target for detection. A major challenge in developing H2S detection methods is separating the often similar reactivity of thiols and other nucleophiles from H2S. To address this need, the nucleophilic aromatic substitution (SNAr) reaction of H2S with electron-poor aromatic electrophiles was developed as a strategy to separate H2S and thiol reactivity. Treatment of aqueous solutions of nitrobenzofurazan (7-nitro-1,2,3-benzoxadiazole, NBD) thioethers with H2S resulted in thiol extrusion and formation of nitrobenzofurazan thiol (λmax = 534 nm). This reactivity allows for unwanted thioether products to be converted to the desired nitrobenzofurazan thiol upon reaction with H2S. The scope of the reaction was investigated using a Hammett linear free energy relationship study, and the determined ρ = +0.34 is consistent with the proposed SN2Ar reaction mechanism. The efficacy of the developed probes was demonstrated in buffer and in serum with associated submicromolar detection limits as low as 190 nM (buffer) and 380 nM (serum). Furthermore, the sigmoidal response of nitrobenzofurazan electrophiles with H2S can be fit to accurately quantify H2S. The developed detection strategy offers a manifold for H2S detection that we foresee being applied in various future applications.

DPPH and oxygen free radicals as pro-oxidant of biomolecules.

PubMed

Letelier, María Eugenia; Molina-Berríos, Alfredo; Cortés-Troncoso, Juan; Jara-Sandoval, José; Holst, Marianne; Palma, Karina; Montoya, Margarita; Miranda, Dante; González-Lira, Víctor

2008-03-01

Numerous investigations exist about the alterations that oxygen free radicals can provoke on biomolecules; these modifications can be prevented and/or reversed by different antioxidants agents. On the other hand, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), a stable nitrogen synthetic radical, is used to evaluate the antioxidant capacity of medicinal herbal products; however, the structural changes that this radical provoke on the herbal active principles are not clear yet. In this work, we compared the redox reactivity of oxygen free radicals and DPPH radical on phospholipids and protein thiol groups present in rat liver microsomes. Cu2+/ascorbate was used as generator system of oxygen free radical and as antioxidant, an extract of Buddleja globosa's leaves. Cu2+/ascorbate provoked microsomal lipid peroxidation, microsomal thiols oxidation and oxygen consumption; all of these phenomena were inhibited by B. globosa extract. On the other hand, DPPH was bleached in different extension by the herbal extract and phosphatidyl choline; beside, DPPH decreased microsomal thiols content, but this phenomenon were not prevented by the herbal extract. Furthermore, DPPH did not induce oxygen consumption and neither modified the oxygen consumption induced by Cu2+/ascorbate. Distinct redox mechanisms may explain the differences between the reactivity of DPPH and oxygen free radicals on biomolecules, which is discussed.

Micrometer sized immobilization of protein molecules onto quartz, silicium and gold.

NASA Astrophysics Data System (ADS)

Petersen, Steffen B.; Neves-Petersen, Maria Teresa; Klitgaard, Søren; Duroux, Meg Crookshanks

2006-02-01

We demonstrate that ultraviolet light can be used to make sterically oriented covalent immobilization of a large variety of protein molecules onto either gold or thiolated quartz or silicium. The reaction mechanism behind the reported new technology involves light induced breakage of disulphide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol reactive surfaces. The protein molecules in general retain their function. The size of the immobilization spot is determined by the dimension of the UV beam. In principle, the spot size may be as small as 1 micrometer or less. We have developed the necessary technology for preparing large protein arrays of enzymes and fragments of monoclonal antibodies. Dedicated Image Processing Software has been developed for making quality assessment of the protein arrays. A multitude of important application areas such as drug carriers and drug delivery, bioelectronics, carbon nanotubes, nanoparticles as well as protein glue are discussed.

Gel-based methods in redox proteomics.

PubMed

Charles, Rebecca; Jayawardhana, Tamani; Eaton, Philip

2014-02-01

The key to understanding the full significance of oxidants in health and disease is the development of tools and methods that allow the study of proteins that sense and transduce changes in cellular redox. Oxidant-reactive deprotonated thiols commonly operate as redox sensors in proteins and a variety of methods have been developed that allow us to monitor their oxidative modification. This outline review specifically focuses on gel-based methods used to detect, quantify and identify protein thiol oxidative modifications. The techniques we discuss fall into one of two broad categories. Firstly, methods that allow oxidation of thiols in specific proteins or the global cellular pool to be monitored are discussed. These typically utilise thiol-labelling reagents that add a reporter moiety (e.g. affinity tag, fluorophore, chromophore), in which loss of labelling signifies oxidation. Secondly, we outline methods that allow specific thiol oxidation states of proteins (e.g. S-sulfenylation, S-nitrosylation, S-thionylation and interprotein disulfide bond formation) to be investigated. A variety of different gel-based methods for identifying thiol proteins that are sensitive to oxidative modifications have been developed. These methods can aid the detection and quantification of thiol redox state, as well as identifying the sensor protein. By understanding how cellular redox is sensed and transduced to a functional effect by protein thiol redox sensors, this will help us better appreciate the role of oxidants in health and disease. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. Copyright © 2013 Elsevier B.V. All rights reserved.

Reactivities of some thiol collectors and their interactions with Ag (+1) ion by molecular modeling

NASA Astrophysics Data System (ADS)

Yekeler, Hulya; Yekeler, Meftuni

2004-09-01

The most commonly used collectors for sulfide minerals in the mining industry are the thiol collectors for the recovery of these minerals from their associated gangues by froth flotation. For this reason, a great deal of attention has been paid to understand the attachment mechanism of thiol collectors to metal sulfide surfaces. The density functional theory (DFT) calculations at the B3LYP/3-21G* and B3LYP/6-31++G** levels were employed to propose the flotation responses of these thiol collectors, namely, diethyl dithiocarbamate, ethyl dithiocarbamate, ethyl dithiocarbonate, ethyl trithiocarbonate and ethyl dithiophosphate ions, and to study the interaction energies of these collectors with Ag (+1) ion in connection to acanthite (Ag 2S) mineral. The calculated interaction energies, Δ E, were interpreted in terms of the highest occupied molecular orbital (HOMO) energies of the isolated collector ions. The results show that the HOMOs are strongly localized to the sulfur atoms and the HOMO energies can be used as a reactivity descriptor for the flotation ability of the thiol collectors. Using the HOMO and Δ E energies, the reactivity order of the collectors is found to be (C 2H 5) 2NCS 2- > C 2H 5NHCS 2- > C 2H 5OCS 2- > C 2H 5SCS 2- > (C 2H 5O)(OH)PS 2-. The theoretically obtained results are in good agreement with the experimental data reported.

Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies.

PubMed

Terrill, Jessica R; Radley-Crabb, Hannah G; Iwasaki, Tomohito; Lemckert, Frances A; Arthur, Peter G; Grounds, Miranda D

2013-09-01

The muscular dystrophies comprise more than 30 clinical disorders that are characterized by progressive skeletal muscle wasting and degeneration. Although the genetic basis for many of these disorders has been identified, the exact mechanism for pathogenesis generally remains unknown. It is considered that disturbed levels of reactive oxygen species (ROS) contribute to the pathology of many muscular dystrophies. Reactive oxygen species and oxidative stress may cause cellular damage by directly and irreversibly damaging macromolecules such as proteins, membrane lipids and DNA; another major cellular consequence of reactive oxygen species is the reversible modification of protein thiol side chains that may affect many aspects of molecular function. Irreversible oxidative damage of protein and lipids has been widely studied in Duchenne muscular dystrophy, and we have recently identified increased protein thiol oxidation in dystrophic muscles of the mdx mouse model for Duchenne muscular dystrophy. This review evaluates the role of elevated oxidative stress in Duchenne muscular dystrophy and other forms of muscular dystrophies, and presents new data that show significantly increased protein thiol oxidation and high levels of lipofuscin (a measure of cumulative oxidative damage) in dysferlin-deficient muscles of A/J mice at various ages. The significance of this elevated oxidative stress and high levels of reversible thiol oxidation, but minimal myofibre necrosis, is discussed in the context of the disease mechanism for dysferlinopathies, and compared with the situation for dystrophin-deficient mdx mice. © 2013 The Authors Journal compilation © 2013 FEBS.

Immobilization of Protein A on SAMs for the elaboration of immunosensors.

PubMed

Briand, Elisabeth; Salmain, Michèle; Compère, Chantal; Pradier, Claire-Marie

2006-12-01

Binary mixtures of 11-mercaptoundecanoic acid (MUA) and other thiols of various lengths and terminal functions were chemisorbed on gold-coated surfaces via S-Au bonds to form mixed self-assembled monolayers (SAMs). Several values of the mole fraction of MUA in the thiol mixtures were tested and the structure and composition of the resulted thin films were characterized by X-ray photoelectron spectroscopy (XPS) and polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS). The results made it clear that co-adsorption of MUA with thiols of similar chain length led to well-ordered monolayers whereas the co-adsorption of MUA with shorter thiols yielded less crystalline-like thin films, but with more reactive carboxylic acid terminal groups. This criterion appeared decisive for efficient covalent binding of Staphylococcus aureus Protein A (PrA), a protein that displays high affinity for the constant fragment (Fc) of antibodies of the IgG type from various mammal species. The ability of immobilized Protein A to recognize and bind a model IgG appeared to be optimal for the mixed SAM of MUA and the short-chain, omega-hydroxythiol 6-mercaptohexanol in the proportion 1-3.

Comparison of inhibition of murine leukaemia cell growth by 9-isothiocyanatoacridine and its cytosine adduct: involvement of thiols.

PubMed

Bajdichova, M; Paulikova, H; Jakubikova, J; Sabolova, D

2007-01-01

Cytotoxicity of two fluorescent acridine derivatives - 9-isothiocyanatoacridine (AcITC) and N-(9-acridinylthiocarbamoyl) cytosine (AcTCC) - a novel acridine compound, were investigated. Both substances have cytotoxic activity against the L1210 cellular line, IC50 values were in the micromolar range. Despite the high reactivity of AcITC towards thiols, its effects on leukemia cells were similar to naturally occurring isothiocyanates. AcITC changed the intracellular level of glutathione (GSH), and induced apoptosis. Arrest of cell cycle (G2/M-phase) was also observed. AcITC primarily reacted with -SH groups on cellular surface, and the study of the interaction of the isotiocyanate with human erythrocyte ghosts confirmed that the plasma membrane was the first place where AcITC bound. AcTCC does not react with cellular thiols; images obtained with fluorescent microscopy confirmed interaction of AcTCC with chromatine. Although AcTCC induced cellular arrest in the G2/M phase, apoptosis was not confirmed.

Redox regulation of mammalian sperm capacitation

PubMed Central

O’Flaherty, Cristian

2015-01-01

Capacitation is a series of morphological and metabolic changes necessary for the spermatozoon to achieve fertilizing ability. One of the earlier happenings during mammalian sperm capacitation is the production of reactive oxygen species (ROS) that will trigger and regulate a series of events including protein phosphorylation, in a time-dependent fashion. The identity of the sperm oxidase responsible for the production of ROS involved in capacitation is still elusive, and several candidates are discussed in this review. Interestingly, ROS-induced ROS formation has been described during human sperm capacitation. Redox signaling during capacitation is associated with changes in thiol groups of proteins located on the plasma membrane and subcellular compartments of the spermatozoon. Both, oxidation of thiols forming disulfide bridges and the increase on thiol content are necessary to regulate different sperm proteins associated with capacitation. Reducing equivalents such as NADH and NADPH are necessary to support capacitation in many species including humans. Lactate dehydrogenase, glucose-6-phospohate dehydrogenase, and isocitrate dehydrogenase are responsible in supplying NAD (P) H for sperm capacitation. Peroxiredoxins (PRDXs) are newly described enzymes with antioxidant properties that can protect mammalian spermatozoa; however, they are also candidates for assuring the regulation of redox signaling required for sperm capacitation. The dysregulation of PRDXs and of enzymes needed for their reactivation such as thioredoxin/thioredoxin reductase system and glutathione-S-transferases impairs sperm motility, capacitation, and promotes DNA damage in spermatozoa leading to male infertility. PMID:25926608

Redox Signaling Regulated by Cysteine Persulfide and Protein Polysulfidation.

PubMed

Kasamatsu, Shingo; Nishimura, Akira; Morita, Masanobu; Matsunaga, Tetsuro; Abdul Hamid, Hisyam; Akaike, Takaaki

2016-12-15

For decades, reactive persulfide species including cysteine persulfide (CysSSH) have been known to exist endogenously in organisms. However, the physiological significance of endogenous persulfides remains poorly understood. That cystathionine β-synthase and cystathionine γ-lyase produced CysSSH from cystine was recently demonstrated. An endogenous sulfur transfer system involving CysSSH evidently generates glutathione persulfide (GSSH) that exists at concentrations greater than 100 μM in vivo. Because reactive persulfide species such as CysSSH and GSSH have higher nucleophilicity than parental cysteine (Cys) and glutathione do, these reactive species exhibit strong scavenging activities against oxidants, e.g., hydrogen peroxide, and electrophiles, which contributes to redox signaling regulation. Also, several papers indicated that various proteins and enzymes have Cys polysulfides including CysSSH at their specific Cys residues, which is called protein polysulfidation. Apart from the redox signaling regulatory mechanism, another plausible function of protein polysulfidation is providing protection for protein thiol residues against irreversible chemical modification caused by oxidants and electrophiles. Elucidation of the redox signaling regulatory mechanism of reactive persulfide species including small thiol molecules and thiol-containing proteins should lead to the development of new therapeutic strategies and drug discoveries for oxidative and electrophilic stress-related diseases.

Reactivation of oxidized PTP1B and PTEN by Thioredoxin 1

PubMed Central

Schwertassek, Ulla; Haque, Aftabul; Krishnan, Navasona; Greiner, Romy; Weingarten, Lars; Dick, Tobias P.; Tonks, Nicholas K.

2014-01-01

The transient inactivation of protein phosphatases contributes to the efficiency and temporal control of kinase-dependent signal transduction. In particular, members of the protein tyrosine phosphatase family are known to undergo reversible oxidation of their active site cysteine. The thiol oxidation step requires activation of co-localized NADPH oxidases and is mediated by locally produced ROS, in particular H2O2. How oxidized phosphatases are returned to the reduced active state is less well studied. Both major thiol reductive systems, the thioredoxin and the glutathione systems, have been implicated in the reactivation of phosphatases. Here, we show that the protein tyrosine phosphatase PTP1B and the dual-specificity phosphatase PTEN are preferentially reactivated by the thioredoxin system. We show that inducible depletion of TRX1 slows down PTEN re-activation in intact living cells. Finally, using a mechanism-based trapping approach we demonstrate direct thiol disulfide exchange between the active sites of thioredoxin and either phosphatase. The application of thioredoxin trapping mutants represents a complementary approach to direct assays of PTP oxidation in elucidating the significance of redox regulation of PTP function in the control of cell signaling. PMID:24976139

Reaction of 2-acetylaminofluorene-N-sulfate with RNA and glutathione: evidence for the generation of two reactive intermediates with different reactivities towards RNA and glutathione.

PubMed

van den Goorbergh, J A; Meerman, J H; de Wit, H; Mulder, G J

1985-11-01

The sulfate ester of N-hydroxy-2-acetylaminofluorene (AAF-N-sulfate) is one of the reactive intermediates of this carcinogen. This ester breaks down spontaneously to a very reactive nitrenium ion, which reacts with nucleophilic groups in protein, DNA, RNA and glutathione (GSH). Reactions involving the nitrenium ion with several nucleophiles under various conditions were studied. The adduct formation to RNA was much higher in Tris-HCI buffer than in phosphate buffer (at pH 7.4), while adduct formation to deoxy-guanosine monomers was the same in both buffers. The presence of 150 mM KCI had the same decreasing effect in both cases. Ionic strength effects may be involved in these phenomena. GSH decreased RNA adduct formation by 20-45%, while other thiols were much more effective. On the other hand, RNA did not decrease the formation of GSH conjugates from AAF-N-sulfate. The decrease in RNA adduct formation by thiols corresponded with an increase in the formation of 2-acetylaminofluorene (AAF) from AAF-N-sulfate, while no N-hydroxy-AAF was formed. These results suggest that two independent reactive intermediates are formed from AAF-N-sulfate, with different reactivities towards RNA and glutathione. Possibly these intermediates are the 'hard' triplet state nitrenium ion and the 'soft' singlet state nitrenium ion. Cysteine, cysteamine and penicillamine were most effective in the inhibition of RNA adduct formation; the extent of inhibition correlated with the extent of AAF formation. The mechanisms involved are discussed.

Thiol/disulfide homeostasis in postmenopausal osteoporosis.

PubMed

Korkmaz, V; Kurdoglu, Z; Alisik, M; Turgut, E; Sezgın, O O; Korkmaz, H; Ergun, Y; Erel, O

2017-04-01

To evaluate the impact of postmenopausal osteoporosis on thiol/disulfide homeostasis. A total of 75 participants were divided into two groups: Group 1 (n = 40) was composed of healthy postmenopausal women, and group 2 (n = 35) was composed of women with postmenopausal osteoporosis. Clinical findings and thiol/disulfide homeostasis were compared between the two groups. The disulfide/native thiol ratio was 8.6% ± 3.6 in group 1 and 12.7% ± 8.4 in group 2 (p = 0.04). The disulfide/native thiol percent ratio was significantly higher in group 2 after adjustment for the years since menopause and age (p < 0.05). The native thiol/total thiol percent ratio was 85.6% ± 4.8 in group 1 and 73.8% ± 24.9 in group 2 (p = 0.01). The native thiol/total thiol percent ratio was significantly lower in group 2 after adjustment for the years since menopause and age (p < 0.05). Thiol/disulfide homeostasis shifted to the disulfide side independent of age and years since menopause in postmenopausal osteoporosis.

Kinetic Resolution of sec-Thiols by Enantioselective Oxidation with Rationally Engineered 5-(Hydroxymethyl)furfural Oxidase.

PubMed

Pickl, Mathias; Swoboda, Alexander; Romero, Elvira; Winkler, Christoph K; Binda, Claudia; Mattevi, Andrea; Faber, Kurt; Fraaije, Marco W

2018-03-05

Various flavoprotein oxidases were recently shown to oxidize primary thiols. Herein, this reactivity is extended to sec-thiols by using structure-guided engineering of 5-(hydroxymethyl)furfural oxidase (HMFO). The variants obtained were employed for the oxidative kinetic resolution of racemic sec-thiols, thus yielding the corresponding thioketones and nonreacted R-configured thiols with excellent enantioselectivities (E≥200). The engineering strategy applied went beyond the classic approach of replacing bulky amino acid residues with smaller ones, as the active site was additionally enlarged by a newly introduced Thr residue. This residue established a hydrogen-bonding interaction with the substrates, as verified in the crystal structure of the variant. These strategies unlocked HMFO variants for the enantioselective oxidation of a range of sec-thiols. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reduced antioxidant capacity and increased subclinical inflammation markers in prepubescent obese children and their relationship with nutritional markers and metabolic parameters.

PubMed

Vehapoglu, Aysel; Turkmen, Serdar; Goknar, Nilufer; Özer, Ömer Faruk

2016-11-01

There are associations between some inflammatory and oxidative markers and obesity in adults, but whether prepubescent children of different weights also have such markers has not been studied. We investigated multiple inflammatory markers and levels of erythrocyte oxidant/antioxidant enzymes in prepubescent children of different weights. Children aged 2-11 years were divided into three groups: 80 were underweight, 90 were obese but otherwise healthy, and 80 were healthy age- and sex-matched children of normal-weight. We analyzed inflammatory markers and the total oxidant status, total antioxidant status (TAS), and total thiol level were also determined, and the oxidative stress index was calculated as an indicator of the degree of oxidative stress. The obese group exhibited higher levels of fasting glucose, insulin, total cholesterol, triglycerides, the homeostatic model assessment of insulin resistance (HOMA-IR), and the homeostatic model assessment of β-cell function (HOMA-β), C-reactive protein (CRP), neutrophils, and neutrophil/lymphocyte ratio (NLR), as well as lower TAS and total thiol levels than the other two groups (all P < 0.001). Moreover, TAS and total thiols were negatively correlated with age in the obese group (r = -0.212, P = 0.001; r = -0.231, P < 0.001, respectively). CRP levels in plasma were positively correlated with the body mass index (BMI), insulin and glucose levels, HOMA-IR, HOMA-β, WBC and neutrophil counts, and the NLR, and were negatively correlated with TAS and total thiol levels in the overall studied population. The coexistence of increased obesity-related subclinical inflammation and decreased antioxidant capacity can be observed even in prepubescence, and may eventually increase the risk of long-term vascular damage.

Impaired Thiol-Disulfide Balance in Acute Brucellosis.

PubMed

Kolgelier, Servet; Ergin, Merve; Demir, Lutfi Saltuk; Inkaya, Ahmet Cagkan; Aktug Demir, Nazlim; Alisik, Murat; Erel, Ozcan

2017-05-24

The objective of this study was to examine a novel profile: thiol-disulfide homeostasis in acute brucellosis. The study included 90 patients with acute brucellosis, and 27 healthy controls. Thiol-disulfide profile tests were analyzed by a recently developed method, and ceruloplasmin levels were determined. Native thiol levels were 256.72 ± 48.20 μmol/L in the acute brucellosis group and 461.13 ± 45.37 μmol/L in the healthy group, and total thiol levels were 298.58 ± 51.78 μmol/L in the acute brucellosis group and 504.83 ± 51.05 μmol/L in the healthy group (p < 0.001, for both). The disulfide/native thiol ratios and disulfide/total thiol ratios were significantly higher, and native thiol/total thiol ratios were significantly lower in patients with acute brucellosis than in the healthy controls (p < 0.001, for all ratios). There were either positive or negative relationships between ceruloplasmin levels and thiol-disulfide parameters. The thiol-disulfide homeostasis was impaired in acute brucellosis. The strong associations between thiol-disulfide parameters and a positive acute-phase reactant reflected the disruption of the balance between the antioxidant and oxidant systems. Since thiol groups act as anti-inflammatory mediators, the alteration in the thiol-disulfide homeostasis may be involved in brucellosis.

Method for detecting the reactivity of chemicals towards peptides as an alternative test method for assessing skin sensitization potential.

PubMed

Cho, Sun-A; Jeong, Yun Hyeok; Kim, Ji Hoon; Kim, Seoyoung; Cho, Jun-Cheol; Heo, Yong; Heo, Young; Suh, Kyung-Do; Shin, Kyeho; An, Susun

2014-02-10

Cosmetics are normally composed of various ingredients. Some cosmetic ingredients can act as chemical haptens reacting toward proteins or peptides of human skin and they can provoke an immunologic reaction, called as skin sensitization. This haptenation process is very important step of inducing skin sensitization and evaluating the sensitizing potentials of cosmetic ingredients is very important for consumer safety. Therefore, animal alternative methods focusing on monitoring haptenation potential are undergoing vigorous research. To examine the further usefulness of spectrophotometric methods to monitor reactivity of chemicals toward peptides for cosmetic ingredients. Forty chemicals (25 sensitizers and 15 non-sensitizers) were reacted with 2 synthetic peptides, e.g., the cysteine peptides (Ac-RFAACAA-COOH) with free thiol group and the lysine peptides (Ac-RFAAKAA-COOH) with free amine group. Unreacted peptides can be detected after incubating with 5,5'-dithiobis-2-nitrobenzoic acid or fluorescamine™ as detection reagents for free thiol and amine group, respectively. Chemicals were categorized as sensitizers when they induced more than 10% depletion of cysteine peptides or more than 30% depletion of lysine peptides. The sensitivity, specificity, and accuracy were 80.0%, 86.7% and 82.5%, respectively. These results demonstrate that spectrophotometric methods can be an easy, fast, and high-throughput screening tools predicting the skin sensitization potential of chemical including cosmetic ingredient. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duan, Jicheng; Gaffrey, Matthew J.; Qian, Wei-Jun

Protein cysteine thiols play a crucial role in redox signaling, regulation of enzymatic activity and protein function, and maintaining redox homeostasis in living systems. The unique chemical reactivity of thiol groups makes cysteine susceptible to oxidative modifications by reactive oxygen and nitrogen species to form a broad array of reversible and irreversible protein post-translational modifications (PTMs). The reversible modifications in particular are one of the major components of redox signaling and are involved in regulation of various cellular processes under physiological and pathological conditions. The biological significance of these redox PTMs in health and diseases has been increasingly recognized. Herein,more » we review the recent advances of quantitative proteomic approaches for investigating redox PTMs in complex biological systems, including the general considerations of sample processing, various chemical or affinity enrichment strategies, and quantitative approaches. We also highlight a number of redox proteomic approaches that enable effective profiling of redox PTMs for addressing specific biological questions. Although some technological limitations remain, redox proteomics is paving the way towards a better understanding of redox signaling and regulation in human health and diseases.« less

Principles in redox signaling: from chemistry to functional significance.

PubMed

Bindoli, Alberto; Rigobello, Maria Pia

2013-05-01

Reactive oxygen and nitrogen species are currently considered not only harmful byproducts of aerobic respiration but also critical mediators of redox signaling. The molecules and the chemical principles sustaining the network of cellular redox regulated processes are described. Special emphasis is placed on hydrogen peroxide (H(2)O(2)), now considered as acting as a second messenger, and on sulfhydryl groups, which are the direct targets of the oxidant signal. Cysteine residues of some proteins, therefore, act as sensors of redox conditions and are oxidized in a reversible reaction. In particular, the formation of sulfenic acid and disulfide, the initial steps of thiol oxidation, are described in detail. The many cell pathways involved in reactive oxygen species formation are reported. Central to redox signaling processes are the glutathione and thioredoxin systems controlling H(2)O(2) levels and, hence, the thiol/disulfide balance. Lastly, some of the most important redox-regulated processes involving specific enzymes and organelles are described. The redox signaling area of research is rapidly expanding, and future work will examine new pathways and clarify their importance in cellular pathophysiology.

Oriented and covalent immobilization of target molecules to solid supports: synthesis and application of a light-activatable and thiol-reactive cross-linking reagent.

PubMed

Collioud, A; Clémence, J F; Sänger, M; Sigrist, H

1993-01-01

Light-dependent oriented and covalent immobilization of target molecules has been achieved by combining two modification procedures: light-dependent coupling of target molecules to inert surfaces and thiol-selective reactions occurring at macromolecule or substrate surfaces. For immobilization purposes the heterobifunctional reagent N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyr amide was synthesized and chemically characterized. The photosensitivity of the carbene-generating reagent and its reactivity toward thiols were ascertained. Light-induced cross-linking properties of the reagent were documented (i) by reacting first the maleimide function with a thiolated surface, followed by carbene insertion into applied target molecules, (ii) by photochemical coupling of the reagent to an inert support followed by thermochemical reactions with thiol functions, and (iii) by thermochemical modification of target molecules prior to carbene-mediated insertion into surface materials. Procedures mentioned led to light-dependent covalent immobilization of target molecules including amino acids, a synthetic peptide, and antibody-derived F(ab') fragments. Topically selective, light-dependent immobilization was attained with the bifunctional reagent by irradiation of coated surfaces through patterned masks. Glass and polystyrene served as substrates. Molecular orientation is asserted by inherently available or selectively introduced terminal thiol functions in F(ab') fragments and synthetic polypeptides, respectively.

Development of photopolymerizable clay nanocomposites utilizing reactive dispersants

NASA Astrophysics Data System (ADS)

Owusu-Adom, Kwame

Nanocomposites hold tremendous promise for expanding the utility of polymeric materials. However, accessing particulate sizes in the nanoscale domain continues to be a scientific challenge, especially in highly cross-linked photopolymerizable systems. In this study, photopolymerizable nanocomposites utilizing clay nanoparticles and reactive dispersants have been developed. The influence of particle size, dispersant-clay interactions, and surfactant concentration on photopolymerization behavior and nanoparticle dispersion has been elucidated. Clay particles serve as templates upon which surfactants aggregate during photopolymerization. This results in higher photopolymerization rates with addition of increasing concentrations of polymerizable surfactants. Furthermore, polymerizable surfactants induce faster photopolymerization rates compared to non-polymerizable analogues in systems that have ionically-bound dispersants on the particle surface. Utilizing reactive organoclays induces significant changes to the photopolymerization behavior depending on the choice of reactive functionality employed. Faster acrylate photopolymerization rates occur in photopolymer systems containing thiol-modified clays, while much slower rates occur for nonpolymerizable organoclay systems. In addition, chemical compatibility between monomer and clay dispersant (based on chemical similarity or polarity) allows enhancement of exfoliation in photopolymerizable formulations. With polymerizable dispersants, exfoliation is readily achieved in various multifunctional acrylate systems. The degree of exfoliation depends on the position of the reactive group relative to the surfactant's cationic site and the type of functionality. Thiolated organoclays exfoliate during polymerization, while methacrylated clays show substantially less dependence on polymerization behavior. Interestingly, changes in the physical properties of the resulting nanocomposite are independent of the degree of exfoliation in polymerizable organoclay systems. The polymer cross-link density dictates the magnitude of change in both modulus and glass transition temperature of the nanocomposite. Substantial increases in modulus and Tg occur in elastomeric and low cross-link density polymers, while decreases occur in the modulus and Tg of highly cross-linked polymer networks. Finally, these parameters have formed a basis for developing nanocomposites with higher moduli and lower volumetric shrinkage. The photopolymerization rates of these systems are controllable and increase substantially with addition of polymerizable organoclays. Such properties occur in traditional multifunctional acrylate photopolymer systems as well as new binary thiol-(meth)acrylate and ternary thiol-ene-(meth)acrylate photopolymers.

Proteins oxidation and autoantibodies' reactivity against hydrogen peroxide and malondialdehyde -oxidized thyroid antigens in patients' plasmas with Graves' disease and Hashimoto Thyroiditis.

PubMed

Mseddi, Malek; Ben Mansour, Riadh; Gargouri, Bochra; Mnif, Fatma; El Ghawi, Samir; Hammami, Boutheina; Ghorbel, Abdelmonem; Abid, Mohamed; Lassoued, Saloua

2017-06-25

The aim of this study was to evaluate proteins oxidation in plasmas of two autoimmune thyroid diseases (AITD): Graves' disease (GD) and Hashimoto Thyroiditis (HT), and to determine whether oxidative modification of thyroid antigens (T.Ag) enhanced the reactivity of autoantibodies in plasmas of AITD patients compared with the reactivity towards native T.Ag. Carbonyl and thiol groups and MDA-protein adducts were assessed spectrophotometric methods in plasmas of 74 AITD patients and 65 healthy controls. The reactivities immunoglobulin (Ig)G autoantibodies towards malondialdéhyde (MDA)-modified T.Ag, hydrogen peroxide (H 2 O 2 )-modified T.Ag, native T.Ag and native derm were checked by enzyme-linked immunosorbent assay (ELISA). Evaluation of oxidized proteins exhibited high levels of MDA bound to proteins and carbonyl groups, as well as reduced thiol level in plasmas of AITD patients by comparison to healthy controls (p < 0.05). The ELISA test showed that AITD patients' plasmas' reactivity to native T.Ag was significantly increased to the reactivity towards native derm, whereas, no differences were found in the reactivity to native T.Ag and derm in controls plasmas. In addition, treatment of T.Ag by oxidants revealed enhanced reactivity of IgG circulating autoantibodies against H 2 O 2 -oxidized T.Ag compared to native ones (p < 0.001) in plasmas of both AITD. Also, reactivity's to MDA-oxidized T.Ag in GD plasmas decreased compared to native ones (p < 0.05) and no changes were noted for HT. Pearson correlation study resulted in positive correlation between reactivity's to H 2 O 2 -oxidized T.Ag and free triodotyronine level in GD patients (r = 0.42, p < 0.05) in one hand and thyroid stimulating hormone level in HT patients in the other (r = 0.65, p < 0.001). The data suggest that high production of H 2 O 2 probably occurred during hormone synthesis could contribute to protein oxidation in AITD and to create neoepitopes responsible for autoantibody reactivity's to H 2 O 2 -oxidized T.Ag enhancement. These results provide support to the involvement of oxidative stress in AITD development and/or exacerbation. Copyright © 2017 Elsevier B.V. All rights reserved.

Selenite Protection of Tellurite Toxicity Toward Escherichia coli

PubMed Central

Vrionis, Helen A.; Wang, Siyuan; Haslam, Bronwyn; Turner, Raymond J.

2015-01-01

In this work the influence of selenite on metal resistance in Escherichia coli was examined. Both synergistic and antagonistic resistance and toxicities were found upon co exposure with selenite. In wild type cells co-exposure to selenite had little effect on arsenic resistance, decreased resistance to cadmium and mercury but led to a dramatically increased resistance to tellurite of 32-fold. Due to the potential importance of thiol chemistry in metal biochemistry, deletion strains in γ-glutamylcysteine synthetase (key step in glutathione biosynthesis, encoded by gshA), thioredoxin (trxA), glutaredoxin (grxA), glutathione oxidoreductase (gor), and the periplasmic glutathione transporter (cydD) were also evaluated for resistance to various metals in the presence of selenite. The protective effect of selenite on tellurite toxicity was seen in several of the mutants and was pronounced in the gshA mutant were resistance to tellurite was increased up to 1000-fold relative to growth in the absence of selenite. Thiol oxidation studies revealed a faster rate of loss of reduced thiol content in the cell with selenite than with tellurite, indicating differential thiol reactivity. Selenite addition resulted in reactive oxygen species (ROS) production equivalent to levels associated with H2O2 addition. Tellurite addition resulted in considerably lower ROS generation while vanadate and chromate treatment did not increase ROS production above that of background. This work shows increased resistance toward most oxyanions in mutants of thiol redox suggesting that metalloid reaction with thiol components such as glutathione actually enhances toxicity of some metalloids. PMID:26732755

Chemiluminescence analysis of antioxidant capacity for serum albumin isolated from healthy or uremic volunteers.

PubMed

Huang, Chih-Yang; Liou, Show-Yih; Kuo, Wei-Wen; Wu, Hsi-Chin; Chang, Yen-Lin; Chen, Tung-Sheng

2016-12-01

Regular hemodialysis treatment induces an elevation in oxidative stress in patients with end-stage renal failure, resulting in oxidative damage of the most abundant serum protein, albumin. Oxidation of serum albumin causes depletion of albumin reactive thiols, leading to oxidative modification of serum albumin. The aim of this study was to screen the antioxidant capacity of albumins isolated from uremic patients (HD-ALB) or healthy volunteers (N-ALB). From high-performance liquid chromatography spectra, we observed that one uremic solute binds to HD-ALB via the formation of disulfide bonds between HD-ALB and the uremic solute. Furthermore, we found using chemiluminescent analysis that the antioxidant capacities for N-ALB to scavenge reactive oxygen species including singlet oxygen, hypochlorite and hydrogen peroxide were higher than HD-ALB. Our results suggest that protein-bound uremic solute binds to albumin via formation of disulfide bonds, resulting in the depletion of albumin reactive thiols. The depletion of albumin reactive thiols leads to a reduced antioxidant capacity of HD-ALB, implying postmodification of albumin. This situation may reduce the antioxidant capacity of albumin and increase oxidative stress, resulting in increase in complications related to oxidative damage in uremic patients. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Selenocysteine in thiol/disulfide-like exchange reactions.

PubMed

Hondal, Robert J; Marino, Stefano M; Gladyshev, Vadim N

2013-05-01

Among trace elements used as cofactors in enzymes, selenium is unique in that it is incorporated into proteins co-translationally in the form of an amino acid, selenocysteine (Sec). Sec differs from cysteine (Cys) by only one atom (selenium versus sulfur), yet this switch dramatically influences important aspects of enzyme reactivity. The main focus of this review is an updated and critical discussion on how Sec might be used to accelerate thiol/disulfide-like exchange reactions in natural selenoenzymes, compared with their Cys-containing homologs. We discuss in detail three major aspects associated with thiol/disulfide exchange reactions: (i) nucleophilicity of the attacking thiolate (or selenolate); (ii) electrophilicity of the center sulfur (or selenium) atom; and (iii) stability of the leaving group (sulfur or selenium). In all these cases, we analyze the benefits that selenium might provide in these types of reactions. It is the biological thiol oxidoreductase-like function that benefits from the use of Sec, since Sec functions to chemically accelerate the rate of these reactions. We review various hypotheses that could help explain why Sec is used in enzymes, particularly with regard to competitive chemical advantages provided by the presence of the selenium atom in enzymes. Ultimately, these chemical advantages must be connected to biological functions of Sec.

Design strategies of fluorescent probes for selective detection among biothiols.

PubMed

Niu, Li-Ya; Chen, Yu-Zhe; Zheng, Hai-Rong; Wu, Li-Zhu; Tung, Chen-Ho; Yang, Qing-Zheng

2015-10-07

Simple thiol derivatives, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play key roles in biological processes, and the fluorescent probes to detect such thiols in vivo selectively with high sensitivity and fast response times are critical for understanding their numerous functions. However, the similar structures and reactivities of these thiols pose considerable challenges to the development of such probes. This review focuses on various strategies for the design of fluorescent probes for the selective detection of biothiols. We classify the fluorescent probes for discrimination among biothiols according to reaction types between the probes and thiols such as cyclization with aldehydes, conjugate addition-cyclization with acrylates, native chemical ligation, and aromatic substitution-rearrangement.

Deficient expression of aldehyde dehydrogenase 1A1 is consistent with increased sensitivity of Gorlin syndrome patients to radiation carcinogenesis

DOE PAGES

Wright, Aaron T.; Magnaldo, Thierry; Sontag, Ryan L.; ...

2013-11-27

Human phenotypes that are highly susceptible to radiation carcinogenesis have been identified. Sensitive phenotypes often display robust regulation of molecular features that modify biological response, which can facilitate identification of relevant pathways/networks. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol-reactive probes with a flexible click chemistry functional group to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. We observe qualitative differences in protein thiol profilesmore » by SDS-PAGE analysis when detection by iodoacetamide vs maleimide probe chemistries are compared, and pretreatment of cells with hydrogen peroxide eliminates detection of the majority of SDS-PAGE bands. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent donors, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and this deficiency was confirmed by Western blot. Redox probes revealed additional protein thiol differences between GDFs and NHDFs, including radiation responsive annexin family members. Our results indicate a multifactorial basis for the unusual sensitivity of Gorlin syndrome to radiation carcinogenesis, and the pathways identified have plausible implications for radiation health effects.« less

Deficient expression of aldehyde dehydrogenase 1A1 is consistent with increased sensitivity of Gorlin syndrome patients to radiation carcinogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wright, Aaron T.; Magnaldo, Thierry; Sontag, Ryan L.

Human phenotypes that are highly susceptible to radiation carcinogenesis have been identified. Sensitive phenotypes often display robust regulation of molecular features that modify biological response, which can facilitate identification of relevant pathways/networks. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol-reactive probes with a flexible click chemistry functional group to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. We observe qualitative differences in protein thiol profilesmore » by SDS-PAGE analysis when detection by iodoacetamide vs maleimide probe chemistries are compared, and pretreatment of cells with hydrogen peroxide eliminates detection of the majority of SDS-PAGE bands. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent donors, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and this deficiency was confirmed by Western blot. Redox probes revealed additional protein thiol differences between GDFs and NHDFs, including radiation responsive annexin family members. Our results indicate a multifactorial basis for the unusual sensitivity of Gorlin syndrome to radiation carcinogenesis, and the pathways identified have plausible implications for radiation health effects.« less

Insulin-induced hypoglycemia and stress oxidative state in healthy people.

PubMed

Yousefzade, Gholamreza; Nakhaee, Akram

2012-12-01

The purpose of this study is to assess the immediate effects of insulin-induced hypoglycemia on the natural antioxidant superoxide dismutase activity, malondialdehyde concentration, total antioxidative capacity and total thiol group concentration in young healthy subjects. In this clinical trial, 16 healthy men with the mean age of 29.3 ± 5.3 years (range 21-39 years) became volunteers to participate the study. Hypoglycemia was induced by intravenous administration of regular insulin 0.1 U/kg. Before and after inducing hypoglycemia, SOD activity was determined in red blood cells, whereas the MDA concentration was determined by thiobarbituric acid reactive substance method, total thiol groups by high-performance liquid chromatography method and total antioxidant capacity by ferric reducing/antioxidant power. A significant increase was seen in the TBARS levels following insulin-induced hypoglycemia (0.19 ± 0.07 vs. 0.38 ± 0.16 nmol/g, P < 0.001), while a significant decrement occurred in the antioxidant power (FRAP value) (321.4 ± 63.4 vs. 231.4 ± 57.5, P < 0.001), total thiol concentration (2.3 ± 0.8 vs. 1.3 ± 0.5, P = 0.001) and SOD enzyme activity (29.4 ± 8.2 vs. 23.1 ± 6.1, P < 0.001) subsequent the hypoglycemia with insulin.

Thiol Reactivity of Curcumin and Its Oxidation Products.

PubMed

Luis, Paula B; Boeglin, William E; Schneider, Claus

2018-04-16

The polypharmacological effects of the turmeric compound curcumin may be partly mediated by covalent adduction to cellular protein. Covalent binding to small molecule and protein thiols is thought to occur through a Michael-type addition at the enone moiety of the heptadienedione chain connecting the two methoxyphenol rings of curcumin. Here we show that curcumin forms the predicted thiol-Michael adducts with three model thiols, glutathione, N-acetylcysteine, and β-mercaptoethanol. More abundant, however, are respective thiol adducts of the dioxygenated spiroepoxide intermediate of curcumin autoxidation. Two electrophilic sites at the quinone-like ring of the spiroepoxide are identified. Addition of β-mercaptoethanol at the 5'-position of the ring gives a 1,7-dihydroxycyclopentadione-5' thioether, and addition at the 1'-position results in cleavage of the aromatic ring from the molecule, forming methoxyphenol-thioether and a tentatively identified cyclopentadione aldehyde. The curcuminoids demethoxy- and bisdemethoxycurcumin do not form all of the possible thioether adducts, corresponding with their increased stability toward autoxidation. RAW264.7 macrophage-like cells activated with phorbol ester form curcumin-glutathionyl and the 1,7-dihydroxycyclopentadione-5'-glutathionyl adducts. These studies indicate that the enone of the parent compound is not the only functional electrophile in curcumin, and that its oxidation products provide additional electrophilic sites. This suggests that protein binding by curcumin may involve oxidative activation into reactive quinone methide and spiroepoxide electrophiles.

Thiol surface functionalization via continuous phase plasma polymerization of allyl mercaptan, with subsequent maleimide-linked conjugation of collagen.

PubMed

Stynes, Gil D; Gengenbach, Thomas R; Kiroff, George K; Morrison, Wayne A; Kirkland, Mark A

2017-07-01

Thiol groups can undergo a large variety of chemical reactions and are used in solution phase to conjugate many bioactive molecules. Previous research on solid substrates with continuous phase glow discharge polymerization of thiol-containing monomers may have been compromised by oxidation. Thiol surface functionalization via glow discharge polymerization has been reported as requiring pulsing. Herein, continuous phase glow discharge polymerization of allyl mercaptan (2-propene-1-thiol) was used to generate significant densities of thiol groups on a mixed macrodiol polyurethane and tantalum. Three general classes of chemistry are used to conjugate proteins to thiol groups, with maleimide linkers being used most commonly. Here the pH specificity of maleimide reactions was used effectively to conjugate surface-bound thiol groups to amine groups in collagen. XPS demonstrated surface-bound thiol groups without evidence of oxidation, along with the subsequent presence of maleimide and collagen. Glow discharge reactor parameters were optimized by testing the resistance of bound collagen to degradation by 8 M urea. The nature of the chemical bonding of collagen to surface thiol groups was effectively assessed by colorimetric assay (ELISA) of residual collagen after incubation in 8 M urea over 8 days and after incubation with keratinocytes over 15 days. The facile creation of useable solid-supported thiol groups via continuous phase glow discharge polymerization of allyl mercaptan opens a route for attaching a vast array of bioactive molecules. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1940-1948, 2017. © 2017 Wiley Periodicals, Inc.

Design of water-soluble, thiol-reactive polymers of controlled molecular weight: a novel multivalent scaffold

NASA Astrophysics Data System (ADS)

Carrillo, Alvaro; Gujraty, Kunal V.; Rai, Prakash R.; Kane, Ravi S.

2005-07-01

Multivalent molecules, i.e. scaffolds presenting multiple copies of a suitable ligand, constitute an emerging class of nanoscale therapeutics. We present a novel approach for the design of multivalent ligands, which allows the biofunctionalization of polymers with proteins or peptides in a controlled orientation. It consists of the synthesis of water-soluble, activated polymer scaffolds of controlled molecular weight, which can be biofunctionalized with various thiolated ligands in aqueous media under mild conditions. These polymers were synthesized by ring-opening metathesis polymerization (ROMP) and further modified to make them water-soluble. The incorporation of chloride groups activated the polymers to react with thiol-containing peptides or proteins, and the formation of multivalent ligands in aqueous media was demonstrated. This strategy represents a convenient route for synthesizing multivalent ligands of controlled dimensions and valency.

The role of oxidative stress in streptozotocin-induced diabetic nephropathy in rats.

PubMed

Fernandes, Sheila Marques; Cordeiro, Priscilla Mendes; Watanabe, Mirian; Fonseca, Cassiane Dezoti da; Vattimo, Maria de Fatima Fernandes

2016-10-01

The objective of this study was to evaluate the role of oxidative stress in an experimental model of streptozotocin-induced diabetic nephropathy in rats. Wistar, adult, male rats were used in the study. Animals were divided in the following groups: Citrate (control, citrate buffer 0.01M, pH 4.2 was administrated intravenously - i.v - in the caudal vein), Uninephrectomy+Citrate (left uninephrectomy-20 days before the study), DM (streptozotocin, 65 mg/kg, i.v, on the 20th day of the study), Uninephrectomy+DM. Physiological parameters (water and food intake, body weight, blood glucose, kidney weight, and relative kidney weight); renal function (creatinine clearance), urine albumin (immunodiffusion method); oxidative metabolites (urinary peroxides, thiobarbituric acid reactive substances, and thiols in renal tissue), and kidney histology were evaluated. Polyphagia, polydipsia, hyperglycemia, and reduced body weight were observed in diabetic rats. Renal function was reduced in diabetic groups (creatinine clearance, p < 0.05). Uninephrectomy potentiated urine albumin and increased kidney weight and relative kidney weight in diabetic animals (p < 0.05). Urinary peroxides and thiobarbituric acid reactive substances were increased, and the reduction in thiol levels demonstrated endogenous substrate consumption in diabetic groups (p < 0.05). The histological analysis revealed moderate lesions of diabetic nephropathy. This study confirms lipid peroxidation and intense consumption of the antioxidant defense system in diabetic rats. The association of hyperglycemia and uninephrectomy resulted in additional renal injury, demonstrating that the model is adequate for the study of diabetic nephropathy.

Determination of acidity and nucleophilicity in thiols by reaction with monobromobimane and fluorescence detection.

PubMed

Sardi, Florencia; Manta, Bruno; Portillo-Ledesma, Stephanie; Knoops, Bernard; Comini, Marcelo A; Ferrer-Sueta, Gerardo

2013-04-01

A method based on the differential reactivity of thiol and thiolate with monobromobimane (mBBr) has been developed to measure nucleophilicity and acidity of protein and low-molecular-weight thiols. Nucleophilicity of the thiolate is measured as the pH-independent second-order rate constant of its reaction with mBBr. The ionization constants of the thiols are obtained through the pH dependence of either second-order rate constant or initial rate of reaction. For readily available thiols, the apparent second-order rate constant is measured at different pHs and then plotted and fitted to an appropriate pH function describing the observed number of ionization equilibria. For less available thiols, such as protein thiols, the initial rate of reaction is determined in a wide range of pHs and fitted to the appropriate pH function. The method presented here shows excellent sensitivity, allowing the use of nanomolar concentrations of reagents. The method is suitable for scaling and high-throughput screening. Example determinations of nucleophilicity and pK(a) are presented for captopril and cysteine as low-molecular-weight thiols and for human peroxiredoxin 5 and Trypanosoma brucei monothiol glutaredoxin 1 as protein thiols. Copyright © 2013 Elsevier Inc. All rights reserved.

Thiol/disulphide homeostasis in celiac disease

PubMed Central

Kaplan, Mustafa; Ates, Ihsan; Yuksel, Mahmut; Ozderin Ozin, Yasemin; Alisik, Murat; Erel, Ozcan; Kayacetin, Ertugrul

2017-01-01

AIM To determine dynamic thiol/disulphide homeostasis in celiac disease and to examine the associate with celiac autoantibodies and gluten-free diet. METHODS Seventy three patients with celiac disease and 73 healthy volunteers were enrolled in the study. In both groups, thiol/disulphide homeostasis was examined with a new colorimetric method recently developed by Erel and Neselioglu. RESULTS In patients with celiac disease, native thiol (P = 0.027) and total thiol (P = 0.031) levels were lower, while disulphide (P < 0.001) level, disulphide/native thiol (P < 0.001) and disulphide/total thiol (P < 0.001) ratios were higher compared to the control group. In patients who do not comply with a gluten-free diet, disulphide/native thiol ratio was found higher compared to the patients who comply with the diet (P < 0.001). In patients with any autoantibody-positive, disulphide/native thiol ratio was observed higher compared to the patients with autoantibody-negative (P < 0.05). It is found that there is a negative correlation between celiac autoantibodies, and native thiol, total thiol levels and native thiol/total thiol ratio, while a positive correlation is observed between disulphide, disulphide/native thiol and disulphide/total thiol levels. CONCLUSION This study is first in the literature which found that the patients with celiac disease the dynamic thiol/disulphide balance shifts through disulphide form compared to the control group. PMID:28533921

Facile quantitation of free thiols in a recombinant monoclonal antibody by reversed-phase high performance liquid chromatography with hydrophobicity-tailored thiol derivatization.

PubMed

Welch, Leslie; Dong, Xiao; Hewitt, Daniel; Irwin, Michelle; McCarty, Luke; Tsai, Christina; Baginski, Tomasz

2018-06-02

Free thiol content, and its consistency, is one of the product quality attributes of interest during technical development of manufactured recombinant monoclonal antibodies (mAbs). We describe a new, mid/high-throughput reversed-phase-high performance liquid chromatography (RP-HPLC) method coupled with derivatization of free thiols, for the determination of total free thiol content in an E. coli-expressed therapeutic monovalent monoclonal antibody mAb1. Initial selection of the derivatization reagent used an hydrophobicity-tailored approach. Maleimide-based thiol-reactive reagents with varying degrees of hydrophobicity were assessed to identify and select one that provided adequate chromatographic resolution and robust quantitation of free thiol-containing mAb1 forms. The method relies on covalent derivatization of free thiols in denatured mAb1 with N-tert-butylmaleimide (NtBM) label, followed by RP-HPLC separation with UV-based quantitation of native (disulfide containing) and labeled (free thiol containing) forms. The method demonstrated good specificity, precision, linearity, accuracy and robustness. Accuracy of the method, for samples with a wide range of free thiol content, was demonstrated using admixtures as well as by comparison to an orthogonal LC-MS peptide mapping method with isotope tagging of free thiols. The developed method has a facile workflow which fits well into both R&D characterization and quality control (QC) testing environments. The hydrophobicity-tailored approach to the selection of free thiol derivatization reagent is easily applied to the rapid development of free thiol quantitation methods for full-length recombinant antibodies. Copyright © 2018 Elsevier B.V. All rights reserved.

Proteomic analysis of UVB-induced protein expression- and redox-dependent changes in skin fibroblasts using lysine- and cysteine-labeling two-dimensional difference gel electrophoresis.

PubMed

Wu, Chieh-Lin; Chou, Hsiu-Chuan; Cheng, Chao-Sheng; Li, Ji-Min; Lin, Szu-Ting; Chen, Yi-Wen; Chan, Hong-Lin

2012-04-03

UVB is the most energetic and DNA-damaging to humans in ultraviolet radiation. Previous research has suggested that exposure to UVB causes skin pathologies because of direct DNA damage and the generation of reactive oxygen species (ROS). However, the detailed molecular mechanisms by which UVB leads to skin cancer have yet to be clarified. In the current study, normal skin fibroblast cells (CCD-966SK) were exposed to various doses of UVB, and the changes in protein expression and thiol reactivity were monitored with lysine- and cysteine-labeling 2D-DIGE and MALDI-TOF mass spectrometry. Our proteomic analysis revealed that 89 identified proteins showed significant changes in protein expression, and 37 in thiol reactivity. Many proteins that are known to be involved in protein folding, redox regulation and nucleotide biosynthesis were up-regulated under UVB irradiation. In contrast, proteins responsible for biosynthesis and protein degradation were down-regulated. In addition, the thiol-reactivity of proteins involving cytoskeleton, metabolism, and signal transduction were altered by UVB. In summary, these UVB-modulated cellular proteins and redox-regulated proteins might play important roles in the early stages of skin cancer formation and photoaging induced by UVB-irradiation. Such proteins might provide a potential target for the rational design of drugs to prevent UVB-induced diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

Novel photonic technique creates micrometer resolution protein arrays and provides a new approach to coupling of genes, peptide hormones and drugs to nanoparticle carriers

NASA Astrophysics Data System (ADS)

Duroux, M.; Duroux, L.; Neves-Petersen, M. T.; Skovsen, E.; Petersen, S. B.

2007-07-01

We demonstrate that ultraviolet light can be used to make sterically oriented covalent immobilization of a large variety of protein molecules onto either thiolated quartz, gold or silicon. The reaction mechanism behind the reported new technology involves light-induced breakage of disulphide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol reactive surfaces. In general, the protein molecules retain their function. The size of the immobilization spot is limited to the focal point of illumination being as small as a few micrometers. This new technology allows for dense packing of different bio-molecules on a surface, allowing the creation of multi-potent functionalised new materials, such as nano-biosensors. We have developed the necessary technology for preparing large protein arrays of enzymes and fragments of monoclonal antibodies. Dedicated image processing software has been developed for making quality assessment of the protein arrays. This novel technology is ideal to couple drugs and other bio-molecules to nanoparticles which can be used as carriers into cells for therapeutic purposes.

S-protected thiolated chitosan for oral delivery of hydrophilic macromolecules: evaluation of permeation enhancing and efflux pump inhibitory properties.

PubMed

Dünnhaupt, Sarah; Barthelmes, Jan; Rahmat, Deni; Leithner, Katharina; Thurner, Clemens C; Friedl, Heike; Bernkop-Schnürch, Andreas

2012-05-07

The objective of this study was the investigation of permeation enhancing and P-glycoprotein (P-gp) inhibition effects of a novel thiolated chitosan, the so-named S-protected thiolated chitosan. Mediated by a carbodiimide, increasing amounts of thioglycolic acid (TGA) were covalently bound to chitosan (CS) in the first step of modification. In the second step, these thiol groups of thiolated chitosan were protected by disulfide bond formation with the thiolated aromatic residue 6-mercaptonicotinamide (6-MNA). Mucoadhesive properties of all conjugates were evaluated in vitro on porcine intestinal mucosa based on tensile strength investigations. Permeation enhancing effects were evaluated ex vivo using rat intestinal mucosa and in vitro via Caco-2 cells using the hydrophilic macromolecule FD(4) as the model drug. Caco-2 cells were further used to show P-gp inhibition effects by using Rho-123 as P-gp substrate. Apparent permeability coefficients (P(app)) were calculated and compared to values obtained from each buffer control. Three different thiolated chitosans were generated in the first step of modification, which displayed increasing amounts of covalently attached free thiol groups on the polymer backbone. In the second modification step, more than 50% of these free thiol groups were covalently linked with 6-MNA. Within 3 h of permeation studies on excised rat intestine, P(app) values of all S-protected chitosans were at least 1.3-fold higher compared to those of corresponding thiomers and more than twice as high as that of unmodified chitosan. Additional permeation studies on Caco-2 cells confirmed these results. Because of the chemical modification and higher amount of reactive thiol groups, all S-protected thiolated chitosans exhibit at least 1.4-fold pronounced P-gp inhibition effects in contrast to their corresponding thiomers. These features approve S-protected thiolated chitosan as a promising excipient for various drug delivery systems providing improved permeation enhancing and efflux inhibition effects.

Selenocysteine in Thiol/Disulfide-Like Exchange Reactions

PubMed Central

Marino, Stefano M.

2013-01-01

Abstract Significance: Among trace elements used as cofactors in enzymes, selenium is unique in that it is incorporated into proteins co-translationally in the form of an amino acid, selenocysteine (Sec). Sec differs from cysteine (Cys) by only one atom (selenium versus sulfur), yet this switch dramatically influences important aspects of enzyme reactivity. Recent Advances: The main focus of this review is an updated and critical discussion on how Sec might be used to accelerate thiol/disulfide-like exchange reactions in natural selenoenzymes, compared with their Cys-containing homologs. Critical Issues: We discuss in detail three major aspects associated with thiol/disulfide exchange reactions: (i) nucleophilicity of the attacking thiolate (or selenolate); (ii) electrophilicity of the center sulfur (or selenium) atom; and (iii) stability of the leaving group (sulfur or selenium). In all these cases, we analyze the benefits that selenium might provide in these types of reactions. Future Directions: It is the biological thiol oxidoreductase-like function that benefits from the use of Sec, since Sec functions to chemically accelerate the rate of these reactions. We review various hypotheses that could help explain why Sec is used in enzymes, particularly with regard to competitive chemical advantages provided by the presence of the selenium atom in enzymes. Ultimately, these chemical advantages must be connected to biological functions of Sec. Antioxid. Redox Signal. 18, 1675–1689. PMID:23121622

High throughput assay for evaluation of reactive carbonyl scavenging capacity.

PubMed

Vidal, N; Cavaille, J P; Graziani, F; Robin, M; Ouari, O; Pietri, S; Stocker, P

2014-01-01

Many carbonyl species from either lipid peroxidation or glycoxidation are extremely reactive and can disrupt the function of proteins and enzymes. 4-hydroxynonenal and methylglyoxal are the most abundant and toxic lipid-derived reactive carbonyl species. The presence of these toxics leads to carbonyl stress and cause a significant amount of macromolecular damages in several diseases. Much evidence indicates trapping of reactive carbonyl intermediates may be a useful strategy for inhibiting or decreasing carbonyl stress-associated pathologies. There is no rapid and convenient analytical method available for the assessment of direct carbonyl scavenging capacity, and a very limited number of carbonyl scavengers have been identified to date, their therapeutic potential being highlighted only recently. In this context, we have developed a new and rapid sensitive fluorimetric method for the assessment of reactive carbonyl scavengers without involvement glycoxidation systems. Efficacy of various thiol- and non-thiol-carbonyl scavenger pharmacophores was tested both using this screening assay adapted to 96-well microplates and in cultured cells. The scavenging effects on the formation of Advanced Glycation End-product of Bovine Serum Albumin formed with methylglyoxal, 4-hydroxynonenal and glucose-glycated as molecular models were also examined. Low molecular mass thiols with an α-amino-β-mercaptoethane structure showed the highest degree of inhibitory activity toward both α,β-unsaturated aldehydes and dicarbonyls. Cysteine and cysteamine have the best scavenging ability toward methylglyoxal. WR-1065 which is currently approved for clinical use as a protective agent against radiation and renal toxicity was identified as the best inhibitor of 4-hydroxynonenal.

Synthesis and crystallographic characterization of a mononuclear cobalt(III) complex possessing both thiol­ate and thio­ether donors: reactivity of an thiol­ate-bridged penta­nuclear Co2Ag3 complex with iodo­methane

PubMed Central

Fukuda, Yosuke; Yoshinari, Nobuto; Konno, Takumi

2017-01-01

Treatment of an S-bridged penta­nuclear AgI 3CoIII 2 complex, [Ag3{Co(L)}2]3+ [L 3– = N(CH2NHCH2CH2S−)3], in which two tris­(thiol­ate)-type mononuclear CoIII units ([Co(L)]) are bridged by three AgI ions through S atoms, with iodo­methane (CH3I) gave a new CoIII mononuclear complex, [Co(LMe2)]2+ [LMe2 − = N(CH2NHCH2CH2S−)(CH2NHCH2CH2SCH3)2], systematic name: {2-[(bis{[2-(methylsulfanyl)ethyl]aminomethyl}aminomethyl)amino]ethanethiolato}cobalt(III) bis(hexafluoridophosphate). This cationic complex was crystallized with PF6 − anions to form the title compound, [Co(LMe2)](PF6)2. In the [Co(LMe2)]2+ cation, two of three thiol­ate groups in [Co(L)] are methyl­ated while one thiol­ate group remains unreacted. Although a total of eight stereoisomers are possible for [Co(LMe2)]2+, only a pair of enanti­omers {ΛRR- and ΔSS-[Co(LMe2)]2+} are selectively formed. In the crystal, the complex cations and the PF­6 − anions are connected through weak N—H⋯F, C—H⋯F and C—H⋯S hydrogen bonds into a three-dimensional structure. Two F atoms in one PF6 anion are disordered over two sets of sites with refined occupancies of 0.61 (4) and 0.39 (4) and two F atoms in the other PF6 − anion are disordered over two sets of sites with occupancies of 0.5. PMID:28529774

Redox Signaling Mediated by Thioredoxin and Glutathione Systems in the Central Nervous System.

PubMed

Ren, Xiaoyuan; Zou, Lili; Zhang, Xu; Branco, Vasco; Wang, Jun; Carvalho, Cristina; Holmgren, Arne; Lu, Jun

2017-11-01

The thioredoxin (Trx) and glutathione (GSH) systems play important roles in maintaining the redox balance in the brain, a tissue that is prone to oxidative stress due to its high-energy demand. These two disulfide reductase systems are active in various areas of the brain and are considered to be critical antioxidant systems in the central nervous system (CNS). Various neuronal disorders have been characterized to have imbalanced redox homeostasis. Recent Advances: In addition to their detrimental effects, recent studies have highlighted that reactive oxygen species/reactive nitrogen species (ROS/RNS) act as critical signaling molecules by modifying thiols in proteins. The Trx and GSH systems, which reversibly regulate thiol modifications, regulate redox signaling involved in various biological events in the CNS. In this review, we focus on the following: (i) how ROS/RNS are produced and mediate signaling in CNS; (ii) how Trx and GSH systems regulate redox signaling by catalyzing reversible thiol modifications; (iii) how dysfunction of the Trx and GSH systems causes alterations of cellular redox signaling in human neuronal diseases; and (iv) the effects of certain small molecules that target thiol-based signaling pathways in the CNS. Further study on the roles of thiol-dependent redox systems in the CNS will improve our understanding of the pathogenesis of many human neuronal disorders and also help to develop novel protective and therapeutic strategies against neuronal diseases. Antioxid. Redox Signal. 27, 989-1010.

Markers of oxidative/nitrative damage of plasma proteins correlated with EDSS and BDI scores in patients with secondary progressive multiple sclerosis.

PubMed

Morel, Agnieszka; Bijak, Michał; Niwald, Marta; Miller, Elżbieta; Saluk, Joanna

2017-11-01

The objective of the present study was to evaluate oxidative/nitrative stress in the plasma of 50 patients suffering from the secondary progressive course of multiple sclerosis (MS), and to verify its correlation with physical and mental disability as assessed by the Expanded Disability Status Scale (EDSS), and the Beck Depression Inventory (BDI). Oxidative and nitrative damage to proteins was determined by the level of carbonyl groups and 3-nitrotyrosine using ELISA test. Based on the reaction with Ellman's reagent, we estimated the concentration of oxidized thiol groups. Additionally, we measured the level of lipid peroxidation. In plasma drawn from MS patients, we observed a significantly higher level of 3-NT (92%; P < 0.0003), carbonyl groups (29%; P < 0.0001) and thiobarbituric acid reactive substances (73%; P < 0.0001), as well as a lower concentration of thiol groups (33%; P < 0.0001), in comparison to healthy subjects. We noted positive correlations between the level of carbonyl groups or 3-NT and both diagnostic parameters, EDSS and BDI. Negative correlations were observed between concentration of -SH groups and EDSS and BDI. Our results indicate that impaired red-ox balance can significantly promote neurodegeneration in secondary progressive MS.

Characterization of acute phase proteins and oxidative stress response to road transportation in the dog.

PubMed

Fazio, Francesco; Casella, Stefania; Giannetto, Claudia; Giudice, Elisabetta; Piccione, Giuseppe

2015-01-01

Haptoglobin (Hp), serum amyloid A (SAA), C-reactive protein (CRP), white blood cells (WBC), reactive oxygen metabolites (ROMs), the antioxidant barrier (Oxy-adsorbent) and thiol groups of plasma compounds (SHp) were measured in ten dogs that had been transported a distance of about 230 km within 2 h (experimental group) and in ten dogs that had not been subjected to road transportation (control group). Blood was collected via cephalic venipuncture before road transportation (T0), after road transportation (T1), and more than 6 (T6) and 24 (T24) hours after road transportation in the experimental group (Group A) and at the same time points in the control group (Group B). The GLM (general linear model) Repeated Measures procedure showed a significant difference between the two groups (P<0.0001) and a significant rise (P<0.0001) in the concentrations of Hp, SAA, CRP, WBC, ROMs, Oxy-adsorbent and SHp after road transportation in Group A, underlining that physiological and homeostatic mechanisms are modified differently at various sampling times.

Applications of reversible covalent chemistry in analytical sample preparation.

PubMed

Siegel, David

2012-12-07

Reversible covalent chemistry (RCC) adds another dimension to commonly used sample preparation techniques like solid-phase extraction (SPE), solid-phase microextraction (SPME), molecular imprinted polymers (MIPs) or immuno-affinity cleanup (IAC): chemical selectivity. By selecting analytes according to their covalent reactivity, sample complexity can be reduced significantly, resulting in enhanced analytical performance for low-abundance target analytes. This review gives a comprehensive overview of the applications of RCC in analytical sample preparation. The major reactions covered include reversible boronic ester formation, thiol-disulfide exchange and reversible hydrazone formation, targeting analyte groups like diols (sugars, glycoproteins and glycopeptides, catechols), thiols (cysteinyl-proteins and cysteinyl-peptides) and carbonyls (carbonylated proteins, mycotoxins). Their applications range from low abundance proteomics to reversible protein/peptide labelling to antibody chromatography to quantitative and qualitative food analysis. In discussing the potential of RCC, a special focus is on the conditions and restrictions of the utilized reaction chemistry.

Inhibition of Urease by Disulfiram, an FDA-Approved Thiol Reagent Used in Humans.

PubMed

Díaz-Sánchez, Ángel Gabriel; Alvarez-Parrilla, Emilio; Martínez-Martínez, Alejandro; Aguirre-Reyes, Luis; Orozpe-Olvera, Jesica Aline; Ramos-Soto, Miguel Armando; Núñez-Gastélum, José Alberto; Alvarado-Tenorio, Bonifacio; de la Rosa, Laura Alejandra

2016-11-26

Urease is a nickel-dependent amidohydrolase that catalyses the decomposition of urea into carbamate and ammonia, a reaction that constitutes an important source of nitrogen for bacteria, fungi and plants. It is recognized as a potential antimicrobial target with an impact on medicine, agriculture, and the environment. The list of possible urease inhibitors is continuously increasing, with a special interest in those that interact with and block the flexible active site flap. We show that disulfiram inhibits urease in Citrullus vulgaris (CVU), following a non-competitive mechanism, and may be one of this kind of inhibitors. Disulfiram is a well-known thiol reagent that has been approved by the FDA for treatment of chronic alcoholism. We also found that other thiol reactive compounds (l-captopril and Bithionol) and quercetin inhibits CVU. These inhibitors protect the enzyme against its full inactivation by the thiol-specific reagent Aldrithiol (2,2'-dipyridyl disulphide, DPS), suggesting that the three drugs bind to the same subsite. Enzyme kinetics, competing inhibition experiments, auto-fluorescence binding experiments, and docking suggest that the disulfiram reactive site is Cys592, which has been proposed as a "hinge" located in the flexible active site flap. This study presents the basis for the use of disulfiram as one potential inhibitor to control urease activity.

Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

PubMed

Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

2011-06-01

Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Chloronitroimidazoles as radiosensitizers of hypoxic cells in vitro.

PubMed

Wideł, M; Watras, J; Suwiński, J; Salwińska, E

1987-01-01

Some results of the first more complex studies in vitro on radio-sensitizing efficiency, cytotoxicity and reactivity with blood-thiols of a series of 4- or 5-nitroimidazoles substituted in the 5, 4 or 2 position with chlorine are presented. The derivatives of 4-nitroimidazole substituted in 5 position ("ortho" position) with Cl show higher radiosensitizing efficiency than one may expect from their reduction potential, E1/2. At the same time they are extremely toxic, especially for aerobic cells. It is considered that high biological activity of ortho-substituted 4-nitroimidazoles is connected with their considerable chemical reactivity towards thiols and suppression of those natural protective compounds in the cells. The corresponding 5-nitro isomers are about tenfold weaker sensitizers, and simultaneously much less cytotoxic, either in aerobic or in hypoxic conditions. The chloro-4(5)-nitroimidazoles nonsubstituted at N-1 and ionizable in aqueous solution are relatively weaker at the same time less toxic radiosensitizers. It is evaluated that potential application in radiotherapy may have those chloronitroimidazoles which show low aerobic cytotoxicity, moderate radiosensitizing ability and no reactivity towards thiols. On the basis of the study in vitro, we have selected such a compound: 1-methyl-2-chloro-4-nitroimidazole (P13) for screening in vivo.

Real Time-NIR/MIR-Photorheology: A Versatile Tool for the in Situ Characterization of Photopolymerization Reactions.

PubMed

Gorsche, Christian; Harikrishna, Reghunathan; Baudis, Stefan; Knaack, Patrick; Husar, Branislav; Laeuger, Joerg; Hoffmann, Helmuth; Liska, Robert

2017-05-02

In photopolymerization reactions, mostly multifunctional monomers are employed, as they ensure fast reaction times and good final mechanical properties of the cured materials. Drawing conclusions about the influence of the components and curing conditions on the mechanical properties of the subsequently formed insoluble networks is challenging. Therefore, an in situ observation of chemical and mechanical characteristics during the photopolymerization reaction is desired. By coupling of an infrared spectrometer with a photorheometer, a broad spectrum of different photopolymerizable formulations can be analyzed during the curing reaction. The rheological information (i.e., time to gelation, final modulus, shrinkage force) can be derived from a parallel plate rheometer equipped with a UV- and IR-translucent window (glass for NIR and CaF 2 window for MIR). Chemical information (i.e., conversion at the gel point and final conversion) is gained by monitoring the decrease of the corresponding IR-peak for the reactive monomer unit (e.g., C═C double bond peak for (meth)acrylates, H-S thiol and C═C double bond peak in thiol-ene systems, C-O epoxy peak for epoxy resins). Depending on the relative concentration of reactive functional groups in the sample volume and the intensity of the IR signal, the conversion can be monitored in the near-infrared region (e.g., acrylate double bonds, epoxy groups) or the MIR region (e.g., thiol signal). Moreover, an integrated Peltier element and external heating hood enable the characterization of photopolymerization reactions at elevated temperatures, which also widens the window of application to resins that are waxy or solid at ambient conditions. By switching from water to heavy water, the chemical conversion during photopolymerization of hydrogel precursor formulations can also be examined. Moreover, this device could also represent an analytical tool for a variety of thermally and redox initiated systems.

Benzofurazan Sulfides for Thiol Imaging and Quantification in Live Cells through Fluorescence Microscopy

PubMed Central

Li, Yinghong; Yang, Yang; Guan, Xiangming

2012-01-01

Thiol groups play a significant role in various cellular functions. Cellular thiol concentrations can be affected by various physiological or pathological factors. A fluorescence imaging agent that can effectively and specifically image thiols in live cells through fluorescence microscopy is desirable for live cell thiol monitoring. Benzofurazan sulfides 1a–e were synthesized and found to be thiol specific fluorogenic agents except 1d. They are not fluorescent but form strong fluorescent thiol adducts after reacting with thiols through a sulfide-thiol exchange reaction. On the other hand, they exhibit no reaction with other biologically relevant nucleophilic functional groups such as -NH2, -OH, or -COOH revealing the specificity for the detection of thiols. Sulfide 1a was selected to confirm its ability to image cellular thiols through fluorescence microscopy. The compound was demonstrated to effectively image and quantify thiol changes in live cells through fluorescence microscopy using 430 nm and 520 nm as the excitation and emission wavelengths respectively. The quantification results of total thiol in live cells obtained from fluorescence microscopy were validated by an HPLC/UV total thiol assay method. The reagents and method will be of a great value to thiol redox-related research. PMID:22794193

The Role of Follicular Fluid Thiol/Disulphide Homeostasis in Polycystic Ovary Syndrome.

PubMed

Tola, Esra Nur; Köroğlu, Nadiye; Ergin, Merve; Oral, Hilmi Baha; Turgut, Abdülkadir; Erel, Özcan

2018-04-04

Oxidative stress is suggested as a potential triggering factor in the etiopathogenesis of Polycystic ovary syndrome related infertility. Thiol/disulphide homeostasis, a recently oxidative stress marker, is one of the antioxidant mechanism in human which have critical roles in folliculogenesis and ovulation. The aim of our study is to investigate follicular fluid thiol/disulphide homeostasis in the etiopathogenesis of Polycystic ovary syndrome and to determine its' association with in vitro fertilization outcome. The study procedures were approved by local ethic committee. Cross sectional design Methods: Follicular fluid of twenty-two Polycystic ovary syndrome women and twenty ovulatory controls undergoing in vitro fertilization treatment were recruited. Thiol/disulphide homeostasis was analyzed via a novel spectrophotometric method. Follicular native thiol levels were found to be lower in Polycystic ovary syndrome group than non- Polycystic ovary syndrome group (p=0.041) as well as native thiol/total thiol ratio (p<0.0001). Disulphide level, disulphide/native thiol and disulphide/total thiol ratios were increased in Polycystic ovary syndrome group (p<0.0001). A positive correlation between fertilization rate and native thiol (p=0.01, r=0.53) and total thiol (p=0.01, r=0.052) among Polycystic ovary syndrome patients was found. A positive predictive effect of native thiol on fertilization rate among Polycystic ovary syndrome group was also found (p=0.03, β=0.45, 95% CI=0.031-0.643). Deterioration in thiol/disulphide homeostasis, especially elevated disulphide levels could be one of the etiopathogenetic mechanism in Polycystic ovary syndrome. Increased native thiol levels is related to fertilization rate among Polycystic ovary syndrome patients and also positive predictor marker of fertilization rate among Polycystic ovary syndrome patients. Improvement of thiol/disulphide homeostasis could be of importance in the treatment of Polycystic ovary syndrome to increase in vitro fertilization success in Polycystic ovary syndrome.

Intercalation of gaseous thiols and sulfides into Ag+ ion-exchanged aluminum dihydrogen triphosphate.

PubMed

Hayashi, Aki; Saimen, Hiroki; Watanabe, Nobuaki; Kimura, Hitomi; Kobayashi, Ayumi; Nakayama, Hirokazu; Tsuhako, Mitsutomo

2005-08-02

Ag(+) ion-exchanged layered aluminum dihydrogen triphosphate (AlP) with the interlayer distance of 0.85 nm was synthesized by the ion-exchange of proton in triphosphate with Ag(+) ion. The amount of exchanged Ag(+) ion depended on the concentration of AgNO(3) aqueous solution. Ag(+) ion-exchanged AlP adsorbed gaseous thiols and sulfides into the interlayer region. The adsorption amounts of thiols were more than those of sulfides, thiols with one mercapto group > thiol with two mercapto groups > sulfides, and depended on the amount of exchanged Ag(+) ion in the interlayer region. The thiols with one mercapto group were intercalated to expand the interlayer distance of Ag(+) ion-exchanged AlP, whereas there was no expansion in the adsorption of sulfide. In the case of thiol with two mercapto groups, there was observed contraction of the interlayer distance through the bridging with Ag(+) ions of the upper and lower sides of the interlayer region.

A Diversity-Oriented Library of Fluorophore-Modified Receptors Constructed from a Chemical Library of Synthetic Fluorophores.

PubMed

Nakano, Shun; Tamura, Tomoki; Das, Raj Kumar; Nakata, Eiji; Chang, Young-Tae; Morii, Takashi

2017-11-16

The practical application of biosensors can be determined by evaluating the sensing ability of fluorophore-modified derivatives of a receptor with appropriate recognition characteristics for target molecules. One of the key determinants for successfully obtaining a useful biosensor is wide variation in the fluorophores attached to a given receptor. Thus, using a larger fluorophore-modified receptor library provides a higher probability of obtaining a practically useful biosensor. However, no effective method has yet been developed for constructing such a diverse library of fluorophore-modified receptors. Herein, we report a method for constructing fluorophore-modified receptors by using a chemical library of synthetic fluorophores with a thiol-reactive group. This library was converted into a library of fluorophore-modified adenosine-binding ribonucleopeptide (RNP) receptors by introducing the fluorophores to the Rev peptide of the RNP complex by alkylation of the thiol group. This method enabled the construction of 263 fluorophore-modified ATP-binding RNP receptors and allowed the selection of suitable receptor-based fluorescent sensors that target ATP. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Substrate and pH-Dependent Kinetic Profile of 3-Mercaptopropionate Dioxygenase from Pseudomonas aeruginosa.

PubMed

Fellner, Matthias; Aloi, Sekotilani; Tchesnokov, Egor P; Wilbanks, Sigurd M; Jameson, Guy N L

2016-03-08

Thiol dioxygenases catalyze the synthesis of sulfinic acids in a range of organisms from bacteria to mammals. A thiol dioxygenase from the bacterium Pseudomonas aeruginosa oxidizes both 3-mercaptopropionic acid and cysteine, with a ∼70 fold preference for 3-mercaptopropionic acid over all pHs. This substrate reactivity is widened compared to other thiol dioxygenases and was exploited in this investigation of the residues important for activity. A simple model incorporating two protonation events was used to fit profiles of the Michaelis-Menten parameters determined at different pH values for both substrates. The pKs determined using plots of k(cat)/Km differ at low pH, but not in a way easily attributable to protonation of the substrate alone and share a common value at higher pH. Plots of k(cat) versus pH are also quite different at low pH showing the monoprotonated ES complexes with 3-mercaptopropionic acid and cysteine have different pKs. At higher pH, k(cat) decreases sigmoidally with a similar pK regardless of substrate. Loss of reactivity at high pH is attributed to deprotonation of tyrosine 159 and its influence on dioxygen binding. A mechanism is proposed by which deprotonation of tyrosine 159 both blocks oxygen binding and concomitantly promotes cystine formation. Finally, the role of tyrosine 159 was further probed by production of a G95C variant that is able to form a cysteine-tyrosine crosslink homologous to that found in mammalian cysteine dioxygenases. Activity of this variant is severely impaired. Crystallography shows that when un-crosslinked, the cysteine thiol excludes tyrosine 159 from its native position, while kinetic analysis shows that the thioether bond impairs reactivity of the crosslinked form.

Chemistry of the 8-Nitroguanine DNA Lesion: Reactivity, Labelling and Repair.

PubMed

Alexander, Katie J; McConville, Matthew; Williams, Kathryn R; Luzyanin, Konstantin V; O'Neil, Ian A; Cosstick, Richard

2018-02-26

The 8-nitroguanine lesion in DNA is increasingly associated with inflammation-related carcinogenesis, whereas the same modification on guanosine 3',5'-cyclic monophosphate generates a second messenger in NO-mediated signal transduction. Very little is known about the chemistry of 8-nitroguanine nucleotides, despite the fact that their biological effects are closely linked to their chemical properties. To this end, a selection of chemical reactions have been performed on 8-nitroguanine nucleosides and oligodeoxynucleotides. Reactions with alkylating reagents reveal how the 8-nitro substituent affects the reactivity of the purine ring, by significantly decreasing the reactivity of the N2 position, whilst the relative reactivity at N1 appears to be enhanced. Interestingly, the displacement of the nitro group with thiols results in an efficient and specific method of labelling this lesion and is demonstrated in oligodeoxynucleotides. Additionally, the repair of this lesion is also shown to be a chemically feasible reaction through a reductive denitration with a hydride source. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Roles of the Yap1 Transcription Factor and Antioxidants in Saccharomyces cerevisiae's Tolerance to Furfural and 5-Hydroxymethylfurfural, Which Function as Thiol-Reactive Electrophiles Generating Oxidative Stress

PubMed Central

Kim, Daehee

2013-01-01

Development of the tolerance of Saccharomyces cerevisiae strains to furfural and 5-hydroxymethylfurfural (HMF) is an important issue for cellulosic ethanol production. Although furfural and HMF are known to induce oxidative stress, the underlying mechanisms are largely unknown. In this study, we show that both furfural and HMF act as thiol-reactive electrophiles, thus directly activating the Yap1 transcription factor via the H2O2-independent pathway, depleting cellular glutathione (GSH) levels, and accumulating reactive oxygen species in Saccharomyces cerevisiae. However, furfural showed higher reactivity than did HMF toward GSH in vitro and in vivo. In line with such toxic mechanisms, overexpression of YAP1C620F, a constitutively active mutant of YAP1, and Yap1 target genes encoding catalases (CTA1 and CTT1) increased tolerance to furfural and HMF. However, increasing GSH levels by overexpression of genes for GSH biosynthesis (GSH1 and GLR1) or by the exogenous addition of GSH to the culture medium enhanced tolerance to furfural but not to HMF. PMID:23793623

Roles of the Yap1 transcription factor and antioxidants in Saccharomyces cerevisiae's tolerance to furfural and 5-hydroxymethylfurfural, which function as thiol-reactive electrophiles generating oxidative stress.

PubMed

Kim, Daehee; Hahn, Ji-Sook

2013-08-01

Development of the tolerance of Saccharomyces cerevisiae strains to furfural and 5-hydroxymethylfurfural (HMF) is an important issue for cellulosic ethanol production. Although furfural and HMF are known to induce oxidative stress, the underlying mechanisms are largely unknown. In this study, we show that both furfural and HMF act as thiol-reactive electrophiles, thus directly activating the Yap1 transcription factor via the H2O2-independent pathway, depleting cellular glutathione (GSH) levels, and accumulating reactive oxygen species in Saccharomyces cerevisiae. However, furfural showed higher reactivity than did HMF toward GSH in vitro and in vivo. In line with such toxic mechanisms, overexpression of YAP1(C620F), a constitutively active mutant of YAP1, and Yap1 target genes encoding catalases (CTA1 and CTT1) increased tolerance to furfural and HMF. However, increasing GSH levels by overexpression of genes for GSH biosynthesis (GSH1 and GLR1) or by the exogenous addition of GSH to the culture medium enhanced tolerance to furfural but not to HMF.

Influence of cysteine doping on photoluminescence intensity from semiconducting single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Kurnosov, N. V.; Leontiev, V. S.; Linnik, A. S.; Karachevtsev, V. A.

2015-03-01

Photoluminescence (PL) from semiconducting single-walled carbon nanotubes can be applied for detection of cysteine. It is shown that cysteine doping (from 10-8 to 10-3 M) into aqueous suspension of nanotubes with adsorbed DNA leads to increase of PL intensity. The PL intensity was enhanced by 27% at 10-3 M cysteine concentration in suspension. Most likely, the PL intensity increases due to the passivation of p-defects on the nanotube by the cysteine containing reactive thiol group. The effect of doping with other amino acids without this group (methionine, serine, aspartic acid, lysine, proline) on the PL intensity is essentially weaker.

Chemical synthesis of oligonucleotides containing a free sulphydryl group and subsequent attachment of thiol specific probes.

PubMed Central

Connolly, B A; Rider, P

1985-01-01

Oligonucleotides containing a free sulphydryl group at their 5'-termini have been synthesised and further derivatised with thiol specific probes. The nucleotide sequence required is prepared using standard solid phase phosphoramidite techniques and an extra round of synthesis is then performed using the S-triphenylmethyl O-methoxymorpholinophosphite derivatives of 2-mercaptoethanol, 3-mercaptopropan (1) ol or 6-mercaptohexan (1) ol. After cleavage from the resin and removal of the phosphate and base protecting groups, this yields an oligonucleotide containing an S-triphenylmethyl group attached to the 5'-phosphate group via a two, three or six carbon chain. The triphenylmethyl group can be readily removed with silver nitrate to give the free thiol. With the three and six carbon chain oligonucleotides, this thiol can be used, at pH 8, for the attachment of thiol specific probes as illustrated by the reaction with fluorescent conjugates of iodoacetates and maleiimides. However, oligonucleotides containing a thiol attached to the 5'-phosphate group via a two carbon chain are unstable at pH 8 decomposing to the free 5'-phosphate and so are unsuitable for further derivatisation. PMID:4011448

Acid-Activatable Michael-Type Fluorescent Probes for Thiols and for Labeling Lysosomes in Live Cells.

PubMed

Dai, Chun-Guang; Du, Xiao-Jiao; Song, Qin-Hua

2015-12-18

A Michael addition is usually taken as a base-catalyzed reaction. Most fluorescent probes have been designed to detect thiols in slightly alkaline solutions (pH 7-9). The sensing reactions of almost all Michael-type fluorescent probes for thiols are faster in a high pH solution than in a low pH solution. In this work, we synthesized a series of 7-substituted 2-(quinolin-2-ylmethylene)malonic acids (QMAs, substituents: NEt2, OH, H, Cl, or NO2) and their ethyl esters (QMEs) as Michael-type fluorescent probes for thiols. The sensing reactions of QMAs and QMEs occur in distinct pH ranges, pH < 7 for QMAs and pH > 7 for QMEs. On the basis of experimental and theoretic studies, we have clarified the distinct pH effects on the sensing reactivity between QMAs and QMEs and demonstrated that two QMAs (NEt2, OH) are highly sensitive and selective fluorescent probes for thiols in acidic solutions (pH < 7) and promising dyes that can label lysosomes in live cells.

The evolution of glutathione metabolism in phototrophic microorganisms

NASA Technical Reports Server (NTRS)

Fahey, R. C.; Buschbacher, R. M.; Newton, G. L.

1987-01-01

Of the many roles ascribed to glutathione (GSH) the one most clearly established is its role in the protection of higher eucaryotes against oxygen toxicity through destruction of thiol-reactive oxygen byproducts. If this is the primary function of GSH then GSH metabolism should have evolved during or after the evolution of oxygenic photosynthesis. That many bacteria do not produce GSH is consistent with this view. In the present study we have examined the low-molecular-weight thiol composition of a variety of phototrophic microorganisms to ascertain how evolution of GSH production is related to evolution of oxygenic photosynthesis. Cells were extracted in the presence of monobromobimane (mBBr) to convert thiols to fluorescent derivatives, which were analyzed by high-pressure liquid chromatography. Significant levels of GSH were not found in the green bacteria (Chlorobium thiosulfatophilum and Chloroflexus aurantiacus). Substantial levels of GSH were present in the purple bacteria (Chromatium vinosum, Rhodospirillum rubrum, Rhodobacter sphaeroides, and Rhodocyclus gelatinosa), the cyanobacteria [Anacystis nidulans, Microcoleus chthonoplastes S.G., Nostoc muscorum, Oscillatoria amphigranulata, Oscillatoria limnetica, Oscillatoria sp. (Stinky Spring, Utah), Oscillatoria terebriformis, Plectonema boryanum, and Synechococcus lividus], and eucaryotic algae (Chlorella pyrenoidsa, Chlorella vulgaris, Euglena gracilis, Scenedesmus obliquus, and Chlamydomonas reinhardtii). Other thiols measured included cysteine, gamma-glutamylcysteine, thiosulfate, coenzyme A, and sulfide; several unidentified thiols were also detected. Many of the organisms examined also exhibited a marked ability to reduce mBBr to syn-(methyl,methyl)bimane, an ability that was quenched by treatment with 2-pyridyl disulfide or 5,5'-bisdithio-(2-nitrobenzoic acid) prior to reaction with mBBr. These observations indicate the presence of a reducing system capable of electron transfer to mBBr and reduction of reactive disulfides. The distribution of GSH in phototrophic eubacteria indicates that GSH synthesis evolved at or around the time that oxygenic photosynthesis evolved.

Sunlight-Triggered Nanoparticle Synergy: Teamwork of Reactive Oxygen Species and Nitric Oxide Released from Mesoporous Organosilica with Advanced Antibacterial Activity.

PubMed

Gehring, Julia; Trepka, Bastian; Klinkenberg, Nele; Bronner, Hannah; Schleheck, David; Polarz, Sebastian

2016-03-09

Colonization of surfaces by microorganisms is an urging problem. In combination with the increasing antibiotic resistance of pathogenic bacteria, severe infections are reported more frequently in medical settings. Therefore, there is a large demand to explore innovative surface coatings that provide intrinsic and highly effective antibacterial activity. Materials containing silver nanoparticles have been developed in the past for this purpose, but this solution has come into criticism due to various disadvantages like notable toxicity against higher organisms, the high price, and low abundance of silver. Here, we introduce a new, sunlight-mediated organosilica nanoparticle (NP) system based on silver-free antibacterial activity. The simultaneous release of nitric oxide (NO) in combination with singlet oxygen and superoxide radicals (O2(•-)) as reactive oxygen species (ROS) leads to the emergence of highly reactive peroxynitrite molecules with significantly enhanced biocidal activity. This special cooperative effect can only be realized, if the ROS-producing moieties and the functional entities releasing NO are spatially separated from each other. In one type of particle, Rose Bengal as an efficient singlet oxygen ((1)O2) producer was covalently bound to SH functionalities applying thiol-ene click chemistry. "Charging" the second type of particles with NO was realized by quantitatively transferring the thiol groups into S-nitrosothiol functionalities. We probed the oxidation power of ROS-NP alone and in combination with NO-NP using sunlight as a trigger. The high antibacterial efficiency of dual-action nanoparticles was demonstrated using disinfection assays with the pathogenic bacterium Pseudomonas aeruginosa.

Thiol/disulfide homeostasis in asphalt workers.

PubMed

Yilmaz, Ömer Hınç; Bal, Ceylan; Neşelioglu, Salim; Büyükşekerci, Murat; Gündüzöz, Meşide; Eren, Funda; Tutkun, Lutfiye; Yilmaz, Fatma Meric

2016-09-02

The aim of this study was to investigate thiol/disulfide homeostasis in asphalt workers who are exposed to polycyclic aromatic hydrocarbons occupationally. The study was carried out in 34 nonsmoker asphalt workers. Additionally, 35 healthy nonsmoker volunteers were recruited as control group. Thiol and disulfide concentrations were determined using the novel automated measurement method. Levels of urinary 1-OH-pyrene were analyzed by liquid chromatography. Disulfide/thiol ratio was significantly higher in exposed group (p = .034). Also, a positive correlation was detected between disulfide/thiol ratio and 1-OH-pyrene values (r = .249, p = .036). Thiol/disulfide homeostasis was found to be disturbed in asphalt workers. The novel test used in this study may be useful for evaluating the oxidative status in polycyclic aromatic hydrocarbon (PAH) exposure.

Evaluation of dynamic thiol/disulphide homeostasis as a novel indicator of oxidative stress in maple syrup urine disease patients under treatment.

PubMed

Zubarioglu, Tanyel; Kiykim, Ertugrul; Cansever, Mehmet Serif; Neselioglu, Salim; Aktuglu-Zeybek, Cigdem; Erel, Ozcan

2017-02-01

Maple syrup urine disease (MSUD) is a metabolic disorder that is caused by deficiency of branched-chain α-keto acid dehydrogenase complex. Although accumulation of toxic metabolites is associated with neurotoxicity, mechanisms underlying brain damage remain unclear. Aim of this study is to evaluate thiol/disulphide homeostasis as a novel indicator of oxidative stress in MSUD patients under treatment. Twenty patients with MSUD and 20 healthy individuals were included in study. All patients were under regular follow-up and had a good metabolic control. Serum native thiol (-SH), total thiol (-SH + -S-S-), disulphide (-S-S) levels were measured in all subjects. Disulphide/native thiol, disulphide/total thiol and native thiol/total thiol ratios were calculated from these values. Simultaneous blood sampling for plasma quantitative amino acid analysis was performed in both groups. Any significant difference was not observed in -SH, -SH + -S-S-, -S-S levels between two groups. In addition no increase of disulphide/native thiol and disulphide/total thiol ratios was detected in patient group. This study is the first study that evaluates dynamic thiol/disulphide homeostasis as an indicator of oxidative stress in MSUD patients. Among previous studies that were made to determine oxidative stress in treated MSUD patients, this study had the largest sample size also. In recent studies, it was claimed that oxidative stress could be responsible from neurotoxicity even in treated patients. Here, dynamic thiol/disulfide homeostasis status showed that providing good metabolic control in MSUD patients prevent oxidative stress. Under regular follow-up and good compliance with diet, additional antioxidant therapies would possibly not be necessary.

Gel Permeation Chromatography Characterization of the Chain Length Distributions in Thiol-Acrylate Photopolymer Networks

PubMed Central

Rydholm, Amber E.; Held, Nicole L.; Bowman, Christopher N.; Anseth, Kristi S.

2008-01-01

Crosslinked, degradable networks formed from the photopolymerization of thiol and acrylate monomers are explored as potential biomaterials. The degradation behavior and material properties of these networks are influenced by the molecular weight of the nondegradable thiol-polyacrylate backbone chains that form during photopolymerization. Here, gel permeation chromatography was used to characterize the thiol-polyacrylate backbone chain lengths in degraded thiol-acrylate networks. Increasing thiol functionality from 1 to 4 increased the backbone molecular weight (M̄w = 2.3 ± 0.07 × 104 Da for monothiol and 3.6 ± 0.1 × 104 Da for tetrathiol networks). Decreasing thiol functional group concentration from 30 to 10 mol% also increased the backbone lengths (M̄w = 7.3 ± 1.1 × 104 Da for the networks containing 10 mol% thiol groups as compared to 3.6 ± 0.1 × 104 Da for 30 mol% thiol). Finally, the backbone chain lengths were probed at various stages of degradation and an increase in backbone molecular weight was observed as mass loss progressed from 10 to 70%. PMID:19079733

Synthesis of cyclic, multivalent Arg-Gly-Asp using sequential thiol-ene/thiol-yne photoreactions

PubMed Central

Aimetti, Alex A.; Feaver, Kristen R.

2014-01-01

A unique method has been developed for the formation of multivalent cyclic peptides. This procedure exploits on-resin peptide cyclization using a photoinitiated thiol-ene click reaction and subsequent clustering using thiol-yne photochemistry. Both reactions utilize the sulfhydryl group on natural cysteine amino acids to participate in the thiol-mediated reactions. PMID:20552127

Purification and characterization of multiple forms of the pineapple-stem-derived cysteine proteinases ananain and comosain.

PubMed Central

Napper, A D; Bennett, S P; Borowski, M; Holdridge, M B; Leonard, M J; Rogers, E E; Duan, Y; Laursen, R A; Reinhold, B; Shames, S L

1994-01-01

A mixture of ananain (EC 3.4.22.31) and comosain purified from crude pineapple stem extract was found to contain numerous closely related enzyme forms. Chromatographic separation of the major enzyme forms was achieved after treatment of the mixture with thiol-modifying reagents: reversible modification with 2-hydroxyethyl disulphide provided enzyme for kinetic studies, and irreversible alkylation with bromotrifluoroacetone or iodoacetamide gave enzyme for structural analyses by 19F-n.m.r. and electrospray mass spectrometry respectively. Structural and kinetic analyses revealed comosain to be closely related to stem bromelain (EC 3.4.22.32), whereas ananain differed markedly from both comosain and stem bromelain. Nevertheless, differences were seen between comosain and stem bromelain in amino acid composition and kinetic specificity towards the epoxide inhibitor E-64. Differences between five isolatable alternative forms of ananain were characterized by amidolytic activity, thiol stoichiometry and accurate mass determinations. Three of the enzyme forms displayed ananain-like amidolytic activity, whereas the other two forms were inactive. Thiol-stoichiometry determinations revealed that the active enzyme forms contained one free thiol, whereas the inactive forms lacked the reactive thiol required for enzyme activity. M.s. provided direct evidence for oxidation of the active-site thiol to the corresponding sulphinic acid. Images Figure 3 Figure 4 PMID:8053898

Thiol-Disulfide Exchange in Peptides Derived from Human Growth Hormone

PubMed Central

Chandrasekhar, Saradha; Epling, Daniel E.; Sophocleous, Andreas M.; Topp, Elizabeth M.

2014-01-01

Disulfide bonds stabilize proteins by crosslinking distant regions into a compact three-dimensional structure. They can also participate in hydrolytic and oxidative pathways to form non-native disulfide bonds and other reactive species. Such covalent modifications can contribute to protein aggregation. Here we present experimental data for the mechanism of thiol-disulfide exchange in tryptic peptides derived from human growth hormone in aqueous solution. Reaction kinetics were monitored to investigate the effect of pH (6.0-10.0), temperature (4-50 °C), oxidation suppressants (EDTA and N2 sparging) and peptide secondary structure (amide cyclized vs. open form). The concentrations of free thiol containing peptides, scrambled disulfides and native disulfide-linked peptides generated via thiol-disulfide exchange and oxidation reactions were determined using RP-HPLC and LC-MS. Concentration vs. time data were fitted to a mathematical model using non-linear least squares regression analysis. At all pH values, the model was able to fit the data with R2≥0.95. Excluding oxidation suppressants (EDTA and N2 sparging) resulted in an increase in the formation of scrambled disulfides via oxidative pathways but did not influence the intrinsic rate of thiol-disulfide exchange. In addition, peptide secondary structure was found to influence the rate of thiol-disulfide exchange. PMID:24549831

Involvement of calcium and calmodulin in oxidative and temperature stress of Amaranthus lividus L. during early germination.

PubMed

Bhattacharjee, Soumen

2009-07-01

Both heat and chilling caused reduction in membrane protein thiol level and increased accumulation of thiobarbituric acid reactive substances in 72 hr old germinating tissues (indicators of oxidative stress) and reduced germination and early growth performances. Calcium chelator EGTA [Ethylene glycol-bis (2-aminoethylether)-N, N,N',N, tetra acetic acid] calcium channel blocker LaCI3 (Lanthanum chloride) and calmodulin inhibitor TFP (trifluroperazine) aggravated these effects of heat and chilling and added calcium reversed them. Imposition of heat and chilling stress during early germination also causes accumulation of reactive oxygen species (ROS) like 02(-) and H2O2. Calcium treatment significantly reduced the accumulation of both the toxic ROS, while EGTA, LaCl3 and TFP treatment enhanced the accumulation. Activities of antioxidative enzymes catalase (CAT), ascorbate peroxidase (APOX) and glutathione reductase (GR) and total thiol content decreased significantly under both heat and chilling stress in germinating Amaranthus seedlings. Seedlings raised with Ca2+ treatment under heat and chilling stress exhibit higher activities of CAT7 GR and APOX and total thiol level than the untreated plants. EGTA, LaCl3 and TFP treatment, on the other hand significantly reduce the activities of all anti-oxidative enzymes and total thiol level. The work clearly supports the view that Ca2+-signalling pathway plays significant role in limiting heat and chilling induced oxidative stress by upregulating antioxidative defense during recovery phase of post-germination event in Amaranthus lividus.

The First MS-Cleavable, Photo-Thiol-Reactive Cross-Linker for Protein Structural Studies

NASA Astrophysics Data System (ADS)

Iacobucci, Claudio; Piotrowski, Christine; Rehkamp, Anne; Ihling, Christian H.; Sinz, Andrea

2018-04-01

Cleavable cross-linkers are gaining increasing importance for chemical cross-linking/mass spectrometry (MS) as they permit a reliable and automated data analysis in structural studies of proteins and protein assemblies. Here, we introduce 1,3-diallylurea (DAU) as the first CID-MS/MS-cleavable, photo-thiol-reactive cross-linker. DAU is a commercially available, inexpensive reagent that efficiently undergoes an anti-Markovnikov hydrothiolation with cysteine residues in the presence of a radical initiator upon UV-A irradiation. Radical cysteine cross-linking proceeds via an orthogonal "click reaction" and yields stable alkyl sulfide products. DAU reacts at physiological pH and cross-linking reactions with peptides, and proteins can be performed at temperatures as low as 4 °C. The central urea bond is efficiently cleaved upon collisional activation during tandem MS experiments generating characteristic product ions. This improves the reliability of automated cross-link identification. Different radical initiators have been screened for the cross-linking reaction of DAU using the thiol-containing compounds cysteine and glutathione. Our concept has also been exemplified for the biologically relevant proteins bMunc13-2 and retinal guanylyl cyclase-activating protein-2. [Figure not available: see fulltext.

Highly Reactive Thiol-Norbornene Photo-Click Hydrogels: Toward Improved Processability.

PubMed

Van Hoorick, Jasper; Gruber, Peter; Markovic, Marica; Rollot, Mélanie; Graulus, Geert-Jan; Vagenende, Maxime; Tromayer, Maximilian; Van Erps, Jürgen; Thienpont, Hugo; Martins, José C; Baudis, Stefan; Ovsianikov, Aleksandr; Dubruel, Peter; Van Vlierberghe, Sandra

2018-06-10

In the present work, gelatin type B is modified with highly reactive norbornene functionalities (Gel-NB) following a one-pot synthesis approach to enable subsequent thiol-ene photo-click crosslinking. The modification strategy displays close control over the amount of introduced functionalities. Additionally, Gel-NB exhibits considerably improved processing capabilities in terms of two-photon polymerization when benchmarked to earlier-reported crosslinkable gelatin derivatives (e.g., gelatin-methacrylamide (Gel-MOD) and gelatin-methacrylamide-aminoethylmethacrylate (Gel-MOD-AEMA)). The improvement is especially apparent in terms of minimally required laser power (20 mW vs ≥60 mW (Gel-MOD) vs ≥40 mW (Gel-MOD-AEMA) at 100 mm s -1 scan speed) and processable concentration range (≥5 w/v% vs ≥10 w/v% (Gel-MOD/Gel-MOD-AEMA)). Furthermore, the proposed functionalization scheme maintains the excellent biocompatibility and cell interactivity of gelatin. Additionally, the norbornene functionalities have potential for straightforward postprocessing "thiol-ene" surface grafting of active molecules. As a consequence, a very promising material toward tissue engineering applications and more specifically, biofabrication, is presented. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Furoxans (1,2,5-Oxadiazole-N-Oxides) as Novel NO Mimetic Neuroprotective and Procognitive Agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schiefer, Isaac T.; VandeVrede, Lawren; Fa

2012-08-31

Furoxans (1,2,5-oxadiazole-N-oxides) are thiol-bioactivated NO-mimetics that have not hitherto been studied in the CNS. Incorporation of varied substituents adjacent to the furoxan ring system led to modulation of reactivity toward bioactivation, studied by HPLC-MS/MS analysis of reaction products. Attenuated reactivity unmasked the cytoprotective actions of NO in contrast to the cytotoxic actions of higher NO fluxes reported previously for furoxans. Neuroprotection was observed in primary neuronal cell cultures following oxygen glucose deprivation (OGD). Neuroprotective activity was observed to correlate with thiol-dependent bioactivation to produce NO{sub 2}{sup -}, but not with depletion of free thiol itself. Neuroprotection was abrogated upon cotreatmentmore » with a sGC inhibitor, ODQ, thus supporting activation of the NO/sGC/CREB signaling cascade by furoxans. Long-term potentiation (LTP), essential for learning and memory, has been shown to be potentiated by NO signaling, therefore, a peptidomimetic furoxan was tested in hippocampal slices treated with oligomeric amyloid-{beta} peptide (A{beta}) and was shown to restore synaptic function. The novel observation of furoxan activity of potential therapeutic use in the CNS warrants further studies.« less

Redox-Active Sensing by Bacterial DksA Transcription Factors Is Determined by Cysteine and Zinc Content

PubMed Central

Crawford, Matthew A.; Tapscott, Timothy; Fitzsimmons, Liam F.; Liu, Lin; Reyes, Aníbal M.; Libby, Stephen J.; Trujillo, Madia; Fang, Ferric C.; Radi, Rafael

2016-01-01

ABSTRACT The four-cysteine zinc finger motif of the bacterial RNA polymerase regulator DksA is essential for protein structure, canonical control of the stringent response to nutritional limitation, and thiol-based sensing of oxidative and nitrosative stress. This interdependent relationship has limited our understanding of DksA-mediated functions in bacterial pathogenesis. Here, we have addressed this challenge by complementing ΔdksA Salmonella with Pseudomonas aeruginosa dksA paralogues that encode proteins differing in cysteine and zinc content. We find that four-cysteine, zinc-bound (C4) and two-cysteine, zinc-free (C2) DksA proteins are able to mediate appropriate stringent control in Salmonella and that thiol-based sensing of reactive species is conserved among C2 and C4 orthologues. However, variations in cysteine and zinc content determine the threshold at which individual DksA proteins sense and respond to reactive species. In particular, zinc acts as an antioxidant, dampening cysteine reactivity and raising the threshold of posttranslational thiol modification with reactive species. Consequently, C2 DksA triggers transcriptional responses in Salmonella at levels of oxidative or nitrosative stress normally tolerated by Salmonella expressing C4 orthologues. Inappropriate transcriptional regulation by C2 DksA increases the susceptibility of Salmonella to the antimicrobial effects of hydrogen peroxide and nitric oxide, and attenuates virulence in macrophages and mice. Our findings suggest that the redox-active sensory function of DksA proteins is finely tuned to optimize bacterial fitness according to the levels of oxidative and nitrosative stress encountered by bacterial species in their natural and host environments. PMID:27094335

Application of Atmospheric Pressure Photoionization H/D-exchange Mass Spectrometry for Speciation of Sulfur-containing Compounds.

PubMed

Acter, Thamina; Kim, Donghwi; Ahmed, Arif; Ha, Ji-Hyoung; Kim, Sunghwan

2017-08-01

Herein we report the observation of atmospheric pressure in-source hydrogen-deuterium exchange (HDX) of thiol group for the first time. The HDX for thiol group was optimized for positive atmospheric pressure photoionization (APPI) mass spectrometry (MS). The optimized HDX-MS was applied for 31 model compounds (thiols, thiophenes, and sulfides) to demonstrate that exchanged peaks were observed only for thiols. The optimized method has been successfully applied to the isolated fractions of sulfur-rich oil samples. The exchange of one and two thiol hydrogens with deuterium was observed in the thiol fraction; no HDX was observed in the other fractions. Thus, the results presented in this study demonstrate that the HDX-MS method using APPI ionization source can be effective for speciation of sulfur compounds. This method has the potential to be used to access corrosion problems caused by thiol-containing compounds. Graphical Abstract ᅟ.

Surface functionalization of gold nanoparticles using hetero-bifunctional poly(ethylene glycol) spacer for intracellular tracking and delivery

PubMed Central

Shenoy, Dinesh; Fu, Wei; Li, Jane; Crasto, Curtis; Jones, Graham; DiMarzio, Charles; Sridhar, Srinivas; Amiji, Mansoor

2006-01-01

For the development of surface-functionalized gold nanoparticles as cellular probes and delivery agents, we have synthesized hetero-bifunctional poly(ethylene glycol) (PEG, MW 1500) having a thiol group on one terminus and a reactive functional group on the other for use as a flexible spacer. Coumarin, a model fluorescent dye, was conjugated to one end of the PEG spacer and gold nanoparticles were modified with coumarin-PEG-thiol. Surface attachment of coumarin through the PEG spacer decreased the fluorescence quenching effect of gold nanoparticles. The results of cellular cytotoxicity and fluorescence confocal analyses showed that the PEG spacer-modified nanoparticles were essentially non-toxic and could be efficiently internalized in the cells within 1 hour of incubation. Intracellular particle tracking using a Keck 3-D Fusion Microscope System showed that the functionalized gold nanoparticles were rapidly internalized in the cells and localized in the peri-nuclear region. Using the PEG spacer, the gold nano-platform can be conjugated with a variety of biologically relevant ligands such as fluorescent dyes, antibodies, etc in order to target, probe, and induce a stimulus at the target site. PMID:16467923

Rethinking Cysteine Protective Groups: S-Alkylsulfonyl-l-Cysteines for Chemoselective Disulfide Formation.

PubMed

Schäfer, Olga; Huesmann, David; Muhl, Christian; Barz, Matthias

2016-12-12

The ability to reversibly cross-link proteins and peptides grants the amino acid cysteine its unique role in nature as well as in peptide chemistry. We report a novel class of S-alkylsulfonyl-l-cysteines and N-carboxy anhydrides (NCA) thereof for peptide synthesis. The S-alkylsulfonyl group is stable against amines and thus enables its use under Fmoc chemistry conditions and the controlled polymerization of the corresponding NCAs yielding well-defined homo- as well as block co-polymers. Yet, thiols react immediately with the S-alkylsulfonyl group forming asymmetric disulfides. Therefore, we introduce the first reactive cysteine derivative for efficient and chemoselective disulfide formation in synthetic polypeptides, thus bypassing additional protective group cleavage steps. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pyridoxylamine reactivity kinetics as an amine based nucleophile for screening electrophilic dermal sensitizers

PubMed Central

Chipinda, Itai; Mbiya, Wilbes; Adigun, Risikat Ajibola; Morakinyo, Moshood K.; Law, Brandon F.; Simoyi, Reuben H.; Siegel, Paul D.

2015-01-01

Chemical allergens bind directly, or after metabolic or abiotic activation, to endogenous proteins to become allergenic. Assessment of this initial binding has been suggested as a target for development of assays to screen chemicals for their allergenic potential. Recently we reported a nitrobenzenethiol (NBT) based method for screening thiol reactive skin sensitizers, however, amine selective sensitizers are not detected by this assay. In the present study we describe an amine (pyridoxylamine (PDA)) based kinetic assay to complement the NBT assay for identification of amine-selective and non-selective skin sensitizers. UV-Vis spectrophotometry and fluorescence were used to measure PDA reactivity for 57 chemicals including anhydrides, aldehydes, and quinones where reaction rates ranged from 116 to 6.2 × 10−6 M−1 s−1 for extreme to weak sensitizers, respectively. No reactivity towards PDA was observed with the thiol-selective sensitizers, non-sensitizers and prohaptens. The PDA rate constants correlated significantly with their respective murine local lymph node assay (LLNA) threshold EC3 values (R2 = 0.76). The use of PDA serves as a simple, inexpensive amine based method that shows promise as a preliminary screening tool for electrophilic, amine-selective skin sensitizers. PMID:24333919

Synchrotron radiation based STXM analysis and micro-XRF mapping of differential expression of extracellular thiol groups by Acidithiobacillus ferrooxidans grown on Fe(2+) and S(0).

PubMed

Xia, Jin-Lan; Liu, Hong-Chang; Nie, Zhen-Yuan; Peng, An-An; Zhen, Xiang-Jun; Yang, Yun; Zhang, Xiu-Li

2013-09-01

The differential expression of extracellular thiol groups by Acidithiobacillus ferrooxidans grown on substrates Fe(2+) and S(0) was investigated by using synchrotron radiation based scanning transmission X-ray microscopy (STXM) imaging and microbeam X-ray fluorescence (μ-XRF) mapping. The extracellular thiol groups (SH) were first alkylated by iodoacetic acid forming Protein-SCH2COOH and then the P-SCH2COOH was marked by calcium ions forming P-SCH2COOCa. The STXM imaging and μ-XRF mapping of SH were based on analysis of SCH2COO-bonded Ca(2+). The results indicated that the thiol group content of A. ferrooxidans grown on S(0) is 3.88 times to that on Fe(2+). Combined with selective labeling of SH by Ca(2+), the STXM imaging and μ-XRF mapping provided an in situ and rapid analysis of differential expression of extracellular thiol groups. © 2013.

Investigation of thiol-disulphide balance in patients with acute urticaria and chronic spontaneous urticaria.

PubMed

Akbas, Ayse; Kilinc, Fadime; Sener, Sertac; Aktaş, Akın; Baran, Pervin; Ergin, Merve

2017-09-01

Thiol-disulphide balance plays a major role in health and diseases. This balance may be disrupted by various diseases. We aimed to determine status of the effect of thiol-disulphide balance in urticaria. We aimed to investigate the thiol-disulphide balance in patients with acute urticaria (AUP) and chronic spontaneous urticaria (CSU). Study included 53 AUP and 47 healthy controls plus 57 patients with chronic spontaneous urticaria (CSUP) and 57 healthy controls. Levels of native thiols, disulphides and total thiols were evaluated in plasma using a new and automated spectrophotometric method. Ratios of disulphides/total thiols, disulphides/native thiols and native thiols/total thiols were calculated. For AU, there was no statistical difference compared to control group in levels of native thiols, disulphides and total thiols. For CSU, however, there was an increase in levels of native thiols, disulphides and total thiols and the ratio of thiol/disulphide in favour of disulphide. Thiol-disulphide balance was not affected by AU but shifted towards to disulphide in CSU indicating the presence of oxidative stress (OS).

Mangiferin, a natural occurring glucosyl xanthone, increases susceptibility of rat liver mitochondria to calcium-induced permeability transition.

PubMed

Andreu, Gilberto Lázaro Pardo; Delgado, René; Velho, Jesus Antonio; Curti, Carlos; Vercesi, Anibal E

2005-07-15

Mitochondrial permeability transition (MPT) is a Ca(2+)-dependent, cyclosporine A-sensitive, non-selective inner membrane permeabilization induced by a wide range of agents or conditions, which has often been associated with necrotic or apoptotic cell death. When mitochondria isolated from livers of rats treated with the natural occurring glucosyl xanthone mangiferin (40 mg/kg body weight) were exposed in vitro to Ca(2+), they underwent CsA, NEM, and ADP-sensitive high amplitude swelling and associated membrane potential dissipation, release of pre-accumulated Ca(2+), oxidation of thiol groups, and depletion of GSH, without changes in the NAD(P)H redox state. The same treatment reduced the phosphorylation rate of mitochondria and the resting respiration by around 4 and 11%, respectively, as well as generation of reactive oxygen species (ROS) by organelle. The in vitro exposure of untreated mitochondria to mangiferin plus Ca(2+) also resulted in oxidation of thiol groups, in the same way that the compound inhibited the Ca(2+)-induced peroxidation of mitochondrial membrane lipids. The spectrum of mangiferin during its oxidation by the H(2)O(2)/HRP system showed a characteristic absorption peak at 380 nm, which decreased immediately after reaction was started; two isosbestic points at around 336 and 412 nm, with a blue shift in the position of the maxima absorption of mangiferin were observed, suggesting their conversion into one oxidation product. Glutathione abolished this decrease of absorbance, suggesting that the oxidation product of mangiferin forms adducts with GSH. We propose that Ca(2+) increases levels of mitochondria-generated ROS, which reacts with mangiferin producing quinoid derivatives, which in turn react with the most accessible mitochondrial thiol groups, thus triggering MPT. It seems probable that the free radical scavenging activity of mangiferin shifts its anti-oxidant protection to the thiol arylation. An interesting proposition is that accumulation of mangiferin quinoid products would take place in cells exposed to an overproduction of ROS, such as cancer cells, where the occurrence of MPT-mediated apoptosis may be a cellular defence mechanism against excessive ROS formation.

Promoting Thiol Expression Increases The Durability of Antitumor T cell Functions

PubMed Central

Scurti, Gina; Thyagarajan, Krishnamurthy; Kaur, Navtej; Husain, Shahid; Fang, Quan; Naga, Osama S.; Simms, Patricia; Beeson, Gyda; Voelkel-Johnson, Christina; Garrett-Mayer, Elizabeth; Beeson, Craig C.; Nishimura, Michael I.; Mehrotra, Shikhar

2014-01-01

Ex vivo-expanded CD8+ T cells used for adoptive immunotherapy generally acquire an effector memory-like phenotype (TEM cells). With regard to therapeutic applications, two undesired features of this phenotype in vivo are limited persistence and reduced anti-tumor efficacy, relative to CD8+ T cells with a central memory-like phenotype (TCM cells). Further, there is incomplete knowledge about all the differences between TEM and TCM cells that may influence tumor treatment outcomes. Given that TCM cells survive relatively longer in oxidative tumor microenvironments, we investigated the hypothesis that TCM possess relatively greater anti-oxidative capacity than TEM cells. Here we report that TCM cells exhibit a relative increase compared to TEM cells in expression of cell surface thiols, a key target of cellular redox controls, along with other antioxidant molecules. Increased expression of redox regulators in TCM cells inversely correlated with the generation of reactive oxygen and nitrogen species, proliferative capacity and glycolytic enzyme levels. Notably, TCR-transduced T cells pretreated with thiol donors, such as N-acetyl cysteine or rapamycin, up-regulated thiol levels and antioxidant genes. A comparison of anti-tumor CD8+ T cell populations on the basis of surface thiol expression showed that thiol-high cells persisted longer in vivo and exerted superior tumor control. Our results suggest that higher levels of reduced cell surface thiols are a key characteristic of T cells that can control tumor growth, and that profiling this biomarker may have benefits to T cell adoptive immunotherapy protocols. PMID:25164014

Thiol/disulfide homeostasis in patients with ankylosing spondylitis

PubMed Central

Dogru, Atalay; Balkarli, Ayse; Cetin, Gozde Yildirim; Neselioglu, Salim; Erel, Ozcan; Tunc, Sevket Ercan; Sahin, Mehmet

2016-01-01

Ankylosing spondylitis (AS) is a chronic inflammatory disease. In many inflammatory diseases, increased production of pro-inflammatory cytokines is associated with an increase in oxidative stress mediators. Thiol/disulfide homeostasis is a marker for oxidative stress. The aim of this study was to examine the dynamic thiol/disulfide homeostasis in AS. Sixty-nine patients with AS and 60 age- and sex-matched controls were included in the study. The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and visual analogue scale (VAS) were used to determine the disease activity. Native thiol, total thiol, and disulfide levels were measured with a novel automated method recently described by Erel and Neselioglu. The aforementioned method is also optionally manual spectrophotometric assay. The total thiol levels were significantly lower in the AS group compared with the control group (p = 0.03). When the patients were divided into active (n = 35) and inactive (n = 34) subgroups using BASDAI scores, the native plasma thiol and total thiol levels were significantly lower in the active AS patients compared to the inactive AS patients (p = 0.02, p = 0.03 respectively). There was a negative correlation between the plasma native thiol levels and VAS, BASDAI scores. Thiol/disulfide homeostasis may be used for elucidating the effects of oxidative stress in AS. Understanding the role of thiol/disulfide homeostasis in AS might provide new therapeutic intervention strategies for patients. PMID:27186972

Effects of Gold Salt Speciation and Structure of Human and Bovine Serum Albumins on the Synthesis and Stability of Gold Nanostructures

PubMed Central

Miranda, Érica G. A.; Tofanello, Aryane; Brito, Adrianne M. M.; Lopes, David M.; Albuquerque, Lindomar J. C.; de Castro, Carlos E.; Costa, Fanny N.; Giacomelli, Fernando C.; Ferreira, Fabio F.; Araújo-Chaves, Juliana C.; Nantes, Iseli L.

2016-01-01

The present study aimed to investigate the influence of albumin structure and gold speciation on the synthesis of gold nanoparticles (GNPs). The strategy of synthesis was the addition of HAuCl4 solutions at different pH values (3–12) to solutions of human and bovine serum albumins (HSA and BSA) at the same corresponding pH values. Different pH values influence the GNP synthesis due to gold speciation. Besides the inherent effect of pH on the native structure of albumins, the use N-ethylmaleimide (NEM)-treated and heat-denaturated forms of HSA and BSA provided additional insights about the influence of protein structure, net charge, and thiol group approachability on the GNP synthesis. NEM treatment, heating, and the extreme values of pH promoted loss of the native albumin structure. The formation of GNPs indicated by the appearance of surface plasmon resonance (SPR) bands became detectable from 15 days of the synthesis processes that were carried out with native, NEM-treated and heat-denaturated forms of HSA and BSA, exclusively at pH 6 and 7. After 2 months of incubation, SPR band was also detected for all synthesis carried out at pH 8.0. The mean values of the hydrodynamic radius (RH) were 24 and 34 nm for GNPs synthesized with native HSA and BSA, respectively. X-ray diffraction (XRD) revealed crystallites of 13 nm. RH, XRD, and zeta potential values were consistent with GNP capping by the albumins. However, the GNPs produced with NEM-treated and heat-denaturated albumins exhibited loss of protein capping by lowering the ionic strength. This result suggests a significant contribution of non-electrostatic interactions of albumins with the GNP surface, in these conditions. The denaturation of proteins exposes hydrophobic groups to the solvent, and these groups could interact with the gold surface. In these conditions, the thiol blockage or oxidation, the latter probably favored upon heating, impaired the formation of a stable capping by thiol coordination with the gold surface. Therefore, the cysteine side chain of albumins is important for the colloidal stabilization of GNPs rather than as the reducing agent for the synthesis. Despite the presence of more reactive gold species at more acidic pH values, i.e., below 6.0, in these conditions the loss of native albumin structure impaired GNP synthesis. Alkaline pH values (9–12) combined the unfavorable conditions of denaturated protein structure with less reactive gold species. Therefore, an optimal condition for the synthesis of GNPs using serum albumins involves more reactive gold salt species combined with a reducing and negatively charged form of the protein, all favored at pH 6–7. PMID:27066476

Oxidative stress and decreased thiol level in patients with migraine: cross-sectional study.

PubMed

Eren, Yasemin; Dirik, Ebru; Neşelioğlu, Salim; Erel, Özcan

2015-12-01

Although migraine is a neurological disorder known since long, its physiopathology remains unclear. Recent studies suggest that migraine is associated with oxidative stress; however, they report divergent results. The aim of the present study was to evaluate total antioxidant status (TAS), total oxidant status (TOS), oxidative stress index (OSI), and serum thiol level in migraine patients with or without aura. The study group consisted of 141 migraine patients. The control group included 70 healthy subjects. TAS, TOS, OSI were evaluated using a method developed by Erel. Serum thiol level was measured using the Hu method. No difference was found in TAS, TOS, OSI between the patients and controls. The level of thiol was significantly lower in patients than in controls. Negative correlations were detected between thiol level and Migraine Disability Assessment score in patients. Although TAS, TOS, and OSI were similar to those of the control group, serum thiol level, an important marker of antioxidant capacity, was significantly lower in migraines compared with controls, and caused more serious disability. Novel treatment approaches may be developed based on these data, and compounds containing thiol, such as alpha lipoic acid and N-acetyl cysteine, may be used in prophylaxis.

Hydropersulfides: H-Atom Transfer Agents Par Excellence.

PubMed

Chauvin, Jean-Philippe R; Griesser, Markus; Pratt, Derek A

2017-05-10

Hydropersulfides (RSSH) are formed endogenously via the reaction of the gaseous biotransmitter hydrogen sulfide (H 2 S) and disulfides (RSSR) and/or sulfenic acids (RSOH). RSSH have been investigated for their ability to store H 2 S in vivo and as a line of defense against oxidative stress, from which it is clear that RSSH are much more reactive to two-electron oxidants than thiols. Herein we describe the results of our investigations into the H-atom transfer chemistry of RSSH, contrasting it with the well-known H-atom transfer chemistry of thiols. In fact, RSSH are excellent H-atom donors to alkyl (k ∼ 5 × 10 8 M -1 s -1 ), alkoxyl (k ∼ 1 × 10 9 M -1 s -1 ), peroxyl (k ∼ 2 × 10 6 M -1 s -1 ), and thiyl (k > 1 × 10 10 M -1 s -1 ) radicals, besting thiols by as little as 1 order and as much as 4 orders of magnitude. The inherently high reactivity of RSSH to H-atom transfer is based largely on thermodynamic factors; the weak RSS-H bond dissociation enthalpy (∼70 kcal/mol) and the associated high stability of the perthiyl radical make the foregoing reactions exothermic by 15-34 kcal/mol. Of particular relevance in the context of oxidative stress is the reactivity of RSSH to peroxyl radicals, where favorable thermodynamics are bolstered by a secondary orbital interaction in the transition state of the formal H-atom transfer that drives the inherent reactivity of RSSH to match that of α-tocopherol (α-TOH), nature's premier radical-trapping antioxidant. Significantly, the reactivity of RSSH eclipses that of α-TOH in H-bond-accepting media because of their low H-bond acidity (α 2 H ∼ 0.1). This affords RSSH a unique versatility compared to other highly reactive radical-trapping antioxidants (e.g., phenols, diarylamines, hydroxylamines, sulfenic acids), which tend to have high H-bond acidities. Moreover, the perthiyl radicals that result are highly persistent under autoxidation conditions and undergo very rapid dimerization (k = 5 × 10 9 M -1 s -1 ) in lieu of reacting with O 2 or autoxidizable substrates.

Biological Chemistry and Functionality of Protein Sulfenic Acids and Related Thiol Modifications

PubMed Central

Devarie-Baez, Nelmi O.; Silva Lopez, Elsa I.; Furdui, Cristina M.

2016-01-01

Selective modification of proteins at cysteine residues by reactive oxygen, nitrogen or sulfur species formed under physiological and pathological states is emerging as a critical regulator of protein activity impacting cellular function. This review focuses primarily on protein sulfenylation (-SOH), a metastable reversible modification connecting reduced cysteine thiols to many products of cysteine oxidation. An overview is first provided on the chemistry principles underlining synthesis, stability and reactivity of sulfenic acids in model compounds and proteins, followed by a brief description of analytical methods currently employed to characterize these oxidative species. The following chapters present a selection of redox-regulated proteins for which the -SOH formation was experimentally confirmed and linked to protein function. These chapters are organized based on the participation of these proteins in the regulation of signaling, metabolism and epigenetics. The last chapter discusses the therapeutic implications of altered redox microenvironment and protein oxidation in disease. PMID:26340608

Certain tricyclic and pentacyclic-hetero nitrogen rhodol dyes

DOEpatents

Haugland, Richard P.; Whitaker, James E.

1993-01-01

Novel fluorescent dyes based on the rhodol structure are provided. The new reagents contain functional groups capable of forming a stable fluorescent product with functional groups typically found in biomolecules or polymers including amines, phenols, thiols, acids, aldehydes and ketones. Reactive groups in the rhodol dyes include activated esters, isothiocyanates, amines, hydrazines, halides, acids, azides, maleimides, aldehydes, alcohols, acrylamides and haloacetamides. The products are detected by their absorbance or fluorescence properties. The spectral properties of the fluorescent dyes are sufficiently similar in wavelengths and intensity to fluorescein or rhodamine derivatives as to permit use of the same equipment. The dyes, however, show less spectral sensitivity to pH in the physiological range than does fluorescein, have higher solubility in non-polar solvents and have improved photostability and quantum yields.

Is early cord clamping, delayed cord clamping or cord milking best?

PubMed

Vatansever, Binay; Demirel, Gamze; Ciler Eren, Elif; Erel, Ozcan; Neselioglu, Salim; Karavar, Hande Nur; Gundogdu, Semra; Ulfer, Gozde; Bahadir, Selcen; Tastekin, Ayhan

2018-04-01

To compare the antioxidant status of three cord clamping procedures (early clamping, delayed clamping and milking) by analyzing the thiol-disulfide balance. This randomized controlled study enrolled 189 term infants who were divided into three groups according to the cord clamping procedure: early clamping, delayed clamping and milking. Blood samples were collected from the umbilical arteries immediately after clamping, and the thiol/disulfide homeostasis was analyzed. The native and total thiol levels were significantly (p < .05) lower in the early cord clamping group compared with the other two groups. The disulfide/total thiol ratio was significantly (p = .026) lower in the delayed cord clamping and milking groups compared with the early clamping groups. Early cord clamping causes the production of more disulfide bonds and lower thiol levels, indicating that oxidation reactions are increased in the early cord clamping procedure compared with the delayed cord clamping and milking procedures. The oxidant capacity is greater with early cord clamping than with delayed clamping or cord milking. Delayed cord clamping or milking are beneficial in neonatal care, and we suggest that they be performed routinely in all deliveries.

[Evaluation of the treatment with D-chiro-inositol on levels of oxidative stress in PCOS patients].

PubMed

De Leo, V; La Marca, A; Cappelli, V; Stendardi, A; Focarelli, R; Musacchio, M C; Piomboni, P

2012-12-01

Recent studies on the pathophysiology of infertility have shown that oxidative stress (OS) can be one of the causal factors. The OS is, by definition, an imbalance between the production of reactive oxygen species (ROS) and antioxidant defense systems. It seems that oxidative stress plays an important role in almost all phases of human reproduction. In fact, ROS are involved in the modulation of a large spectrum of reproductive functions such as oocyte maturation, ovarian steroidogenesis, corpus luteum functions and are involved in the processes of fertilization, embryo development and pregnancy, but also in some diseases that cause infertility. Polycystic ovary syndrome (PCOS) has recently been associated with increased oxidative stress, often put in relation to the syndrome's typical metabolic disorder. Inositol is an intracellular mediator of insulin, currently much used as a therapeutic agent in PCOS. While its main action takes place via insulin sensitization, little is known about the possible effects of other disorders, such as oxidative stress, associated with PCOS. The purpose of this study was therefore to assess the effect of D-chiro-inositol on the state of oxidative stress in the follicular fluid of women with PCOS. Follicular fluids were obtained from women who have turned to the Center for Diagnosis and Treatment of Sterility of Obstetrics and Gynecology of the University Hospital of Siena and Modena diagnosed with PCOS. The women were treated with D-chiro-inositol (500 mg x 2 per day) for 3 months before being subjected to cycles of in vitro fertilization (IVF). The state of oxidative stress was measured by marking of free thiol groups of proteins in the follicular fluid with 3-(N-Maleimidopropionyl)-biocytin. In our study we obtained a lesser presence of free thiol protein groups equal to 77.8% in the follicular fluid of women with PCOS not treated with D-chiro-inositolo, compared to patients who instead have carried out such treatment. These results suggest that in PCOS women there is an increase of the oxidation of thiol groups of proteins follicular, correlated to a progressive increase of the oxidative stress and that the administration of D-chiro-inositol in patients with this disease seems to reduce the oxidation of thiol groups.

Selenocysteine in proteins-properties and biotechnological use.

PubMed

Johansson, Linda; Gafvelin, Guro; Arnér, Elias S J

2005-10-30

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK(a) of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of 77Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.

Reaction kinetics and targeting to cellular glutathione S-transferase of the glutathione peroxidase mimetic PhSeZnCl and its D,L-polylactide microparticle formulation.

PubMed

Bartolini, D; Piroddi, M; Tidei, C; Giovagnoli, S; Pietrella, D; Manevich, Y; Tew, K D; Giustarini, D; Rossi, R; Townsend, D M; Santi, C; Galli, F

2015-01-01

Catalytic properties and cellular effects of the glutathione peroxidase (GPx)-mimetic compound PhSeZnCl or its d,l-lactide polymer microencapsulation form (M-PhSeZnCl) were investigated and compared with the prototypical Se-organic compounds ebselen and diselenide (PhSe)2. PhSeZnCl was confirmed to catalyze the ping-pong reaction of GPx with higher Vmax than ebselen and (PhSe)2, but the catalytic efficiency calculated for the cosubstrates glutathione (GSH) and H2O2, and particularly the high reactivity against thiols (lowest KM for GSH in the series of test molecules), suggested poor biological applicability of PhSeZnCl as a GPx mimetic. Cytotoxicity of PhSeZnCl was demonstrated in various cancer cell lines via increased reactive oxygen species (ROS) generation, depletion of intracellular thiols, and induction of apoptosis. Experiments carried out in GSH S-transferase P (GSTP)-overexpressing K562 human erythroleukemia cells and in GSTP1-1-knockout murine embryonic fibroblasts (MEFs) demonstrated that this cytosolic enzyme represents a preferential target of the redox disturbances produced by this Se-compound with a key role in controlling H2O2 generation and the perturbation of stress/survival kinase signaling. Microencapsulation was adopted as a strategy to control the thiol reactivity and oxidative stress effects of PhSeZnCl, then assessing applications alternative to anticancer. The uptake of this "depowered" GPx-mimetic formulation, which occurred through an endocytosis-like mechanism, resulted in a marked reduction of cytotoxicity. In MCF-7 cells transfected with different allelic variants of GSTP, M-PhSeZnCl lowered the burst of cellular ROS induced by the exposure to extracellular H2O2, and the extent of this effect changed between the GSTP variants. Microencapsulation is a straightforward strategy to mitigate the toxicity of thiol-reactive Se-organic drugs that enhanced the antioxidant and cellular protective effects of PhSeZnCl. A mechanistic linkage of these effects with the expression pattern and signaling properties of GSTP . This has overcome the GPx-mimetic paradigm proposed for Se-organic drugs with a more pragmatic concept of GSTP signaling modulators. Copyright © 2014 Elsevier Inc. All rights reserved.

Reaction kinetics and targeting to cellular glutathione S-transferase of the glutathione peroxidase mimetic PhSeZnCl and its d,l-polylactide microparticle formulation

PubMed Central

Bartolini, D.; Piroddi, M.; Tidei, C.; Giovagnoli, S.; Pietrella, D.; Manevich, Y.; Tew, K.D.; Giustarini, D.; Rossi, R.; Townsend, D.M.; Santi, C.; Galli, F.

2015-01-01

Catalytic properties and cellular effects of the glutathione peroxidase (GPx)-mimetic compound PhSeZnCl or its d,l-lactide polymer microencapsulation form (M-PhSeZnCl) were investigated and compared with the prototypical Se-organic compounds ebselen and diselenide (PhSe)2. PhSeZnCl was confirmed to catalyze the ping-pong reaction of GPx with higher Vmax than ebselen and (PhSe)2, but the catalytic efficiency calculated for the cosubstrates glutathione (GSH) and H2O2, and particularly the high reactivity against thiols (lowest KM for GSH in the series of test molecules), suggested poor biological applicability of PhSeZnCl as a GPx mimetic. Cytotoxicity of PhSeZnCl was demonstrated in various cancer cell lines via increased reactive oxygen species (ROS) generation, depletion of intracellular thiols, and induction of apoptosis. Experiments carried out in GSH S-transferase P (GSTP)-overexpressing K562 human erythroleukemia cells and in GSTP1-1-knockout murine embryonic fibroblasts (MEFs) demonstrated that this cytosolic enzyme represents a preferential target of the redox disturbances produced by this Se-compound with a key role in controlling H2O2 generation and the perturbation of stress/survival kinase signaling. Microencapsulation was adopted as a strategy to control the thiol reactivity and oxidative stress effects of PhSeZnCl, then assessing applications alternative to anticancer. The uptake of this “depowered” GPx-mimetic formulation, which occurred through an endocytosis-like mechanism, resulted in a marked reduction of cytotoxicity. In MCF-7 cells transfected with different allelic variants of GSTP, M-PhSeZnCl lowered the burst of cellular ROS induced by the exposure to extracellular H2O2, and the extent of this effect changed between the GSTP variants. Microencapsulation is a straightforward strategy to mitigate the toxicity of thiol-reactive Se-organic drugs that enhanced the antioxidant and cellular protective effects of PhSeZnCl. A mechanistic linkage of these effects with the expression pattern and signaling properties of GSTP. This has overcome the GPx-mimetic paradigm proposed for Se-organic drugs with a more pragmatic concept of GSTP signaling modulators. PMID:25452145

Inhibition of the ATPase from Halobacterium Saccharovorum by Thiol Inhibitors: Evidence for the Presence of More Than One Essential Cysteinyl Residue

NASA Technical Reports Server (NTRS)

Hochstein, Lawrence I.; Emrich, Errol; Stan-Lotter, Helga; DeVincenzi, Donald L. (Technical Monitor)

1995-01-01

The vacuolar-like ATPase from Halobacterium saccha vorum is inhibited by N-ethylmaleimide and p-chloromercudphenylsulfonate. The failure of adenine nucleotides to protect against p-chloromercuriphenyisulfonate inhibition, of p-chloromercuriphenylsulfonate to protect against N-ethylmaleimide inhibition, and the difference in the temperature dependence of inactivation infers that the enzyme contains at least two thiols that are essential for enzyme activity. CNBr cleavage of C-14-N-ethylmaleimide labeled subunit results in two radioactive peptides that locates the N-ethylmaleimide-reactive cysteinyl residue as cysteine-262 in the H. salinarium sequence.

Disulphide bond exchange inhibited by air - kinetic and thermodynamic products in a library of macrocyclic cysteine derivatives.

PubMed

Cholewiak, Agnieszka; Dobrzycki, Łukasz; Jurczak, Janusz; Ulatowski, Filip

2018-04-04

In this paper we present the synthesis and reactivity of dithiols comprising of two cysteine moieties attached to a dipicolinic acid core. Oxidation of these thiols provides oligomeric macrocycles. Monomers with 13-membered rings are kinetic products which are, however, strained and readily transform into higher oligomers under basic conditions or elevated temperature via a disulphide exchange reaction. Dimers, which are the most stable thermodynamic products, equilibrate only under inert conditions with thiolate as a catalyst. Under aerobic conditions, the thiols are oxidised before the equilibrium is reached.

Protective effect of lipoic acid on cyclophosphamide-induced testicular toxicity.

PubMed

Selvakumar, Elangovan; Prahalathan, Chidambaram; Sudharsan, Periyasamy Thandavan; Varalakshmi, Palaninathan

2006-05-01

Cyclophosphamide (CP), a widely used anticancer and immunosuppressive drug causes severe testicular toxicity. We investigated the protective effect of lipoic acid in CP-induced testicular toxicity. Two groups of male Wistar rats (140+/-20 g) were administered CP (15 mg/kg body weight, oral gavage) once a week for 10 weeks to induce testicular toxicity; one of these groups received lipoic acid treatment (35 mg/kg body weight, i.p., 24 h prior to CP administration) once a week for 10 weeks. A vehicle treated control and a lipoic acid control groups were also included. The untreated CP exposed rats showed a significant increase in testicular reactive oxygen species (ROS) level, along with a significant decrease in cellular thiol levels. The activities of testicular marker enzymes such as gamma-glutamyl transferase, beta-glucuronidase, acid phosphatase and alkaline phosphatase were increased whereas the activities of sorbitol dehydrogenase and lactate dehydrogenase-X were decreased significantly in the animals treated with CP. In contrast, rats pretreated with lipoic acid showed normal marker enzymic patterns and normal levels of ROS and thiols. Testicular protection by lipoic acid is further substantiated by the normal histologic findings as against shrunken seminiferous tubules with impaired spermatogenesis in the CP administered rats. By the reversal of biochemical and morphological changes towards normalcy, the cytoprotective role of lipoic acid is illuminated in CP-induced testicular toxicity.

Aluminum exposure for one hour decreases vascular reactivity in conductance and resistance arteries in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmidt, Patrícia Medeiros; Escobar, Alyne Goulart

Aims: Aluminum (Al) is an important environmental contaminant; however, there are not enough evidences of Al-induced cardiovascular dysfunction. We investigated the effects of acute exposure to aluminum chloride (AlCl{sub 3}) on blood pressure, vascular reactivity and oxidative stress. Methods and results: Male Wistar rats were divided into two groups: Untreated: vehicle (ultrapure water, ip) and AlCl{sub 3}: single dose of AlCl{sub 3} (100 mg/kg,ip). Concentration-response curves to phenylephrine in the absence and presence of endothelium, the nitric oxide synthase inhibitor L-NAME, the potassium channel blocker tetraethylammonium, and the NADPH oxidase inhibitor apocynin were performed in segments from aortic and mesentericmore » resistance arteries. NO released was assessed in aorta and reactive oxygen species (ROS), malondialdehyde, non-protein thiol levels, antioxidant capacity and enzymatic antioxidant activities were investigated in plasma, aorta and/or mesenteric arteries. After one hour of AlCl{sub 3} exposure serum Al levels attained 147.7 ± 25.0 μg/L. Al treatment: 1) did not affect blood pressure, heart rate and vasodilator responses induced by acetylcholine or sodium nitroprusside; 2) decreased phenylephrine-induced vasoconstrictor responses; 3) increased endothelial modulation of contractile responses, NO release and vascular ROS production from NADPH oxidase; 4) increased plasmatic, aortic and mesenteric malondialdehyde and ROS production, and 5) decreased antioxidant capacity and affected the antioxidant biomarkers non-protein thiol levels, glutathione peroxidase, glutathione-S-transferase, superoxide dismutase and catalase enzymatic activities. Conclusion: AlCl{sub 3}-acute exposure reduces vascular reactivity. This effect is associated with increased NO production, probably acting on K{sup +} channels, which seems to occur as a compensatory mechanism against Al-induced oxidative stress. Our results suggest that Al exerts toxic effects to the vascular system. - Highlights: • One hour of aluminum exposure decreases vascular reactivity. • Vascular imbalance after aluminum exposure is related to increased oxidative stress. • Enhanced NO release is the first step against the effects of Al at vascular system.« less

Conversion of S–phenylsulfonylcysteine residues to mixed disulfides at pH 4.0: utility in protein thiol blocking and in protein–S–nitrosothiol detection

PubMed Central

Reeves, B. D.; Joshi, N.; Campanello, G. C.; Hilmer, J. K.; Chetia, L.; Vance, J. A.; Reinschmidt, J. N.; Miller, C. G.; Giedroc, D. P.; Dratz, E. A.; Singel, D. J.; Grieco, P. A.

2014-01-01

A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: 1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); 2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and 3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR*-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both the initial blocking step as well as in the conversion of protein-S-sulfonylcysteines to form stable fluorescent disulfides. Labelling was monitored by TOF-MS and gel electrophoresis. Proteolysis and peptide analysis of the resulting digest identified the cysteine residues containing mixed disulfides bearing the fluorescent probe, Rhod-SH. PMID:24986430

Amaranth addition to enzymatically modified wheat flour improves dough functionality, bread immunoreactivity and quality.

PubMed

Heredia-Sandoval, N G; Calderón de la Barca, A M; Carvajal-Millán, E; Islas-Rubio, A R

2018-01-24

Consumers with gluten-related disorders require gluten-free (GF) foods to avoid an immune response. Alternative to the use of non-gluten containing grains to prepare GF bread, the gluten reactivity has been greatly reduced using a proline specific cleavage enzyme, however, the gluten functionality was lost. The aim of this study was to evaluate the effect of adding an amaranth flour blend (AFB) to enzymatically modified wheat-flour proteins on dough functionality and to evaluate the immunoreactivity and acceptability of the prepared bread. First, wheat flour (20% w/v, substrate) was hydrolyzed using 8.4 U mg -1 protein Aspergillus niger prolyl-endopeptidase (AnPEP) for 8 h at 40 °C under constant agitation. Four types of breads were prepared with the same formulation except for the type of flour (14% w.b.): wheat flour (WF), WF-AFB unmodified not incubated, WF-AFB unmodified incubated and WF-AFB modified. The protein composition and free thiols were analyzed before and after amaranth addition, and the flour and bread proteins were run using SDS-PAGE and immune-detected in blots with IgA from celiac disease patients. The immunoreactive gluten content, specific volume and bread acceptability were evaluated. The polymeric proteins and free thiol groups of WF decreased after AnPEP treatment. The electrophoretic patterns of the modified flour and bread proteins were different and the IgA-immunodetection in blots was highly reduced, particularly for the higher molecular weight subunits. The addition of AFB to the modified wheat flour prepared using AnPEP improved the dough functionality by increasing the thiol groups and allowed the preparation of a sensorially acceptable bread with only 60 mg kg -1 immunoreactive gluten.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rouyer-Fessard, P.; Garel, M.C.; Domenget, C.

The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with ({sup 3}H)N-ethylmaleimide. This pool of soluble alpha chains was 0.067 {plus minus} 0.017% of hemoglobin in blood of normal adult, 0.11 {plus minus} 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% inmore » the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using ({sup 3}H)N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.« less

Redox Energy and Sulfur Chemistry in Prebiotic Polymer Synthesis and Replication

NASA Technical Reports Server (NTRS)

Weber, Arthur L.

1997-01-01

In the past year we have made significant progress in three research areas: (1) Most importantly, we discovered a new pathway of prebiotic amino acid synthesis in which formaldehyde and glycolaldehyde (substrates of the formose reaction) react with ammonia yielding alanine and homoserine in the presence of thiol catalysts. This thiol-dependent synthesis of amino acids undoubtedly occurs via amino acid thioester intermediates capable of forming peptides. This 'one-pot' reaction system operates under mild aqueous conditions, and like modern amino acid biosynthesis, uses sugar intermediates which are converted to amino acids by energy-yielding redox disproportionation. (2) Finally, in preparation for the analysis of Martian meteorite samples, we upgraded our HPLC system and developed an improved method capable of detecting a I femtomole of amino acid enantiomers. (3) We completed our analysis of the energetics of metabolism that revealed that life depends on biosynthetic processes driven by chemical energy made available by the redox disproportionation of carbon groups of sugars. We established that the favorable energy of redox disproportionation is based on the universal reduction potentials of carbon groups. We concluded that it is hard to imagine any other organic molecule besides sugars (formaldehyde oligomers) having the energy and reactivity needed to drive either modem biosynthesis or the chemical processes behind its origin.

Arsenolysis and Thiol-Dependent Arsenate Reduction

EPA Science Inventory

Conversion of arsenate to arsenite is a critical event in the pathway that leads from inorganic arsenic to a variety of methylated metabolites. The formation of methylated metabolites influences distribution and retention of arsenic and affects the reactivity and toxicity of thes...

Formation of methemoglobin and phenoxyl radicals from p-hydroxyanisole and oxyhemoglobin.

PubMed

Stolze, K; Nohl, H

1991-01-01

The reaction of p-hydroxyanisole with oxyhemoglobin was investigated using electron spin resonance spectroscopy (ESR) and visible spectroscopy. As a reactive reaction intermediate we found the p-methoxyphenoxyl radical, the one-electron oxidation product of p-hydroxyanisole. Detection of this species required the rapid flow device elucidating the instability of this radical intermediate. The second reaction product formed is methemoglobin. Catalase or SOD had no effect upon the reaction kinetics. Accordingly, reactive oxygen species such as hydroxyl radicals or superoxide could not be observed although the spin trapping agent DMPO was used to make these short-lived species detectable. When the sulfhydryl blocking agents N-ethylmaleimide or mersalyl acid were used, an increase of the methemoglobin formation rate and of the phenoxyl radical concentration were observed. We have interpreted this observation in terms of a side reaction of free radical intermediates with thiol groups.

An evaluation of thiol/disulphide homeostasis in patients with psoriasis

PubMed Central

Yorulmaz, Ahu; Erdogan, Serpil; Cakmak, Seray Kulcu; Guney, Elif; Sen, Orhan; Erel, Ozcan

2017-01-01

Introduction The role of oxidative stress in the pathogenesis of psoriasis has been investigated in previous studies with conflicting results. On the other hand, well-established treatments currently used in psoriasis exert their effects via a boost of oxidative stress. Recently, a strong positive association between psoriasis, metabolic syndrome and dyslipidemia has also been described showing the complex nature of the disease. Aim To examine thiol/disulphide homeostasis, a newly developed homeostasis assay in psoriasis and evaluate the possible association between thiol/disulphide homeostasis and dyslipidemia in psoriasis. Material and methods The study population included 92 psoriasis patients and 71 healthy subjects. Serum native thiol, total thiol and disulphide levels were investigated in patients with psoriasis and in healthy subjects. In addition, lipid profile (serum total cholesterol, triglyceride, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol) levels were investigated in both groups. The association between thiol-disulphide parameters and dyslipidemia was also evaluated. Results Serum total cholesterol and triglyceride levels were found to be higher in patients with psoriasis than in the healthy group. Lower plasma disulphide and higher native thiol levels were found in patients with psoriasis indicating an antioxidant status. Conclusions To our knowledge, this is the first study showing the shift of dynamic thiol/disulphide homeostasis towards the thiol form in psoriasis which indicate higher antioxidant status. PMID:29507562

Thiol dioxygenase turnover yields benzothiazole products from 2-mercaptoaniline and O2-dependent oxidation of primary alcohols.

PubMed

Morrow, William P; Sardar, Sinjinee; Thapa, Pawan; Hossain, Mohammad S; Foss, Frank W; Pierce, Brad S

2017-10-01

Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O 2 -dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO 2 - ). Previous chemical rescue studies identified a putative Fe III -O 2 - intermediate that precedes substrate oxidation in Mus musculus cysteine dioxygenase (Mm CDO). Given that a similar reactive intermediate has been identified in the extradiol dioxygenase 2, 3-HCPD, it is conceivable that these enzymes share other mechanistic features with regard to substrate oxidation. To explore this possibility, enzymatic reactions with Mm CDO (as well as the bacterial 3-mercaptopropionic acid dioxygenase, Av MDO) were performed using a substrate analogue (2-mercaptoaniline, 2ma). This aromatic thiol closely approximates the catecholic substrate of homoprotocatechuate of 2, 3-HPCD while maintaining the 2-carbon thiol-amine separation preferred by Mm CDO. Remarkably, both enzymes exhibit 2ma-gated O 2 -consumption; however, none of the expected products for thiol dioxygenase or intra/extradiol dioxygenase reactions were observed. Instead, benzothiazoles are produced by the condensation of 2ma with aldehydes formed by an off-pathway oxidation of primary alcohols added to aqueous reactions to solubilize the substrate. The observed oxidation of 1º-alcohols in 2ma-reactions is consistent with the formation of a high-valent intermediate similar to what has been reported for cytochrome P450 and mononuclear iron model complexes. Copyright © 2017 Elsevier Inc. All rights reserved.

Synthesis of soybean oil-based thiol oligomers.

PubMed

Wu, Jennifer F; Fernando, Shashi; Weerasinghe, Dimuthu; Chen, Zhigang; Webster, Dean C

2011-08-22

Industrial grade soybean oil (SBO) and thiols were reacted to generate thiol-functionalized oligomers via a thermal, free radical initiated thiol-ene reaction between the SBO double bond moieties and the thiol functional groups. The effect of the reaction conditions, including thiol concentration, catalyst loading level, reaction time, and atmosphere, on the molecular weight and the conversion to the resultant soy-thiols were examined in a combinatorial high-throughput fashion using parallel synthesis, combinatorial FTIR, and rapid gel permeation chromatography (GPC). High thiol functionality and concentration, high thermal free radical catalyst concentration, long reaction time, and the use of a nitrogen reaction atmosphere were found to favor fast consumption of the SBO, and produced high molecular weight products. The thiol conversion during the reaction was inversely affected by a high thiol concentration, but was favored by a long reaction time and an air reaction atmosphere. These experimental observations were explained by the initial low affinity of the SBO and thiol, and the improved affinity between the generated soy-thiol oligomers and unreacted SBO during the reaction. The synthesized soy-thiol oligomers can be used for renewable thiol-ene UV curable materials and high molecular solids and thiourethane thermal cure materials. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamic thiol/disulphide homeostasis in patients with basal cell carcinoma.

PubMed

Demirseren, Duriye Deniz; Cicek, Cagla; Alisik, Murat; Demirseren, Mustafa Erol; Aktaş, Akın; Erel, Ozcan

2017-09-01

The aim of this study is to measure and compare the dynamic thiol/disulphide homeostasis of patients with basal cell carcinoma and healthy subjects with a newly developed and original method. Thirty four patients attending our outpatient clinic and clinically and histopathologically diagnosed as nodular basal cell carcinoma, and age and gender matched 30 healthy individuals have been involved in the study. Thiol/disulphide homeostasis tests have been measured with a novel automatic spectrophotometric method developed and the results have been compared statistically. Serum native thiol and disulphide levels in the patient and control group show a considerable variance statistically (p = 0.028, 0.039, respectively). Total thiol levels do not reveal a considerable variation (p = 0.094). Disulphide/native thiol ratios and native thiol/total thiol ratios also show a considerable variance statistically (p = 0.012, 0.013, 0.010, respectively). Thiol disulphide homeostasis in patients with basal cell carcinoma alters in the way that disulphide gets lower and thiols get higher. Thiol/disulphide level is likely to have a role in basal cell carcinoma pathogenesis.

Functional thiols as repair and doping agents of defective MoS2 monolayers

NASA Astrophysics Data System (ADS)

Förster, Anja; Gemming, Sibylle; Seifert, Gotthard

2018-06-01

Recent experimental and theoretical studies indicate that thiols (R-SH) can be used to repair sulfur vacancy defects in MoS2 monolayers (MLs). This density functional theory study investigates how the thiol repair mechanism process can be used to dope MoS2 MLs. Fluorinated thiols as well as amine-containing ones are used to p- and n-dope the MoS2 ML, respectively. It is shown that functional groups are only physisorbed on the repaired MoS2 surface. This explains the reversible doping with fluorinated thiols.

Quinone-induced protein modifications: Kinetic preference for reaction of 1,2-benzoquinones with thiol groups in proteins.

PubMed

Li, Yuting; Jongberg, Sisse; Andersen, Mogens L; Davies, Michael J; Lund, Marianne N

2016-08-01

Oxidation of polyphenols to quinones serves as an antioxidative mechanism, but the resulting quinones may induce damage to proteins as they react through a Michael addition with nucleophilic groups, such as thiols and amines to give protein adducts. In this study, rate constants for the reaction of 4-methylbenzoquinone (4MBQ) with proteins, thiol and amine compounds were determined under pseudo first-order conditions by UV-vis stopped-flow spectrophotometry. The chemical structures of the adducts were identified by LC-ESI-MS/MS. Proteins with free thiols were rapidly modified by 4MBQ with apparent second order rate constants, k2 of (3.1±0.2)×10(4)M(-1)s(-1) for bovine serum albumin (BSA) and (4.8±0.2)×10(3)M(-1)s(-1) for human serum albumin at pH 7.0. These values are at least 12-fold greater than that for α-lactalbumin (4.0±0.2)×10(2)M(-1)s(-1), which does not contain any free thiols. Reaction of Cys-34 of BSA with N-ethylmaleimide reduced the thiol concentration by ~59%, which resulted in a decrease in k2 by a similar percentage, consistent with rapid adduction at Cys-34. Reaction of 4MBQ with amines (Gly, Nα-acetyl-l-Lys, Nε-acetyl-l-Lys and l-Lys) and the guanidine group of Nα-acetyl-l-Arg was at least 5×10(5) slower than with low-molecular-mass thiols (l-Cys, Nα-acetyl-l-Cys, glutathione). The thiol-quinone interactions formed colorless thiol-phenol products via an intermediate adduct, while the amine-quinone interactions generated colored amine-quinone products that require oxygen involvement. These data provide strong evidence for rapid modification of protein thiols by quinone species which may be of considerable significance for biological and food systems. Copyright © 2016 Elsevier Inc. All rights reserved.

Functionalization of Bambusurils by thiol-ene click reaction and new, facile method for the preparation of anion-free Bambus[6]urils.

PubMed

Heck, Marie-Pierre; Azazna, Djamille; Lafosse, Marine; Wang, Jialan; Rivollier, Julie; Ben Cheikh, Imen; Meyer, Michel; Thuéry, Pierre; Dognon, Jean-Pierre; Huber, Gaspard

2018-05-03

New sulfide-functionalized bambus[4]urils ((RS)8BU[4]) and bambus[6]urils ((RS)12BU[6]) have been synthesized through thiol-ene click coupling reactions (TEC) of allylbambus[n]urils. Synthesis of BU[6] derivatives always requires the use of a template anion (iodide, chloride or bromide) which is enclosed in the cavity of BU[6]. We show that this anion influences the reactivity of bambus[6]urils. An encapsulated iodide makes allyl functions of allyl12BU[6] less reactive towards TEC and hydrogenation reactions in comparison to the corresponding chloride or bromide inclusion complexes. This is critical for the chemical reactivity of BU[6] and even more to determine their anion-binding properties. We report a new, facile and fast method using AgSbF6 to prepare anion-free BU[6]. NMR methods were used to estimate association constants of these new empty BU[6] with different anions. Quantum chemistry calculations were employed to rationalize the observed results. These new functionalized bambusuril scaffolds in alternate conformation could find applications as multivalent binders. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photonics and microarray technology

NASA Astrophysics Data System (ADS)

Skovsen, E.; Duroux, M.; Neves-Petersen, M. T.; Duroux, L.; Petersen, S. B.

2007-05-01

Photonic induced immobilization of biosensor molecules is a novel technology that results in spatially oriented and spatially localized covalent coupling of a large variety of biomolecules onto thiol reactive surfaces, e.g. thiolated glass, quartz, gold or silicon. The reaction mechanism behind the reported new technology involves light-induced breakage of disulphide bridges in proteins upon UV illumination of nearby aromatic amino acids resulting in the formation of reactive molecules that will form covalent bonds with thiol reactive surfaces. This new technology has the potential of replacing present micro dispensing arraying technologies, where the size of the individual sensor spots are limited by the size of the dispensed droplets. Using light-induced immobilization the spatial resolution is defined by the area of the sensor surface that is illuminated by UV light and not by the physical size of the dispensed droplets of sensor molecules. This new technology allows for dense packing of different biomolecules on a surface, allowing the creation of multi-potent functionalized materials, such as biosensors with micrometer sized individual sensor spots. Thus, we have developed the necessary technology for preparing large protein arrays of enzymes and fragments of antibodies, with micrometer resolution, without the need for liquid micro dispensing.

The thiol of human serum albumin: Acidity, microenvironment and mechanistic insights on its oxidation to sulfenic acid.

PubMed

Bonanata, Jenner; Turell, Lucía; Antmann, Laura; Ferrer-Sueta, Gerardo; Botasini, Santiago; Méndez, Eduardo; Alvarez, Beatriz; Coitiño, E Laura

2017-07-01

Human serum albumin (HSA) has a single reduced cysteine residue, Cys34, whose acidity has been controversial. Three experimental approaches (pH-dependence of reactivity towards hydrogen peroxide, ultraviolet titration and infrared spectroscopy) are used to determine that the pK a value in delipidated HSA is 8.1±0.2 at 37°C and 0.1M ionic strength. Molecular dynamics simulations of HSA in the sub-microsecond timescale show that while sulfur exposure to solvent is limited and fluctuating in the thiol form, it increases in the thiolate, stabilized by a persistent hydrogen-bond (HB) network involving Tyr84 and bridging waters to Asp38 and Gln33 backbone. Insight into the mechanism of Cys34 oxidation by H 2 O 2 is provided by ONIOM(QM:MM) modeling including quantum water molecules. The reaction proceeds through a slightly asynchronous S N 2 transition state (TS) with calculated Δ ‡ G and Δ ‡ H barriers at 298K of respectively 59 and 54kJmol -1 (the latter within chemical accuracy from the experimental value). A post-TS proton transfer leads to HSA-SO - and water as products. The structured reaction site cages H 2 O 2 , which donates a strong HB to the thiolate. Loss of this HB before reaching the TS modulates Cys34 nucleophilicity and contributes to destabilize H 2 O 2 . The lack of reaction-site features required for differential stabilization of the TS (positive charges, H 2 O 2 HB strengthening) explains the striking difference in kinetic efficiency for the same reaction in other proteins (e.g. peroxiredoxins). The structured HB network surrounding HSA-SH with sequestered waters carries an entropic penalty on the barrier height. These studies contribute to deepen the understanding of the reactivity of HSA-SH, the most abundant thiol in human plasma, and in a wider perspective, provide clues on the key aspects that modulate thiol reactivity against H 2 O 2 . Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibition of Carbamyl Phosphate Synthetase-I and Glutamine Synthetase by Hepatotoxic Doses of Acetaminophen in Mice

PubMed Central

Gupta, Sanjiv; Rogers, Lynette K.; Taylor, Sarah K.; Smith, Charles V.

2016-01-01

The primary mechanisms proposed for acetaminophen-induced hepatic necrosis should deplete protein thiols, either by covalent binding and thioether formation or by oxidative reactions such as S-thiolations. However, in previous studies we did not detect significant losses of protein thiol contents in response to administration of hepatotoxic doses of acetaminophen in vivo. In the present study we employed derivatization with the thiol-specific agent monobromobimane and separation of proteins by SDS–PAGE to investigate the possible loss of specific protein thiols during the course of acetaminophen-induced hepatic necrosis. Fasted adult male mice were given acetaminophen, and protein thiol status was examined subsequently in subcellular fractions isolated by differential centrifugation. No decreases in protein thiol contents were indicated, with the exception of a marked decrease in the fluorescent intensity, but not of protein content, as indicated by staining with Coomassie blue, of a single band of approximately 130 kDa in the mitochondrial fractions of acetaminophen-treated mice. This protein was identified by isolation and N-terminal sequence analysis as carbamyl phosphate synthetase-I (CPS-I) (EC 6.3.4.16). Hepatic CPS-I activities were decreased in mice given hepatotoxic doses of acetaminophen. In addition, hepatic glutamine synthetase activities were lower, and plasma ammonia levels were elevated in mice given hepatotoxic doses of acetaminophen. The observed hyperammonemia may contribute to the adverse effects of toxic doses of acetaminophen, and elucidation of the specific mechanisms responsible for the hyperammonemia may prove to be useful clinically. However, the preferential depletion of protein thiol content of a mitochondrial protein by chemically reactive metabolites generated in the endoplasmic reticulum presents a challenging and potentially informative mechanistic question. PMID:9344900

Thiol Specific and Mitochondria Selective Fluorogenic Benzofurazan Sulfide for Live Cell Nonprotein Thiol Imaging and Quantification in Mitochondria.

PubMed

Wang, Shenggang; Yin, Huihui; Huang, Yue; Guan, Xiangming

2018-06-11

Cellular thiols are divided into two major categories: nonprotein thiols (NPSH) and protein thiols (PSH). Thiols are unevenly distributed inside the cell and compartmentalized in subcellular structures. Most of our knowledge on functions/dysfunctions of cellular/subcellular thiols is based on the quantification of cellular/subcellular thiols through homogenization of cellular/subcellular structures followed by a thiol quantification method. We would like to report a thiol-specific mitochondria-selective fluorogenic benzofurazan sulfide {7,7'-thiobis( N-rhodamine-benzo[c][1,2,5]oxadiazole-4-sulfonamide) (TBROS)} that can effectively image and quantify live cell NPSH in mitochondria through fluorescence intensity. Limited methods are available for imaging thiols in mitochondria in live cells especially in a quantitative manner. The thiol specificity of TBROS was demonstrated by its ability to react with thiols and inability to react with biologically relevant nucleophilic functional groups other than thiols. TBROS, with minimal fluorescence, formed strong fluorescent thiol adducts (λ ex = 550 nm, λ em = 580 nm) when reacting with NPSH confirming its fluorogenicity. TBROS failed to react with PSH from bovine serum albumin and cell homogenate proteins. The high mitochondrial thiol selectivity of TBROS was achieved by its mitochondria targeting structure and its higher reaction rate with NPSH at mitochondrial pH. Imaging of mitochondrial NPSH in live cells was confirmed by two colocalization methods and use of a thiol-depleting reagent. TBROS effectively imaged NPSH changes in a quantitative manner in mitochondria in live cells. The reagent will be a useful tool in exploring physiological and pathological roles of mitochondrial thiols.

Combinational chelation therapy abrogates lead-induced neurodegeneration in rats.

PubMed

Pachauri, Vidhu; Saxena, Geetu; Mehta, Ashish; Mishra, Deepshikha; Flora, Swaran J S

2009-10-15

Lead, a ubiquitous and potent neurotoxicant causes oxidative stress which leads to numerous neurobehavioral and physiological alterations. The ability of lead to bind sulfhydryl groups or compete with calcium could be one of the reasons for its debilitating effects. In the present study, we addressed: i) if chelation therapy could circumvent the altered oxidative stress and prevent neuronal apoptosis in chronic lead-intoxicated rats, ii) whether chelation therapy could reverse biochemical and behavioral changes, and iii) if mono or combinational therapy with captopril (an antioxidant) and thiol chelating agents (DMSA/MiADMSA) is more effective than individual thiol chelator in lead-exposed rats. Results indicated that lead caused a significant increase in reactive oxygen species, nitric oxide, and intracellular free calcium levels along with altered behavioral abnormalities in locomotor activity, exploratory behavior, learning, and memory that were supported by changes in neurotransmitter levels. A fall in membrane potential, release of cytochrome c, and DNA damage indicated mitochondrial-dependent apoptosis. Most of these alterations showed significant recovery following combined therapy with captopril with MiADMSA and to a smaller extend with captopril+DMSA over monotherapy with these chelators. It could be concluded from our present results that co-administration of a potent antioxidant (like captopril) might be a better treatment protocol than monotherapy to counter lead-induced oxidative stress. The major highlight of the work is an interesting experimental evidence of the efficacy of combinational therapy using an antioxidant with a thiol chelator in reversing neurological dystrophy caused due to chronic lead exposure in rats.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pachauri, Vidhu; Saxena, Geetu; Mehta, Ashish

Lead, a ubiquitous and potent neurotoxicant causes oxidative stress which leads to numerous neurobehavioral and physiological alterations. The ability of lead to bind sulfhydryl groups or compete with calcium could be one of the reasons for its debilitating effects. In the present study, we addressed: i) if chelation therapy could circumvent the altered oxidative stress and prevent neuronal apoptosis in chronic lead-intoxicated rats, ii) whether chelation therapy could reverse biochemical and behavioral changes, and iii) if mono or combinational therapy with captopril (an antioxidant) and thiol chelating agents (DMSA/MiADMSA) is more effective than individual thiol chelator in lead-exposed rats. Resultsmore » indicated that lead caused a significant increase in reactive oxygen species, nitric oxide, and intracellular free calcium levels along with altered behavioral abnormalities in locomotor activity, exploratory behavior, learning, and memory that were supported by changes in neurotransmitter levels. A fall in membrane potential, release of cytochrome c, and DNA damage indicated mitochondrial-dependent apoptosis. Most of these alterations showed significant recovery following combined therapy with captopril with MiADMSA and to a smaller extend with captopril + DMSA over monotherapy with these chelators. It could be concluded from our present results that co-administration of a potent antioxidant (like captopril) might be a better treatment protocol than monotherapy to counter lead-induced oxidative stress. The major highlight of the work is an interesting experimental evidence of the efficacy of combinational therapy using an antioxidant with a thiol chelator in reversing neurological dystrophy caused due to chronic lead exposure in rats.« less

Building thiol and metal-thiolate functions into coordination nets: Clues from a simple molecule

NASA Astrophysics Data System (ADS)

He, Jun; Yang, Chen; Xu, Zhengtao; Zeller, Matthias; Hunter, Allen D.; Lin, Jianhua

2009-07-01

The simple and easy-to-prepare bifunctional molecule 2,5-dimercapto-1,4-benzenedicarboxylic acid (H 4DMBD) interacts with the increasingly harder metal ions of Cu +, Pb 2+ and Eu 3+ to form the coordination networks of Cu 6(DMBD) 3(en) 4(Hen) 6 ( 1), Pb 2(DMBD)(en) 2 ( 2) and Eu 2(H 2DMBD) 3(DEF) 4 ( 3), where the carboxyl and thiol groups bind with distinct preference to the hard and soft metal ions, respectively. Notably, 1 features uncoordinated carboxylate groups and Cu 3 cluster units integrated via the thiolate groups into an extended network with significant interaction between the metal centers and the organic molecules; 2 features a 2D coordination net based on the mercapto and carboxylic groups all bonded to the Pb 2+ ions; 3 features free-standing thiol groups inside the channels of a metal-carboxylate-based network. This study illustrates the rich solid state structural features and potential functions offered by the carboxyl-thiol combination.

Thiolysis and alcoholysis of phosphate tri- and monoesters with alkyl and aryl leaving groups. An ab initio study in the gas phase.

PubMed

Arantes, Guilherme Menegon; Chaimovich, Hernan

2005-06-30

Phosphate esters are important compounds in living systems. Their biological reactions with alcohol and thiol nucleophiles are catalyzed by a large superfamily of phosphatase enzymes. However, very little is known about the intrinsic reactivity of these nucleophiles with phosphorus centers. We have performed ab initio calculations on the thiolysis and alcoholysis at phosphorus of trimethyl phosphate, dimethyl phenyl phosphate, methyl phosphate, and phenyl phosphate. Results in the gas phase are a reference for the study of the intrinsic reactivity of these compounds. Thiolysis of triesters was much slower and less favorable than the corresponding alcoholysis. Triesters reacted through an associative mechanism. Monoesters can react by both associative and dissociative mechanisms. The basicity of the attacking and leaving groups and the possibility of proton transfers can modulate the reaction mechanisms. Intermediates formed along associative reactions did not follow empirically proposed rules for ligand positioning. Our calculations also allow re-interpretation of some experimental results, and new experiments are proposed to trace reactions that are normally not observed, both in the gas phase and in solution.

Chemoselective amide formation using O-(4-nitrophenyl)hydroxylamines and pyruvic acid derivatives.

PubMed

Kumar, Sonali; Sharma, Rashi; Garcia, Megan; Kamel, Joseph; McCarthy, Caroline; Muth, Aaron; Phanstiel, Otto

2012-12-07

A series of O-(4-nitrophenyl)hydroxylamines were synthesized from their respective oximes using a pulsed addition of excess NaBH(3)CN at pH 3 in 65-75% yield. Steric hindrance near the oxime functional group played a key role in both the ease by which the oxime could be reduced and the subsequent reactivity of the respective hydroxylamine. Reaction of the respective hydroxylamines with pyruvic acid derivatives generated the desired amides in good yields. A comparison of phenethylamine systems bearing different leaving groups revealed significant differences in the rates of these systems and suggested that the leaving group ability of the N-OR substituent plays an important role in determining their reactivity with pyruvic acid. Competition experiments (in 68% DMSO/phosphate buffered saline) using 1 equiv of N-phenethyl-O-(4-nitrophenyl)hydroxylamine and 2 equiv of pyruvic acid in the presence of other nucleophiles such as glycine, cysteine, phenol, hexanoic acid, and lysine demonstrated that significant chemoselectivity is present in this reaction. The results suggest that this chemoselective reaction can occur in the presence of excess α-amino acids, phenols, acids, thiols, and amines.

Depletion of Intracellular Thiols and Increased Production of 4-Hydroxynonenal that Occur During Cryopreservation of Stallion Spermatozoa Lead to Caspase Activation, Loss of Motility, and Cell Death.

PubMed

Martin Muñoz, Patricia; Ortega Ferrusola, Cristina; Vizuete, Guillermo; Plaza Dávila, Maria; Rodriguez Martinez, Heriberto; Peña, Fernando J

2015-12-01

Oxidative stress has been linked to sperm death and the accelerated senescence of cryopreserved spermatozoa. However, the molecular mechanisms behind this phenomenon remain poorly understood. Reactive oxygen species (ROS) are considered relevant signaling molecules for sperm function, only becoming detrimental when ROS homeostasis is lost. We hereby hypothesize that a major component of the alteration of ROS homeostasis in cryopreserved spermatozoa is the exhaustion of intrinsic antioxidant defense mechanisms. To test this hypothesis, semen from seven stallions was frozen using a standard technique. The parameters of sperm quality (motility, velocity, and membrane integrity) and markers of sperm senescence (caspase 3, 4-hydroxynonenal, and mitochondrial membrane potential) were assessed before and after cryopreservation. Changes in the intracellular thiol content were also monitored. Cryopreservation caused significant increases in senescence markers as well as dramatic depletion of intracellular thiols to less than half of the initial values (P < 0.001) postthaw. Interestingly, very high and positive correlations were observed among thiol levels with sperm functionality postthaw: total motility (r = 0.931, P < 0.001), progressive motility (r = 0.904, P < 0.001), and percentage of live spermatozoa without active caspase 3 (r = 0.996, P < 0.001). In contrast, negative correlations were detected between active caspase 3 and thiol content both in living (r = -0.896) and dead (r = -0.940) spermatozoa; additionally, 4-hydroxynonenal levels were negatively correlated with thiol levels (r = -0.856). In conclusion, sperm functionality postthaw correlates with the maintenance of adequate levels of intracellular thiols. The accelerated senescence of thawed spermatozoa is related to oxidative and electrophilic stress induced by increased production of 4-hydroxynoneal in thawed samples once intracellular thiols are depleted. © 2015 by the Society for the Study of Reproduction, Inc.

The compromise of dynamic disulfide/thiol homeostasis as a biomarker of oxidative stress in trichloroethylene exposure.

PubMed

Bal, C; Büyükşekerci, M; Koca, C; Ağış, E R; Erdoğan, S; Baran, P; Gündüzöz, M; Yilmaz, Öh

2016-09-01

In this study, we aimed to investigate disulfide/thiol homeostasis in trichloroethylene (TCE) exposure. The study was carried out in 30 nonsmoker TCE-exposed workers with a variety of occupations. Additionally, 30 healthy nonsmoker volunteers were recruited as the control group. TCE exposure was determined by measuring urinary trichloroacetic acid (TCA) concentration. Median urinary TCA levels of exposed workers (20.5 mg/L) were significantly higher than control subjects (5 mg/L). Thiol and disulfide concentrations were determined using a novel automated method. Disulfide/thiol ratio was significantly higher in the exposed group (p < 0.001). Thiol/disulfide homeostasis was found to be disturbed in TCE-exposed workers. We predict that in TCE-exposed workers this disturbance can be a therapeutic target, and the efficiency of the treatment can easily be monitored by the novel method we used. © The Author(s) 2015.

Purification and characterization of O-acetylserine (thiol) lyase from spinach chloroplasts.

PubMed

Droux, M; Martin, J; Sajus, P; Douce, R

1992-06-01

O-Acetylserine (thiol) lyase, the last enzyme in the cysteine biosynthetic pathway, was purified to homogeneity from spinach leaf chloroplasts. The enzyme has a molecular mass of 68,000 and consists of two identical subunits of Mr 35,000. The absorption spectrum obtained at pH 7.5 exhibited a peak at 407 nm due to pyridoxal phosphate, and addition of O-acetylserine induced a considerable modification of the spectrum. The pyridoxal phosphate content was found to be 1.1 per subunit of 35,000, and the chromophore was displaced from the enzyme by O-acetylserine, leading to a progressive inactivation of the holoenzyme. Upon gel filtration chromatography on Superdex 200, part of the chloroplastic O-acetylserine (thiol) lyase eluted in association with serine acetyltransferase at a position corresponding to a molecular mass of 310,000 (such a complex called cysteine synthase has been characterized in bacteria). The activity of O-acetylserine (thiol) lyase was optimum between pH 7.5 and 8.5. The apparent Km for O-acetylserine was 1.3 mM and for sulfide was 0.25 mM. The calculated activation energy was 12.6 kcal/mol at 10 mM O-acetylserine. The overall amino-acid composition of spinach chloroplast O-acetylserine (thiol) lyase was different than that determined for the same enzyme (cytosolic?) obtained from a crude extract of spinach leaves. A polyclonal antibody prepared against the chloroplastic O-acetylserine (thiol) lyase exhibited a very low cross-reactivity with a preparation of mitochondrial matrix and cytosolic proteins suggesting that the chloroplastic isoform was distinct from the mitochondrial and cytosolic counterparts.

Deficient expression of aldehyde dehydrogenase 1A1 is consistent with increased sensitivity of Gorlin syndrome patients to radiation carcinogenesis.

PubMed

Wright, Aaron T; Magnaldo, Thierry; Sontag, Ryan L; Anderson, Lindsey N; Sadler, Natalie C; Piehowski, Paul D; Gache, Yannick; Weber, Thomas J

2015-06-01

Human phenotypes that are highly susceptible to radiation carcinogenesis have been identified. Sensitive phenotypes often display robust regulation of molecular features that modify biological response, which can facilitate identification of the pathways/networks that contribute to pathophysiological outcomes. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced inducible tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol reactive probes to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent Gorlin syndrome patients, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and ALDH1A1 protein deficiency in GDFs was confirmed by Western blot. A number of additional protein thiol differences in GDFs were identified, including radiation responsive annexin family members and lamin A/C. Collectively, candidates identified in our study have plausible implications for radiation health effects and cancer susceptibility. © 2013 Wiley Periodicals, Inc.

Dietary thiols in exercise: oxidative stress defence, exercise performance, and adaptation.

PubMed

McLeay, Yanita; Stannard, Stephen; Houltham, Stuart; Starck, Carlene

2017-01-01

Endurance athletes are susceptible to cellular damage initiated by excessive levels of aerobic exercise-produced reactive oxygen species (ROS). Whilst ROS can contribute to the onset of fatigue, there is increasing evidence that they play a crucial role in exercise adaptations. The use of antioxidant supplements such as vitamin C and E in athletes is common; however, their ability to enhance performance and facilitate recovery is controversial, with many studies suggesting a blunting of training adaptations with supplementation. The up-regulation of endogenous antioxidant systems brought about by exercise training allows for greater tolerance to subsequent ROS, thus, athletes may benefit from increasing these systems through dietary thiol donors. Recent work has shown supplementation with a cysteine donor (N-acetylcysteine; NAC) improves antioxidant capacity by augmenting glutathione levels and reducing markers of oxidative stress, as well as ergogenic potential through association with delayed fatigue in numerous experimental models. However, the use of this, and other thiol donors may have adverse physiological effects. A recent discovery for the use of a thiol donor food source, keratin, to potentially enhance endogenous antioxidants may have important implications for endurance athletes hoping to enhance performance and recovery without blunting training adaptations.

Methyl-esterified 3-hydroxybutyrate oligomers protect bacteria from hydroxyl radicals

USDA-ARS?s Scientific Manuscript database

Bacteria rely mainly on enzymes, glutathione and other low-molecular weight thiols to overcome oxidative stress. However, hydroxyl radicals are the most cytotoxic reactive oxygen species, and no known enzymatic system exists for their detoxification. We now show that methyl-esterified dimers and tri...

S-Nitrosylation and the Development of Pulmonary Hypertension

DTIC Science & Technology

2010-02-01

cleavage, catalyzed by transition metal ions and reactive oxygen species, or through enzymatic degradation. One specific enzyme that regulates S...Hb causes a change in its conformation, resulting in the transfer of nitrosonium (NO+) equivalents from the heme (Fe+2) to the thiol (cysteine β93

A Comparison of Five Bioconjugatable Ferrocenes for Labeling of Biomolecules†

PubMed Central

Ge, Dongbiao; Levicky, Rastislav

2010-01-01

Five electroactive ferrocene tags for labelling of biomolecules are contrasted with regard to conjugation reactivity with amine and thiol moieties, stability to loss of electrochemical activity, and impact of molecular structure on the redox potential of the free and DNA-conjugated forms. PMID:20740226

Enhancement of bismuth antibacterial activity with lipophilic thiol chelators.

PubMed Central

Domenico, P; Salo, R J; Novick, S G; Schoch, P E; Van Horn, K; Cunha, B A

1997-01-01

The antibacterial properties of bismuth are greatly enhanced when bismuth is combined with certain lipophilic thiol compounds. Antibacterial activity was enhanced from 25- to 300-fold by the following seven different thiols, in order of decreasing synergy: 1,3-propanedithiol, dimercaprol (BAL), dithiothreitol, 3-mercapto-2-butanol, beta-mercaptoethanol, 1-monothioglycerol, and mercaptoethylamine. The dithiols produced the greatest synergy with bismuth at optimum bismuth-thiol molar ratios of from 3:1 to 1:1. The monothiols were generally not as synergistic and required molar ratios of from 1:1 to 1:4 for optimum antibacterial activity. The most-active mono- or dithiols were also the most soluble in butanol. The intensity of the yellow formed by bismuth-thiol complexes reflected the degree of chelation and correlated with antibacterial potency at high molar ratios. The bismuth-BAL compound (BisBAL) was active against most bacteria, as assessed by broth dilution, agar diffusion, and agar dilution analyses. Staphylococci (MIC, 5 to 7 microM Bi3+) and Helicobacter pylori (MIC, 2.2 microM) were among the most sensitive bacteria. Gram-negative bacteria were sensitive (MIC, < 17 microM). Enterococci were relatively resistant (MIC, 63 microM Bi3+). The MIC range for anaerobes was 15 to 100 microM Bi3+, except for Clostridium difficile (MIC, 7.5 microM). Bactericidal activity averaged 29% above the MIC. Bactericidal activity increased with increasing pH and/or increasing temperature. Bismuth-thiol solubility, stability, and antibacterial activity depended on pH and the bismuth-thiol molar ratio. BisBAL was stable but ineffective against Escherichia coli at pH 4. Activity and instability (reactivity) increased with increasing alkalinity. BisBAL was acid soluble at a molar ratio of greater than 3:2 and alkaline soluble at a molar ratio of less than 2:3. In conclusion, certain lipophilic thiol compounds enhanced bismuth antibacterial activity against a broad spectrum of bacteria. The activity, solubility, and stability of BisBAL were strongly dependent on the pH, temperature, and molar ratio. Chelation of bismuth with certain thiol agents enhanced the solubility and lipophilicity of this cationic heavy metal, thereby significantly enhancing its potency and versatility as an antibacterial agent. PMID:9257744

Kinetics and Mechanism Study of Competitive Inhibition of Jack-Bean Urease by Baicalin

PubMed Central

Tan, Lirong; Su, Jiyan; Wu, Dianwei; Yu, Xiaodan; Su, Zuqing; Wu, Xiaoli; Kong, Songzhi; Lai, Xiaoping; Lin, Ji; Su, Ziren

2013-01-01

Baicalin (BA) is the principal component of Radix Scutellariae responsible for its pharmacological activity. In this study, kinetics and mechanism of inhibition by BA against jack-bean urease were investigated for its therapeutic potential. It was revealed that the IC50 of BA against jack-bean urease was 2.74 ± 0.51 mM, which was proved to be a competitive and concentration-dependent inhibition with slow-binding progress curves. The rapid formation of initial BA-urease complex with an inhibition constant of K i = 3.89 × 10−3 mM was followed by a slow isomerization into the final complex with an overall inhibition constant of K i* = 1.47 × 10−4 mM. High effectiveness of thiol protectors against BA inhibition indicated that the strategic role of the active-site sulfhydryl group of the urease was involved in the blocking process. Moreover, the inhibition of BA was proved to be reversible due to the fact that urease could be reactivated by dithiothreitol but not reactant dilution. Molecular docking assay suggested that BA made contacts with the important activating sulfhydryl group Cys-592 residues and restricted the mobility of the active-site flap. Taken together, it could be deduced that BA was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for treatments on urease-related diseases. PMID:24198731

Vinyl functionalized silica hybrid monolith-based trypsin microreactor for on line digestion and separation via thiol-ene "click" strategy.

PubMed

Chen, Yingzhuang; Wu, Minghuo; Wang, Keyi; Chen, Bo; Yao, Shouzhuo; Zou, Hanfa; Nie, Lihua

2011-11-04

A novel thiol-ene "click" strategy for the preparation of monolithic trypsin microreactor was proposed. The hybrid organic-inorganic monolithic capillary column with ene-functionality was fabricated by sol-gel process using tetramethoxysilane (TMOS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS) as precursors. The disulfide bonds of trypsin were reduced to form free thiol groups. Then the trypsin containing free thiol groups was attached on the γ-MAPS hybrid monolithic column with ene-functionality via thiol-ene click chemistry to form a trypsin microreactor. The activity of the trypsin microreactor was characterized by detecting the substrate (Nα-p-tosyl-L-arginine methyl ester hydrochloride, TAME) and the product (Nα-p-tosyl-L-arginine, TA) with on-line capillary zone electrophoresis. After investigating various synthesizing conditions, it was found that the microreactor with poly(N,N'-methylenebisacrylamide) as spacer can deliver the highest activity, yielding a rapid reaction rate. After repeatedly sampling and analyzing for 100 times, the monolithic trypsin microreactor still remained 87.5% of its initial activity. It was demonstrated that thiol-ene "click" strategy for the construction of enzyme microreactor is a promising method for the highly selective immobilization of proteins under mild conditions, especially enzymes with free thiol radicals. Copyright © 2011 Elsevier B.V. All rights reserved.

Novel microfabrication stage allowing for one-photon and multi-photon light assisted molecular immobilization and for multi-photon microscope

NASA Astrophysics Data System (ADS)

Gonçalves, Odete; Snider, Scott; Zadoyan, Ruben; Nguyen, Quoc-Thang; Vorum, Henrik; Petersen, Steffen B.; Neves-Petersen, Maria Teresa

2017-02-01

Light Assisted Molecular Immobilization (LAMI) results in spatially oriented and localized covalent coupling of biomolecules onto thiol reactive surfaces. LAMI is possible due to the conserved spatial proximity between aromatic residues and disulfide bridges in proteins. When aromatic residues are excited with UV light (275-295nm), disulphide bridges are disrupted and the formed thiol groups covalently bind to surfaces. Immobilization hereby reported is achieved in a microfabrication stage coupled to a fs-laser, through one- or multi-photon excitation. The fundamental 840nm output is tripled to 280nm and focused onto the sample, leading to one-photon excitation and molecular immobilization. The sample rests on a xyz-stage with micrometer step resolution and is illuminated according to a pattern uploaded to the software controlling the stage and the shutter. Molecules are immobilized according to such pattern, with micrometer spatial resolution. Spatial masks inserted in the light path lead to light diffraction patterns used to immobilize biomolecules with submicrometer spatial resolution. Light diffraction patterns are imaged by an inbuilt microscope. Two-photon microscopy and imaging of the fluorescent microbeads is shown. Immobilization of proteins, e.g. C-reactive protein, and of an engineered molecular beacon has been successfully achieved. The beacon was coupled to a peptide containing a disulfide bridge neighboring a tryptophan residue, being this way possible to immobilize the beacon on a surface using one-photon LAMI. This technology is being implemented in the creation of point-of-care biosensors aiming at the detection of cancer and cardiovascular disease markers.

Synthesis of Diiron(I) Dithiolato Carbonyl Complexes.

PubMed

Li, Yulong; Rauchfuss, Thomas B

2016-06-22

Virtually all organosulfur compounds react with Fe(0) carbonyls to give the title complexes. These reactions are reviewed in light of major advances over the past few decades, spurred by interest in Fe2(μ-SR)2(CO)x centers at the active sites of the [FeFe]-hydrogenase enzymes. The most useful synthetic route to Fe2(μ-SR)2(CO)6 involves the reaction of thiols with Fe2(CO)9 and Fe3(CO)12. Such reactions can proceed via mono-, di-, and triiron intermediates. The reactivity of Fe(0) carbonyls toward thiols is highly chemoselective, and the resulting dithiolato complexes are fairly rugged. Thus, many complexes tolerate further synthetic elaboration directed at the organic substituents. A second major route involves alkylation of Fe2(μ-S2)(CO)6, Fe2(μ-SH)2(CO)6, and Li2Fe2(μ-S)2(CO)6. This approach is especially useful for azadithiolates Fe2[(μ-SCH2)2NR](CO)6. Elaborate complexes arise via addition of the FeSH group to electrophilic alkenes, alkynes, and carbonyls. Although the first example of Fe2(μ-SR)2(CO)6 was prepared from ferrous reagents, ferrous compounds are infrequently used, although the Fe(II)(SR)2 + Fe(0) condensation reaction is promising. Almost invariably low-yielding, the reaction of Fe3(CO)12, S8, and a variety of unsaturated substrates results in C-H activation, affording otherwise inaccessible derivatives. Thiones and related C═S-containing reagents are highly reactive toward Fe(0), often giving complexes derived from substituted methanedithiolates and C-H activation.

The Expanding Landscape of the Thiol Redox Proteome*

PubMed Central

Yang, Jing; Carroll, Kate S.; Liebler, Daniel C.

2016-01-01

Cysteine occupies a unique place in protein chemistry. The nucleophilic thiol group allows cysteine to undergo a broad range of redox modifications beyond classical thiol-disulfide redox equilibria, including S-sulfenylation (-SOH), S-sulfinylation (-SO2H), S-sulfonylation (-SO3H), S-nitrosylation (-SNO), S-sulfhydration (-SSH), S-glutathionylation (-SSG), and others. Emerging evidence suggests that these post-translational modifications (PTM) are important in cellular redox regulation and protection against oxidative damage. Identification of protein targets of thiol redox modifications is crucial to understanding their roles in biology and disease. However, analysis of these highly labile and dynamic modifications poses challenges. Recent advances in the design of probes for thiol redox forms, together with innovative mass spectrometry based chemoproteomics methods make it possible to perform global, site-specific, and quantitative analyses of thiol redox modifications in complex proteomes. Here, we review chemical proteomic strategies used to expand the landscape of thiol redox modifications. PMID:26518762

Pyrimidine Nucleosides with a Reactive (β-Chlorovinyl)sulfone or (β-Keto)sulfone Group at the C5 Position, Their Reactions with Nucleophiles and Electrophiles, and Their Polymerase-Catalyzed Incorporation into DNA

PubMed Central

2018-01-01

Transition-metal-catalyzed chlorosulfonylation of 5-ethynylpyrimidine nucleosides provided (E)-5-(β-chlorovinyl)sulfones A, which undergo nucleophilic substitution with amines or thiols affording B. The treatment of vinyl sulfones A with ammonia followed by acid-catalyzed hydrolysis of the intermediary β-sulfonylvinylamines gave 5-(β-keto)sulfones C. The latter reacts with electrophiles, yielding α-carbon-alkylated or -sulfanylated analogues D. The 5′-triphosphates of A and C were incorporated into double-stranded DNA, using open and one-nucleotide gap substrates, by human or Escherichia coli DNA-polymerase-catalyzed reactions. PMID:29732453

Cysteine-containing peptide tag for site-specific conjugation of proteins

DOEpatents

Backer, Marina V.; Backer, Joseph M.

2008-04-08

The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety bound to the targeting moiety; the biological conjugate having a covalent bond between the thiol group of SEQ ID NO:2 and a functional group in the binding moiety. The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety that comprises an adapter protein, the adapter protein having a thiol group; the biological conjugate having a disulfide bond between the thiol group of SEQ ID NO:2 and the thiol group of the adapter protein. The present invention is also directed to biological sequences employed in the above biological conjugates, as well as pharmaceutical preparations and methods using the above biological conjugates.

Cysteine-containing peptide tag for site-specific conjugation of proteins

DOEpatents

Backer, Marina V.; Backer, Joseph M.

2010-10-05

The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety bound to the targeting moiety; the biological conjugate having a covalent bond between the thiol group of SEQ ID NO:2 and a functional group in the binding moiety. The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety that comprises an adapter protein, the adapter protein having a thiol group; the biological conjugate having a disulfide bond between the thiol group of SEQ ID NO:2 and the thiol group of the adapter protein. The present invention is also directed to biological sequences employed in the above biological conjugates, as well as pharmaceutical preparations and methods using the above biological conjugates.

Electron transport chain dysfunction by H(2)O (2) is linked to increased reactive oxygen species production and iron mobilization by lipoperoxidation: studies using Saccharomyces cerevisiae mitochondria.

PubMed

Cortés-Rojo, Christian; Estrada-Villagómez, Mirella; Calderón-Cortés, Elizabeth; Clemente-Guerrero, Mónica; Mejía-Zepeda, Ricardo; Boldogh, Istvan; Saavedra-Molina, Alfredo

2011-04-01

The mitochondrial electron transport chain (ETC) contains thiol groups (-SH) which are reversibly oxidized to modulate ETC function during H(2)O(2) overproduction. Since deleterious effects of H(2)O(2) are not limited to -SH oxidation, due to the formation of other H(2)O(2)-derived species, some processes like lipoperoxidation could enhance the effects of H(2)O(2) over ETC enzymes, disrupt their modulation by -SH oxidation and increase superoxide production. To verify this hypothesis, we tested the effects of H(2)O(2) on ETC activities, superoxide production and iron mobilization in mitochondria from lipoperoxidation-resistant native yeast and lipoperoxidation-sensitized yeast. Only complex III activity from lipoperoxidation-sensitive mitochondria exhibited a higher susceptibility to H(2)O(2) and increased superoxide production. The recovery of ETC activity by the thiol reductanct β-mercaptoethanol (BME) was also altered at complex III, and a role was attributed to lipoperoxidation, the latter being also responsible for iron release. A hypothetical model linking lipoperoxidation, increased complex III damage, superoxide production and iron release is given.

Synthesis of l-cysteine derivatives containing stable sulfur isotopes and application of this synthesis to reactive sulfur metabolome.

PubMed

Ono, Katsuhiko; Jung, Minkyung; Zhang, Tianli; Tsutsuki, Hiroyasu; Sezaki, Hiroshi; Ihara, Hideshi; Wei, Fan-Yan; Tomizawa, Kazuhito; Akaike, Takaaki; Sawa, Tomohiro

2017-05-01

Cysteine persulfide is an L-cysteine derivative having one additional sulfur atom bound to a cysteinyl thiol group, and it serves as a reactive sulfur species that regulates redox homeostasis in cells. Here, we describe a rapid and efficient method of synthesis of L-cysteine derivatives containing isotopic sulfur atoms and application of this method to a reactive sulfur metabolome. We used bacterial cysteine syntheses to incorporate isotopic sulfur atoms into the sulfhydryl moiety of L-cysteine. We cloned three cysteine synthases-CysE, CysK, and CysM-from the Gram-negative bacterium Salmonella enterica serovar Typhimurium LT2, and we generated their recombinant enzymes. We synthesized 34 S-labeled L-cysteine from O-acetyl-L-serine and 34 S-labeled sodium sulfide as substrates for the CysK or CysM reactions. Isotopic labeling of L-cysteine at both sulfur ( 34 S) and nitrogen ( 15 N) atoms was also achieved by performing enzyme reactions with 15 N-labeled L-serine, acetyl-CoA, and 34 S-labeled sodium sulfide in the presence of CysE and CysK. The present enzyme systems can be applied to syntheses of a series of L-cysteine derivatives including L-cystine, L-cystine persulfide, S-sulfo-L-cysteine, L-cysteine sulfonate, and L-selenocystine. We also prepared 34 S-labeled N-acetyl-L-cysteine (NAC) by incubating 34 S-labeled L-cysteine with acetyl coenzyme A in test tubes. Tandem mass spectrometric identification of low-molecular-weight thiols after monobromobimane derivatization revealed the endogenous occurrence of NAC in the cultured mammalian cells such as HeLa cells and J774.1 cells. Furthermore, we successfully demonstrated, by using 34 S-labeled NAC, metabolic conversion of NAC to glutathione and its persulfide, via intermediate formation of L-cysteine, in the cells. The approach using isotopic sulfur labeling combined with mass spectrometry may thus contribute to greater understanding of reactive sulfur metabolome and redox biology. Copyright © 2017 Elsevier Inc. All rights reserved.

Moderate physical exercise induces the oxidation of human blood protein thiols.

PubMed

Inayama, Takayo; Oka, Jun; Kashiba, Misato; Saito, Makoto; Higuchi, Mitsuru; Umegaki, Keizo; Yamamoto, Yorihiro; Matsuda, Mitsuo

2002-03-15

Exercise is known to induce the oxidation of blood low-molecular-weight (LMW) thiols such as reduced glutathione (GSH). We previously reported that full-marathon running induced a decrease in human plasma levels of protein-bound sulfhydryl groups (p-SHs). Moderate exercise, a 30-min running at the intensity of the individual ventilatory threshold, performed by untrained healthy females caused a significant decrease in erythrocyte levels of p-SHs (mostly hemoglobin cysteine residues) and LMW thiols, but their levels returned to each baseline by 2 h. No significant change in plasma LMW thiols was observed. However, plasma levels of p-SHs significantly decreased after running and remained unchanged after 24 h. These results suggest that moderate exercise causes the oxidation of blood thiols, especially protein-bound thiols.

Detoxification of Atrazine by Low Molecular Weight Thiols in Alfalfa (Medicago sativa).

PubMed

Zhang, Jing Jing; Xu, Jiang Yan; Lu, Feng Fan; Jin, She Feng; Yang, Hong

2017-10-16

Low molecular weight (LMW) thiols in higher plants are a group of sulfur-rich nonprotein compounds and play primary and multiple roles in cellular redox homeostasis, enzyme activities, and xenobiotics detoxification. This study focused on identifying thiols-related protein genes from the legume alfalfa exposed to the herbicide atrazine (ATZ) residues in environment. Using high-throughput RNA-sequencing, a set of ATZ-responsive thiols-related protein genes highly up-regulated and differentially expressed in alfalfa was identified. Most of the differentially expressed genes (DEGs) were involved in regulation of biotic and abiotic stress responses. By analyzing the genes involved in thiols-mediated redox homeostasis, we found that many of them were thiols-synthetic enzymes such as γ-glutamylcysteine synthase (γECS), homoglutathione synthetase (hGSHS), and glutathione synthetase (GSHS). Using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), we further characterized a group of ATZ-thiols conjugates, which are the detoxified forms of ATZ in plants. Cysteine S-conjugate ATZ-HCl+Cys was the most important metabolite detected by MS. Several other ATZ-conjugates were also examined as ATZ-detoxified metabolites. Such results were validated by characterizing their analogs in rice. Our data showed that some conjugates under ATZ stress were detected in both plants, indicating that some detoxified mechanisms and pathways can be shared by the two plant species. Overall, these results indicate that LMW thiols play critical roles in detoxification of ATZ in the plants.

Differential Expression of NADPH Oxidases Depends on Skeletal Muscle Fiber Type in Rats.

PubMed

Loureiro, Adriano César Carneiro; do Rêgo-Monteiro, Igor Coutinho; Louzada, Ruy A; Ortenzi, Victor Hugo; de Aguiar, Angélica Ponte; de Abreu, Ewerton Sousa; Cavalcanti-de-Albuquerque, João Paulo Albuquerque; Hecht, Fabio; de Oliveira, Ariclécio Cunha; Ceccatto, Vânia Marilande; Fortunato, Rodrigo S; Carvalho, Denise P

2016-01-01

NADPH oxidases (NOX) are important sources of reactive oxygen species (ROS) in skeletal muscle, being involved in excitation-contraction coupling. Thus, we aimed to investigate if NOX activity and expression in skeletal muscle are fiber type specific and the possible contribution of this difference to cellular oxidative stress. Oxygen consumption rate, NOX activity and mRNA levels, and the activity of catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase (SOD), as well as the reactive protein thiol levels, were measured in the soleus (SOL), red gastrocnemius (RG), and white gastrocnemius (WG) muscles of rats. RG showed higher oxygen consumption flow than SOL and WG, while SOL had higher oxygen consumption than WG. SOL showed higher NOX activity, as well as NOX2 and NOX4 mRNA levels, antioxidant enzymatic activities, and reactive protein thiol contents when compared to WG and RG. NOX activity and NOX4 mRNA levels as well as antioxidant enzymatic activities were higher in RG than in WG. Physical exercise increased NOX activity in SOL and RG, specifically NOX2 mRNA levels in RG and NOX4 mRNA levels in SOL. In conclusion, we demonstrated that NOX activity and expression differ according to the skeletal muscle fiber type, as well as antioxidant defense.

Recent trends in electrochemical biosensors of superoxide dismutases.

PubMed

Balamurugan, Murugesan; Santharaman, Paulraj; Madasamy, Thangamuthu; Rajesh, Seenivasan; Sethy, Niroj Kumar; Bhargava, Kalpana; Kotamraju, Srigiridhar; Karunakaran, Chandran

2018-09-30

Superoxide dismutases (SODs), a family of ubiquitous enzymes, provide essential protection to biological systems against uncontrolled reactions with oxygen- and nitrogen- based radical species. We review first the role of SODs in oxidative stress and the other biological functions such as peroxidase, nitrite oxidase, thiol oxidase activities etc., implicating its role in neurodegenerative, cardiovascular diseases, and ageing. Also, this review focuses on the development of electrochemical label-free immunosensor for SOD1 and the recent advances in biosensing assay methods based on their catalytic and biological functions with various substrates including reactive oxygen species (superoxide anion radical, hydrogen peroxide), nitric oxide metabolites (nitrite, nitrate) and thiols using thiol oxidase activity. Furthermore, we emphasize the progress made in improving the detection performance through incorporation of the SOD into conducting polymers and nanocomposite matrices. In addition, we address the potential opportunities, challenges, advances in electrochemical-sensing platforms and development of portable analyzer for point-of-care applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Click and chemically triggered declick reactions through reversible amine and thiol coupling via a conjugate acceptor

NASA Astrophysics Data System (ADS)

Diehl, Katharine L.; Kolesnichenko, Igor V.; Robotham, Scott A.; Bachman, J. Logan; Zhong, Ye; Brodbelt, Jennifer S.; Anslyn, Eric V.

2016-10-01

The coupling and decoupling of molecular units is a fundamental undertaking of organic chemistry. Herein we report the use of a very simple conjugate acceptor, derived from Meldrum's acid, for the sequential ‘clicking’ together of an amine and a thiol in aqueous conditions at neutral pH. Subsequently, this linkage can be ‘declicked’ by a chemical trigger to release the original amine and thiol undisturbed. The reactivity differs from that of other crosslinking agents because the selectivity for sequential functionalization derives from an altering of the electrophilicity of the conjugate acceptor on the addition of the amine. We describe the use of the procedure to modify proteins, create multicomponent libraries and synthesize oligomers, all of which can be declicked to their starting components in a controlled fashion when desired. Owing to the mild reaction conditions and ease of use in a variety of applications, the method is predicted to have wide utility.

Neutrophil-generated HOCl leads to non-specific thiol oxidation in phagocytized bacteria

PubMed Central

Degrossoli, Adriana; Müller, Alexandra; Xie, Kaibo; Schneider, Jannis F; Bader, Verian; Winklhofer, Konstanze F; Meyer, Andreas J

2018-01-01

Phagocytic immune cells kill pathogens in the phagolysosomal compartment with a cocktail of antimicrobial agents. Chief among them are reactive species produced in the so-called oxidative burst. Here, we show that bacteria exposed to a neutrophil-like cell line experience a rapid and massive oxidation of cytosolic thiols. Using roGFP2-based fusion probes, we could show that this massive breakdown of the thiol redox homeostasis was dependent on phagocytosis, presence of NADPH oxidase and ultimately myeloperoxidase. Interestingly, the redox-mediated fluorescence change in bacteria expressing a glutathione-specific Grx1-roGFP2 fusion protein or an unfused roGFP2 showed highly similar reaction kinetics to the ones observed with roGFP2-Orp1, under all conditions tested. We recently observed such an indiscriminate oxidation of roGFP2-based fusion probes by HOCl with fast kinetics in vitro. In line with these observations, abating HOCl production in immune cells with a myeloperoxidase inhibitor significantly attenuated the oxidation of all three probes in bacteria. PMID:29506649

Allicin-inspired thiolated fluoroquinolones as antibacterials against ESKAPE pathogens.

PubMed

Sheppard, Jordan G; Long, Timothy E

2016-11-15

Thiolated fluoroquinolones were synthesized from ciprofloxacin and evaluated for antimicrobial activity against a panel of pathogenic bacteria. Gram-positive species including methicillin-resistant Staphylococcus aureus (MRSA) exhibited the highest level of increased sensitivity toward ciprofloxacin bound with a N-propylthio substituent. Evidence was found that the antibiotics form disulfides with low molecular weight thiols in bacteria and potentiate generation of cytosolic reactive oxygen species (ROS). In final analysis, the enhanced anti-MRSA activity of thiolated fluoroquinolones was attributed to increased cell permeability and reaction with cytosolic thiols that yields an inactive disulfide metabolite and the parent drug ciprofloxacin as an inhibitor of DNA synthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

An excited state intramolecular proton transfer dye based fluorescence turn-on probe for fast detection of thiols and its applications in bioimaging

NASA Astrophysics Data System (ADS)

Zhao, Yun; Xue, Yuanyuan; Li, Haoyang; Zhu, Ruitao; Ren, Yuehong; Shi, Qinghua; Wang, Song; Guo, Wei

2017-03-01

In this study, a new fluorescent probe 2-(2‧-hydroxy-5‧-N-maleimide phenyl)-benzothiazole (probe 1), was designed and synthesized by linking the excited state intramolecular proton transfer (ESIPT) fluorophore to the maleimide group for selective detection of thiols in aqueous solution. The fluorescence of probe 1 is strongly quenched by maleimide group through the photo-induced electron transfer (PET) mechanism, but after reaction with thiol, the fluorescence of ESIPT fluorophore is restored, affording a large Stokes shifts. Upon addition of cysteine (Cys), probe 1 exhibited a fast response time (complete within 30 s) and a high signal-to-noise ratio (up to 23-fold). It showed a high selectivity and excellent sensitivity to thiols over other relevant biological species, with a detection limit of 3.78 × 10- 8 M (S/N = 3). Moreover, the probe was successfully applied to the imaging of thiols in living cells.

Dip-pen nanolithography on (bio)reactive monolayer and block-copolymer platforms: deposition of lines of single macromolecules.

PubMed

Salazar, Ramon B; Shovsky, Alexander; Schönherr, Holger; Vancso, G Julius

2006-11-01

The application of atomic force microscopy (AFM) tip-mediated molecular transfer (dip-pen nanolithography or DPN) to fabricate nanopatterned (bio)reactive platforms based on dendrimers on reactive self-assembled monolayer (SAM) and polymer thin films is discussed. The transfer of high-molar-mass polyamidoamine (PAMAM) dendrimers (generation 5) and the rapid in situ covalent attachment of the deposited adsorbates onto reactive N-hydroxysuccinimide (NHS) terminated SAMs on gold and NHS-activated polystyrene-block-poly(tert-butyl acrylate) (PS(690)-b-PtBA(1210)) block copolymer thin films were investigated as strategies to suppress line broadening by surface diffusion in DPN. By exploiting carefully controlled environmental conditions (such as temperature and relative humidity), scan rates, and in particular the covalent attachment of the dendrimers to the reactive films, the observed line broadening and hence the lateral diffusion of dendrimers was substantially less pronounced compared to that observed with DPN of thiols on gold. By this method, high-definition patterns of dendrimers were conveniently fabricated down to 30-nm length scales. The presence of primary amino groups in the deposited dendrimers ultimately offers the possibility to anchor biochemically relevant molecules, such as proteins and polypeptides, to these nanostructured platforms for a wide range of possible applications in the life sciences and in particular for the investigation of controlled cell-surface interactions.

Modulation of oxidative damage by nitroxide free radicals.

PubMed

Dragutan, Ileana; Mehlhorn, Rolf J

2007-03-01

Piperidine nitroxides like 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) are persistent free radicals in non-acidic aqueous solutions and organic solvents that may have value as therapeutic agents in medicine. In biological environments, they undergo mostly reduction to stable hydroxylamines but can also undergo oxidation to reactive oxoammonium compounds. Reactions of the oxoammonium derivatives could have adverse consequences including chemical modification of vital macromolecules and deleterious effects on cell signaling. An examination of their reactivity in aqueous solution has shown that oxoammonium compounds can oxidize almost any organic as well as many inorganic molecules found in biological systems. Many of these reactions appear to be one-electron transfers that reduce the oxoammonium to the corresponding nitroxide species, in contrast to a prevalence of two-electron reductions of oxoammonium in organic solvents. Amino acids, alcohols, aldehydes, phospholipids, hydrogen peroxide, other nitroxides, hydroxylamines, phenols and certain transition metal ions and their complexes are among reductants of oxoammonium, causing conversion of this species to the paramagnetic nitroxide. On the other hand, thiols and oxoammonium yield products that cannot be detected by ESR even under conditions that would oxidize hydroxylamines to nitroxides. These products may include hindered secondary amines, sulfoxamides and sulfonamides. Thiol oxidation products other than disulfides cannot be restored to thiols by common enzymatic reduction pathways. Such products may also play a role in cell signaling events related to oxidative stress. Adverse consequences of the reactions of oxoammonium compounds may partially offset the putative beneficial effects of nitroxides in some therapeutic settings.

4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole as a reactivity probe for the investigation of the thiol proteinases. evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain.

PubMed Central

Shipton, M; Stuchbury, T; Brocklehurst, K

1976-01-01

1. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride) was used as a reactivity probe to characterize the active centres of papin (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). 2. In the pH range 0-8 Nbd chloride probably exists mainly as a monocation, possibly with the proton located on N-1 of the oxadiazole ring. 3. Spectroscopic evidence is presented for the intermediacy of Meisenheimer-type adducts in the reaction of Nbd chloride with nucleophiles. 4. The pH-dependence of the second-order rate constants (k) of the reactions of the three enzymes with Nbd chloride was determined at 25 degrees C, I = 0.1 mol/litre in 6.7% (v/v) ethanol in the pH range 2.5-5, where, at least for papain and ficin, the reactions occur specifically with their active-centre thiol groups. The pH-k profile for the papain reaction is bell-shaped (pKaI = 3.24, pKaII = 3.44 and k = 86M(-1)-s(-1), whereas that for ficin is sigmoidal (pKa = 3.6, k = 0.36M(-1)-s(-1), the rate increasing with increasing pH. The profile for the bromelain reaction appears to resemble that for the ficin reaction, but is complicated by amino-group labelling. 5. The bell-shaped profile of the papain reaction is considered to arise from the reaction of the thiolate ion of cysteine-25, maintained in acidic media by interaction with the side chain of histidine-159, with the Nbd chloride monocation hydrogen-bonded at its nitro group to the un-ionized form of the carboxyl group of aspartic acid-158. The lack of acid catalysis in the corresponding reactions of ficin and probably of bromelain suggests that these enzymes may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain. The possible consequences of this for the catalytic sites of these enzymes is discussed. PMID:11778

Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins

PubMed Central

Yao, Chunxiang; Behring, Jessica B.; Shao, Di; Sverdlov, Aaron L.; Whelan, Stephen A.; Elezaby, Aly; Yin, Xiaoyan; Siwik, Deborah A.; Seta, Francesca; Costello, Catherine E.; Cohen, Richard A.; Matsui, Reiko; Colucci, Wilson S.; McComb, Mark E.; Bachschmid, Markus M.

2015-01-01

Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a ‘Tandem Mass Tag’ (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation. PMID:26642319

Prebiotic Amino Acid Thioester Synthesis: Thiol-Dependent Amino Acid Synthesis from Formose substrates (Formaldehyde and Glycolaldehyde) and Ammonia

NASA Technical Reports Server (NTRS)

Weber, Arthur L.

1998-01-01

Formaldehyde and glycolaldehyde (substrates of the formose autocatalytic cycle) were shown to react with ammonia yielding alanine and homoserine under mild aqueous conditions in the presence of thiol catalysts. Since similar reactions carried out without ammonia yielded alpha-hydroxy acid thioesters, the thiol-dependent synthesis of alanine and homoserine is presumed to occur via amino acid thioesters-intermediates capable of forming peptides. A pH 5.2 solution of 20 mM formaldehyde, 20 mM glycolaldehyde, 20 mM ammonium chloride, 23 mM 3-mercaptopropionic acid, and 23 mM acetic acid that reacted for 35 days at 40 C yielded (based on initial formaldehyde) 1.8% alanine and 0.08% homoserine. In the absence of thiol catalyst, the synthesis of alanine and homoserine was negligible. Alanine synthesis required both formaldehyde and glycolaldehyde, but homoserine synthesis required only glycolaldehyde. At 25 days the efficiency of alanine synthesis calculated from the ratio of alanine synthesized to formaldehyde reacted was 2.1%, and the yield (based on initial formaldehyde) of triose and tetrose intermediates involved in alanine and homoserine synthesis was 0.3 and 2.1%, respectively. Alanine synthesis was also seen in similar reactions containing only 10 mM each of aldehyde substrates, ammonia, and thiol. The prebiotic significance of these reactions that use the formose reaction to generate sugar intermediates that are converted to reactive amino acid thioesters is discussed.

Simultaneous LC/MS/MS determination of thiols and disulfides in urine samples based on differential labeling with ferrocene-based maleimides.

PubMed

Seiwert, Bettina; Karst, Uwe

2007-09-15

A method for the simultaneous determination of a series of thiols and disulfides in urine samples has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. The sample is first exposed to N-(2-ferroceneethyl)maleimide, thus leading to the derivatization of free thiol groups in the sample. After quantitative reaction and subsequent reduction of the disulfide-bound thiols by tris(2-carboxyethyl)phosphine, the newly formed thiol functionalities are reacted with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide. The reaction products are determined by LC/MS/MS in the multiple reaction mode, and precursor ion scan as well as neutral loss scan is applied to detect unknown further thiols. The method was successfully applied to the analysis of free and disulfide-bound thiols in urine samples. Limits of detection are 30 to 110 nM, and the linear range comprises two decades of concentration, thus covering the relevant concentration range of thiols in urine samples. The thiol and disulfide concentrations were referred to the creatinine content to compensate for different sample volumes. As some calibration standards for the disulfides are not commercially available, they were synthesized in an electrochemical flow-through cell. This allowed the synthesis of hetero- and homodimeric disulfides.

Photoreduction of Hg(II) and photodemethylation of methylmercury: the key role of thiol sites on dissolved organic matter

USGS Publications Warehouse

Jeremiason, Jeffrey D.; Portner, Joshua C.; Aiken, George R.; Hiranaka, Amber J.; Dvorak, Michelle T.; Tran, Khuyen T.; Latch, Douglas E.

2015-01-01

This study examined the kinetics of photoreduction of Hg(II) and photodemethylation of methylmercury (MeHg+) attached to, or in the presence of, dissolved organic matter (DOM). Both Hg(II) and MeHg+ are principally bound to reduced sulfur groups associated with DOM in many freshwater systems. We propose that a direct photolysis mechanism is plausible for reduction of Hg(II) bound to reduced sulfur groups on DOM while an indirect mechanism is supported for photodemethylation of MeHg+ bound to DOM. UV spectra of Hg(II) and MeHg+ bound to thiol containing molecules demonstrate that the Hg(II)–S bond is capable of absorbing UV-light in the solar spectrum to a much greater extent than MeHg+–S bonds. Experiments with chemically distinct DOM isolates suggest that concentration of DOM matters little in the photochemistry if there are enough reduced S sites present to strongly bind MeHg+ and Hg(II); DOM concentration does not play a prominent role in photodemethylation other than to screen light, which was demonstrated in a field experiment in the highly colored St. Louis River where photodemethylation was not observed at depths ≥10 cm. Experiments with thiol ligands yielded slower photodegradation rates for MeHg+ than in experiments with DOM and thiols; rates in the presence of DOM alone were the fastest supporting an intra-DOM mechanism. Hg(II) photoreduction rates, however, were similar in experiments with only DOM, thiols plus DOM, or only thiols suggesting a direct photolysis mechanism. Quenching experiments also support the existence of an intra-DOM photodemethylation mechanism for MeHg+. Utilizing the difference in photodemethylation rates measured for MeHg+ attached to DOM or thiol ligands, the binding constant for MeHg+ attached to thiol groups on DOM was estimated to be 1016.7.

9-Fluorenylmethyl (Fm) Disulfides: Biomimetic Precursors for Persulfides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Chung-Min; Johnson, Brett A.; Duan, Jicheng

Protein S-sulfhydration has been recognized as an important post-translational modification that regulates H2S signals. However, the reactivity and biological implications of the products of S-sulfhydration, i.e. persulfides, are still unclear. This is mainly due to the instability of persulfides and difficulty to access these molecules. Under physiological conditions persulfides mainly exist in anionic forms because of their low pKa values. However, current methods do not allow for the direct generation of persulfide anions under biomimetic and non-H2S conditions. Herein we report the development of a functional disulfide, FmSSPy-A (Fm =9-fluorenylmethyl; Py = pyridinyl). This reagent can effectively convert both smallmore » molecule and protein thiols (-SH) to form –S-SFm adducts under mild conditions. It allows for a H2S-free and biomimetic protocol to generate highly reactive persulfides (in their anionic forms). We also demonstrated the high nucleophilicity of persulfides toward a number of thiol-blocking reagents. This method holds promise for further understanding the chemical biology of persulfides and S-sulfhydration.« less

Thiol reduction of arsenite and selenite: DFT modeling of the pathways to an as-se bond.

PubMed

Harper, Lenora K; Antony, Sonia; Bayse, Craig A

2014-12-15

The reactivity of arsenite and selenite with biological thiols plays an important role in the toxicity of these elements. However, toxic effects are eliminated when the species are coadministered, due to the antagonistic relationship between selenium and arsenic. The reduction of arsenous acid and selenious acid by thiol and the formation of an As-Se species have been modeled using density functional theory (DFT) and solvent-assisted proton exchange (SAPE), a microsolvation technique that uses a network of water molecules to mimic the participation of bulk solvent in proton transfer processes. Activation barriers and relative energies were calculated for the stepwise thiol reduction of arsenite to form As(SR)3 and selenious acid to first form a selenotrisulfide (Se(SR)2) and then H2Se. Several pathways were explored for the formation of an As-Se bond: the nucleophilic attack of selenide or selenopersulfide on As(OH)3, (RS)As(OH)2, and (RS)2AsOH to form (RS)2AsSeH. On the basis of the lower activation barrier and bioavailability of (RS)2AsOH, the reaction of H2Se with (RS)2AsOH is deemed the most favorable, consistent with previous experimental studies.

Electrochemical and spectroscopic characterization of surface sol-gel processes.

PubMed

Chen, Xiaohong; Wilson, George S

2004-09-28

(3-Mercaptopropyl)trimethoxysilane (MTS) forms a unique film on a platinum substrate by self-assembly and sol-gel cross-linking. The gelating and drying states of the self-assembled MTS sol-gel films were probed by use of electrochemical and spectroscopic methods. The thiol moiety was the only active group within the sol-gel network. Gold nanoparticles were employed to detect the availability of the thiol group and their interaction further indicated the physicochemical states of the sol-gel inner structure. It was found that the thiol groups in the open porous MTS aerogel matrix were accessible to the gold nanoparticles while thiol groups in the compact MTS xerogel network were not accessible to the gold nanoparticles. The characteristics of the sol-gel matrix change with time because of its own irreversible gelating and drying process. The present work provides direct evidence of gold nanoparticle binding with thiol groups within the sol-gel structures and explains the different permeability of "aerogel" and "xerogel" films of MTS on the basis of electrochemical and spectroscopic results. Two endogenous species, hydrogen peroxide and ascorbic acid, were used to test the permeability of the self-assembled sol-gel film in different states. The MTS xerogel film on the platinum electrode was extremely selective against ascorbic acid while maintaining high sensitivity to hydrogen peroxide in contrast to the relatively high permeability of ascorbic acid in the MTS aerogel film. This study showed the potential of the MTS sol-gel film as a nanoporous material in biosensor development.

Bromine catalyzed conversion of S-tert-butyl groups into versatile and, for self-assembly processes accessible, acetyl-protected thiols.

PubMed

Blaszczyk, Alfred; Elbing, Mark; Mayor, Marcel

2004-10-07

The facile and efficient conversion of a tert-butyl protecting group to an acetyl protecting group for thiols by catalytic amounts of bromine in acetyl chloride and the presence of acetic acid has been developed. The fairly mild reaction conditions are of particular interest for new protecting group strategies for sulfur functionalised target structures. Copyright 2004 The Royal Society of Chemistry

Au99(SPh)42 nanomolecules: aromatic thiolate ligand induced conversion of Au144(SCH2CH2Ph)60.

PubMed

Nimmala, Praneeth Reddy; Dass, Amala

2014-12-10

A new aromatic thiolate protected gold nanomolecule Au99(SPh)42 has been synthesized by reacting the highly stable Au144(SCH2CH2Ph)60 with thiophenol, HSPh. The ubiquitous Au144(SR)60 is known for its high stability even at elevated temperature and in the presence of excess thiol. This report demonstrates for the first time the reactivity of the Au144(SCH2CH2Ph)60 with thiophenol to form a different 99-Au atom species. The resulting Au99(SPh)42 compound, however, is unreactive and highly stable in the presence of excess aromatic thiol. The molecular formula of the title compound is determined by high resolution electrospray mass spectrometry (ESI-MS) and confirmed by the preparation of the 99-atom nanomolecule using two ligands, namely, Au99(SPh)42 and Au99(SPh-OMe)42. This mass spectrometry study is an unprecedented advance in nanoparticle reaction monitoring, in studying the 144-atom to 99-atom size evolution at such high m/z (∼12k) and resolution. The optical and electrochemical properties of Au99(SPh)42 are reported. Other substituents on the phenyl group, HS-Ph-X, where X = -F, -CH3, -OCH3, also show the Au144 to Au99 core size conversion, suggesting minimal electronic effects for these substituents. Control experiments were conducted by reacting Au144(SCH2CH2Ph)60 with HS-(CH2)n-Ph (where n = 1 and 2), bulky ligands like adamantanethiol and cyclohexanethiol. It was observed that conversion of Au144 to Au99 occurs only when the phenyl group is directly attached to the thiol, suggesting that the formation of a 99-atom species is largely influenced by aromaticity of the ligand and less so on the bulkiness of the ligand.

Chemical copatterning strategies using azlactone-based block copolymers

DOE PAGES

Masigol, Mohammadali; Barua, Niloy; Retterer, Scott T.; ...

2017-09-01

Interfaces can be modified with azlactone-functional polymers in order to manipulate the chemical surface reactivity. Azlactone groups are highly reactive toward amine, thiol, and alcohol nucleophiles, providing a versatile coupling chemistry for secondary surface modification. Azlactone-based surface polymers have been explored in numerous applications, including chemical and biological capture, sensing, and cell culture. These applications often require that the polymer is copatterned within a chemically or biologically inert background; however, common fabrication methods degrade azlactone groups during processing steps or result in polymer films with poorly controlled thicknesses. Here, the authors develop fabrication strategies using parylene lift-off and interface-directed assemblymore » methods to generate microscale patterns of azlactone-based block copolymer in chemically or biologically inert backgrounds. The functionality of azlactone groups was preserved during fabrication, and patterned films appeared as uniform, 80–120nm brushlike films. The authors also develop a patterning approach that uses a novel microcontact stamping method to generate cross-linked, three-dimensional structures of azlactone-based polymers with controllable, microscale thicknesses. The authors identify the benefits of each approach and expect these polymers and patterning strategies to provide a versatile toolbox for developing synthetic interfaces with tuned chemical and physical features for sensing, cell culture, or material capture applications.« less

Chemical copatterning strategies using azlactone-based block copolymers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Masigol, Mohammadali; Barua, Niloy; Retterer, Scott T.

Interfaces can be modified with azlactone-functional polymers in order to manipulate the chemical surface reactivity. Azlactone groups are highly reactive toward amine, thiol, and alcohol nucleophiles, providing a versatile coupling chemistry for secondary surface modification. Azlactone-based surface polymers have been explored in numerous applications, including chemical and biological capture, sensing, and cell culture. These applications often require that the polymer is copatterned within a chemically or biologically inert background; however, common fabrication methods degrade azlactone groups during processing steps or result in polymer films with poorly controlled thicknesses. Here, the authors develop fabrication strategies using parylene lift-off and interface-directed assemblymore » methods to generate microscale patterns of azlactone-based block copolymer in chemically or biologically inert backgrounds. The functionality of azlactone groups was preserved during fabrication, and patterned films appeared as uniform, 80–120nm brushlike films. The authors also develop a patterning approach that uses a novel microcontact stamping method to generate cross-linked, three-dimensional structures of azlactone-based polymers with controllable, microscale thicknesses. The authors identify the benefits of each approach and expect these polymers and patterning strategies to provide a versatile toolbox for developing synthetic interfaces with tuned chemical and physical features for sensing, cell culture, or material capture applications.« less

Modification of the mitochondrial sulfonylurea receptor by thiol reagents.

PubMed

Szewczyk, A; Wójcik, G; Lobanov, N A; Nalecz, M J

1999-08-19

The purpose of this study was to investigate the effects exerted by thiol-modifying reagents on themitochondrial sulfonylurea receptor. The thiol-oxidizing agents (timerosal and 5, 5'-dithio-bis(2-nitrobenzoic acid)) were found to produce a large inhibition (70% to 80%) of specific binding of [(3)H]glibenclamide to the beef heart mitochondrial membrane. Similar effects were observed with membrane permeable (N-ethylmaleimide) and non-permeable (mersalyl) thiol modifying agents. Glibenclamide binding was also decreased by oxidizing agents (hydrogen peroxide) but not by reducing agents (reduced gluthatione, dithiothreitol and the 2,3-dihydroxy-1,4-dithiolbutane). The results suggest that intact thiol groups, facing the mitochondrial matrix, are essential for glibenclamide binding to the mitochondrial sulfonylurea receptor. Copyright 1999 Academic Press.

Association between biomarkers of carbonyl stress with increased systemic inflammatory response in different stages of chronic kidney disease and after renal transplantation.

PubMed

Aveles, Paulo R; Criminácio, Ciro R; Gonçalves, Simone; Bignelli, Alexandre T; Claro, Ligia Maria; Siqueira, Sérgio S; Nakao, Lia S; Pecoits-Filho, Roberto

2010-01-01

Chronic kidney disease (CKD) is characterized by progressive kidney dysfunction accompanied by accumulation of uremic toxins and a potential disequilibrium between the redox status and the generation of prooxidants, resulting in oxidative stress and chronic inflammation which is associated with complications (particularly cardiovascular disease) in this population. We aimed to analyze the concentration of total plasma thiols (indicator of antioxidant capacity) and the protein carbonyl content (a marker of carbonyl stress) in relation to kidney function and inflammation in a group of patients with CKD. A group of 68 patients with CKD (stages 2-5; mean age 57 ± 12 years, 46% male, 34% diabetics) and another group of 21 patients who underwent living donor kidney transplantation (mean age 36 ± 17 years, 50% male, 10% diabetics, and 9 ± 2 months after renal transplantation) were included in the study. Total plasma thiol and protein carbonyl levels were determined by the DTNB and DNPH methods, respectively, and were adjusted to the plasma albumin concentrations. Plasma levels of fibrinogen and C-reactive protein (CRP) were measured by routine methods and used as markers of inflammation. Mean glomerular filtration rate (GFR) was 48 ml/min, and there was a positive correlation between GFR and thiol (r = 0.25, p < 0.05) and a negative correlation between GFR and carbonyl (r = -0.26, p < 0.05), fibrinogen (r = -0.45, p < 0.0001) and CRP (r = -0.14, p = ns). Carbonyl strongly correlated with CRP (0.49, p < 0.0001) and fibrinogen (0.30, p < 0.01). There was a significant reduction in plasma carbonyl after renal transplantation (1.4 ± 0.4 nmol/mg albumin), compared with the levels before the procedure (2.0 ± 1.4 nmol/mg albumin, p < 0.05), which parallels an improvement in thiol levels (15 ± 4 vs. 21 ± 5 nmol/mg albumin, p < 0.001). In addition, there was a significant correlation between CRP and carbonyl after the transplantation (r = 0.65; p < 0.005). We observed that patients with CKD present an altered redox status and increased signs of carbonyl stress and inflammatory activity as kidney function deteriorates, which was partially but significantly improved after renal transplantation. These findings indicate the importance of renal function in the complications of CKD related to oxidative stress and inflammation. Copyright © 2010 S. Karger AG, Basel.

Investigation of a thiolated polymer in gene delivery

NASA Astrophysics Data System (ADS)

Bacalocostantis, Irene

Thiol-containing bioreducible polymers show significant potential as delivery vectors in gene therapy, a rapidly growing field which seeks to treat genetic-based disorders by delivering functional synthetic genes to diseased cells. Studies have shown that thiolated polymers exhibit improved biodegradability and prolonged in vivo circulation times over non-thiolated polymers. However, the extent to which thiol concentrations impact the carrier's delivery potential has not been well explored. The aim of this dissertation is to investigate how relative concentrations of free thiols and disulfide crosslinks impact a polymeric carriers delivery performance with respect to DNA packaging, complex stability, cargo protection, gene release, internalization efficiency and cytotoxicity. To accomplish this goal, several fluorescent polymers containing varying concentrations of thiol groups were synthesized by conjugating thiol-pendant chains onto the primary amines of cationic poly(allylamine). In vitro delivery assays and characterization techniques were employed to assess the effect of thiols in gene delivery.

Reactivity of Biliatresone, a Natural Biliary Toxin, with Glutathione, Histamine, and Amino Acids

PubMed Central

Koo, Kyung A.; Waisbourd-Zinman, Orith; Wells, Rebecca G.; Pack, Michael; Porter, John R.

2016-01-01

In our previous work, we identified a natural toxin, biliatresone, from Dysphania glomulifera and D. littoralis, endemic plants associated with outbreaks of biliary atresia in Australian neonatal livestock. Biliatresone is a very rare isoflavonoid with an α-methylene ketone between two phenyls, 1,2-diaryl-2-propenone, along with methylenedioxy, dimethoxyl, and hydroxyl functional groups, that causes extrahepatic biliary toxicity in zebrafish. The toxic core of biliatresone is a methylene in the α-position relative to the ketone of 1,2-diaryl-2-propenone that serves as an electrophilic Michael acceptor. The α-methylene of biliatresone spontaneously conjugated with water and methanol (MeOH), respectively, via Michael addition in a reverse phase high-performance liquid chromatography (RP-HPLC) analysis. We here report the reactivity of biliatresone toward glutathione (GSH), several amino acids, and other thiol- or imidazole-containing biomolecules. LC-MS and HPLC analysis of the conjugation reaction showed the reactivity of biliatresone to be in the order histidine > N-acetyl-d-cysteine (D-NAC) = N-acetyl-l-cysteine (L-NAC) > histamine > glutathione ≥ cysteine ≫ glycine > glutamate > phenylalanine, while serine and adenine had no reactivity due to intramolecular hydrogen bonding in the protic solvents. The reactivity of ethyl vinyl ketone (EVK, 1-penten-3-one), an example of a highly reactive α,ß-unsaturated ketone, toward GSH gave a 6.7-fold lower reaction rate constant than that of biliatresone. The reaction rate constant of synthetic 1,2-diaryl-2-propen-1-one (DP), a core structure of the toxic molecule, was 10-fold and 1.5-fold weaker in potency compared to the reaction rate constants of biliatresone and EVK, respectively. These results demostrated that the methylenedioxy, dimethoxyl, and hydroxyl functional groups of biliatresone contribute to the stronger reactivity of the Michael acceptor α-methylene ketone toward nucleophiles compared to that of DP and EVK. PMID:26713899

Reactivity of Biliatresone, a Natural Biliary Toxin, with Glutathione, Histamine, and Amino Acids.

PubMed

Koo, Kyung A; Waisbourd-Zinman, Orith; Wells, Rebecca G; Pack, Michael; Porter, John R

2016-02-15

In our previous work, we identified a natural toxin, biliatresone, from Dysphania glomulifera and D. littoralis, endemic plants associated with outbreaks of biliary atresia in Australian neonatal livestock. Biliatresone is a very rare isoflavonoid with an α-methylene ketone between two phenyls, 1,2-diaryl-2-propenone, along with methylenedioxy, dimethoxyl, and hydroxyl functional groups, that causes extrahepatic biliary toxicity in zebrafish. The toxic core of biliatresone is a methylene in the α-position relative to the ketone of 1,2-diaryl-2-propenone that serves as an electrophilic Michael acceptor. The α-methylene of biliatresone spontaneously conjugated with water and methanol (MeOH), respectively, via Michael addition in a reverse phase high-performance liquid chromatography (RP-HPLC) analysis. We here report the reactivity of biliatresone toward glutathione (GSH), several amino acids, and other thiol- or imidazole-containing biomolecules. LC-MS and HPLC analysis of the conjugation reaction showed the reactivity of biliatresone to be in the order histidine > N-acetyl-d-cysteine (D-NAC) = N-acetyl-l-cysteine (L-NAC) > histamine > glutathione ≥ cysteine ≫ glycine > glutamate > phenylalanine, while serine and adenine had no reactivity due to intramolecular hydrogen bonding in the protic solvents. The reactivity of ethyl vinyl ketone (EVK, 1-penten-3-one), an example of a highly reactive α,ß-unsaturated ketone, toward GSH gave a 6.7-fold lower reaction rate constant than that of biliatresone. The reaction rate constant of synthetic 1,2-diaryl-2-propen-1-one (DP), a core structure of the toxic molecule, was 10-fold and 1.5-fold weaker in potency compared to the reaction rate constants of biliatresone and EVK, respectively. These results demostrated that the methylenedioxy, dimethoxyl, and hydroxyl functional groups of biliatresone contribute to the stronger reactivity of the Michael acceptor α-methylene ketone toward nucleophiles compared to that of DP and EVK.

Thiols in the alphaIIbbeta3 integrin are necessary for platelet aggregation.

PubMed

Manickam, Nagaraj; Sun, Xiuhua; Hakala, Kevin W; Weintraub, Susan T; Essex, David W

2008-07-01

Sulfhydryl groups of platelet surface proteins are important in platelet aggregation. While p-chloromercuribenzene sulphonate (pCMBS) has been used in most studies on platelet surface thiols, the specific thiol-proteins that pCMBS reacts with to inhibit aggregation have not been well defined. Since the thiol-containing P2Y(12) ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y(12) receptor antagonist 2-MeSAMP to examine thiol-dependent reactions in the absence of contributions from this receptor. We provide evidence for a non-P2Y(12) thiol-dependent reaction near the final alphaIIbbeta3-dependent events of aggregation. We then used 3-(N-maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in alphaIIbbeta3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the alphaIIb and beta3 subunits was confirmed by identification of the purified proteins by mass spectrometry and by inhibition of labelling with 5,5'-dithiobis-(2-nitrobenzoic acid). In contrast to MPB, pCMBS preferentially reacted with thiols in alphaIIbbeta3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling-independent activation of alphaIIbbeta3 by Mn(2+). Our results suggest that the thiols in alphaIIbbeta3 that are blocked by pCMBS are important in the activation of this integrin.

Conducting Polymers Crosslinked with Sulfur as Cathode Materials for High-Rate, Ultralong-Life Lithium-Sulfur Batteries.

PubMed

Zeng, Shuaibo; Li, Ligui; Xie, Lihong; Zhao, Dengke; Wang, Nan; Chen, Shaowei

2017-09-11

Low electrical conductivity and a lack of chemical confinement are two major factors that limit the rate performances and cycling stabilities of cathode materials in lithium-sulfur (Li-S) batteries. Herein, sulfur is copolymerized with poly(m-aminothiophenol) (PMAT) nanoplates through inverse vulcanization to form the highly crosslinked copolymer cp(S-PMAT) in which approximately 80 wt % of the feed sulfur is bonded chemically to the thiol groups of PMAT. A cp(S-PMAT)/C-based cathode exhibits a high discharge capacity of 1240 mAh g -1 at 0.1 C and remarkable rate capacities of 880 mAh g -1 at 1 C and 600 mAh g -1 at 5 C. Moreover, it can retain a capacity of 495 mAh g -1 after 1000 deep discharge-charge cycles at 2 C; this corresponds to a retention of 66.9 % and a decay rate of only 0.040 % per cycle. Such a remarkable rate performance is attributed to the highly conductive pathways of PMAT nanoplates, and the excellent cycling stability is ascribed mainly to the chemical confinement of sulfur through a large number of stable covalent bonds between sulfur and the thiol groups of PMAT. The results suggest that this strategy is a viable paradigm for the design and engineering of conducting polymers with reactive functional groups as effective electrode materials for high-performance Li-S batteries. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Resistance exercise training attenuates exercise-induced lipid peroxidation in the elderly.

PubMed

Vincent, Kevin R; Vincent, Heather K; Braith, Randy W; Lennon, Shannon L; Lowenthal, David T

2002-08-01

This study examined the effects of 6 months of resistance exercise (RX) on basal and post-aerobic exercise lipid peroxidation (LIPOX). Men and women [n = 62, mean (SD) age 68.4 (6) years] were divided randomly into either a control (n = 16, CON), low-intensity training [LEX n = 24; 50% one-repetition maximum (1RM), 13 repetitions/exercise], or high-intensity training (HEX n = 22, 80% 1RM, 8 repetitions/exercise) group. Pre- and post-training, subjects performed a graded aerobic exercise test (GXT). Blood samples were collected prior to and 10 min following each GXT. Subjects trained 3 times per week for 6 months using 12 RX machines. LIPOX was determined by measuring levels of thiobarbituric reactive acid substances (TBARS) and lipid hydroperoxides (PEROX). RX had no effect on resting LIPOX. Post-training, post-GXT TBARS were lower in the LEX and HEX groups by 14% and 18%, respectively, compared to CON (P < 0.05). Post-GXT PEROX levels were lower (P < 0.05) in LEX and HEX compared to CON [CON 3.51 (0.56) nmol/ml, LEX 2.89 (0.80) nmol/ml, HEX 2.99 (0.63) nmol/ml]. Serum total and non-protein (glutathione) thiols were higher in the LEX and HEX groups following training compared to CON (P < 0.05). These data suggest that RX can (1) reduce serum LIPOX, (2) provide protection against oxidizing agents in vitro, and (3) provide a "cross-protection" against the oxidative stress generated by aerobic exercise, perhaps mediated by improvements in the thiol portion of the antioxidant defense.

Changes in Thiol-Disulfide Homeostasis of the Body to Surgical Trauma in Laparoscopic Cholecystectomy Patients.

PubMed

Polat, Murat; Ozcan, Onder; Sahan, Leyla; Üstündag-Budak, Yasemin; Alisik, Murat; Yilmaz, Nigar; Erel, Özcan

2016-12-01

We aimed to investigate the short-term effect of laparoscopic surgery on serum thiol-disulfide homeostasis levels as a marker of oxidant stress of surgical trauma in elective laparoscopic cholecystectomy patients. Venous blood samples were collected, and levels of native thiols, total thiols, and disulfides were determined with a novel automated assay. Total antioxidant capacity (measured as the ferric-reducing ability of plasma) and serum ischemia modified albumin, expressed as absorbance units assayed by the albumin cobalt binding test, were determined. The major findings of the present study were that native thiol (283 ± 45 versus 241 ± 61 μmol/L), total thiol (313 ± 49 versus 263 ± 67 μmol/L), and disulfide (14.9 ± 4.6 versus 11.0 ± 6.1 μmol/L) levels were decreased significantly during operation and although they increased, they did not return to preoperation levels 24 hours after laparoscopic surgery compared to the levels at baseline. Disulfide/native thiol and disulfide/total thiol levels did not change during laparoscopic surgery. The decrease in plasma level of native and total thiol groups suggests impairment of the antioxidant capacity of plasma; however, the delicate balance between the different redox forms of thiols was maintained during surgery.

Natural structural variation in enzymes as a tool in the study of mechanism exemplified by a comparison of the catalytic-site structure and characteristics of cathepsin B and papain. pH-dependent kinetics of the reactions of cathepsin B from bovine spleen and from rat liver with a thiol-specific two-protonic-state probe (2,2'-dipyridyl disulphide) and with a specific synthetic substrate (N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide).

PubMed Central

Willenbrock, F; Brocklehurst, K

1984-01-01

Cathepsin B (EC 3.4.22.1) from bovine spleen and the analogous enzyme from rat liver were investigated at 25 degrees C at I0.1 in acidic media by kinetic study of (a) the reactions of their catalytic-site thiol groups towards the two-protonic-state reactivity probe 2,2'-dipyridyl disulphide and (b) their catalysis of the hydrolysis of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide. Reactivity-probe kinetics showed that nucleophilic character is generated in the sulphur atom of cathepsin B by protonic dissociation with pKa 3.4, presumably to form an S-/ImH+ ion-pair. Substrate-catalysis kinetics showed that ion-pair formation is not sufficient to generate catalytic competence in cathepsin B, because catalytic activity is not generated as the pH is raised across pKa 3.4 but rather as it is raised across pKa 5-6 (5.1 for kcat; 5.6 for kcat./Km for the bovine spleen enzyme and 5.8 for kcat./Km for the rat liver enzyme). The implications of these results and of known structural differences between the catalytic sites of the rat liver enzyme and papain (EC 3.4.22.2) for the mechanism of cysteine-proteinase-catalysed hydrolysis are discussed. PMID:6534384

N-acetylcysteine does not influence the activity of endothelium-derived relaxing factor in vivo.

PubMed

Creager, M A; Roddy, M A; Boles, K; Stamler, J S

1997-02-01

Nitric oxide forms complexes with an array of biomolecular carriers that retain biological activity. This reactivity of nitric oxide in physiological systems has led to some dispute as to whether endothelium-derived relaxing factors nitric oxide or a closely related adduct thereof, such as a nitrosothiol. In vitro bioassays used to address this question are limited by the exclusion of biological thiols that are requisite for nitrosothiol formation. Thus, the purpose of this study was to obtain insight into the identity of endothelium-derived relaxing factor in vivo. We reasoned that if endothelium-derived relaxing factor in nitric oxide, infusion of physiological concentrations of thiol would potentiate its bioactivity by analogy with effects seen in vitro, whereas nitrosothiol would be resistant to such modulation. We used venous-occlusion plethysmography to study forearm blood flow in normal subjects. Methacholine (0.3 to 10 micrograms/min) and nitroglycerin (1 to 30 micrograms/min) were infused via the brachial artery to elicit endothelium-dependent and endothelium-independent vasodilation, respectively. Dose-response determinations were made for each drug before and after an intra-arterial infusion of the reduced thiol, N-acetylcysteine, at rates estimated to achieve a physiological concentration of 1 mmol/L. Methacholine increased forearm blood flow in a dose-dependent manner. Infusion of N-acetylcysteine did not change the sensitivity (ED50, 1.7 versus 1.7 micrograms/min, P = NS) or maximal response to methacholine. In contrast, thiol increased the sensitivity to nitroglycerin (ED50, 4.7 versus 2.8 micrograms/min, P < .01). Thus, conflicting with reports in vitro, thiol does not modulate endothelium-derived relaxing factor responses in vivo. These data indicate that sulfhydryl groups are not a limiting factor for endothelium-derived relaxing factor responses in forearm resistance vessels in normal humans and are in keeping with reports that nitrosothiol contributes to endothelium-derived relaxing factor bioactivity in plasma and vascular smooth muscle. Potentiation of the effects of nitroglycerin by N-acetylcysteine can be attributed to its enhanced biotransformation to an endothelium-derived relaxing factor equivalent, such as nitrosothiol. These observations support the notion of an equilibrium between nitric oxide and nitrosothiol in biological systems that may be influenced by redox state.

Engineering surfaces for bioconjugation: developing strategies and quantifying the extent of the reactions.

PubMed

Gauvreau, Virginie; Chevallier, Pascale; Vallières, Karine; Petitclerc, Eric; Gaudreault, René C; Laroche, Gaétan

2004-01-01

This study presents two-step and multistep reactions for modifying the surface of plasma-functionalized poly(tetrafluoroethylene) (PTFE) surfaces for subsequent conjugation of biologically relevant molecules. First, PTFE films were treated by a radiofrequency glow discharge (RFGD) ammonia plasma to introduce amino groups on the fluoropolymer surface. This plasma treatment is well optimized and allows the incorporation of a relative surface concentration of approximately 2-3.5% of amino groups, as assessed by chemical derivatization followed by X-ray photoelectron spectroscopy (XPS). In a second step, these amino groups were further reacted with various chemical reagents to provide the surface with chemical functionalities such as maleimides, carboxylic acids, acetals, aldehydes, and thiols, that could be used later on to conjugate a wide variety of biologically relevant molecules such as proteins, DNA, drugs, etc. In the present study, glutaric and cis-aconitic anhydrides were evaluated for their capability to provide carboxylic functions to the PTFE plasma-treated surface. Bromoacetaldehyde diethylacetal was reacted with the aminated PTFE surface, providing a diethylacetal function, which is a latent form of aldehyde functionality. Reactions with cross-linkers such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB) were evaluated to provide a highly reactive maleimide function suitable for further chemical reactions with thiolated molecules. Traut reagent (2-iminothiolane) was also conjugated to introduce a thiol group onto the fluoropolymer surface. PTFE-modified surfaces were analyzed by XPS with a particular attention to quantify the extent of the reactions that occurred on the polymer. Finally, surface immobilization of fibronectin performed using either glutaric anhydride or sulfo-SMPB activators demonstrated the importance of selecting the appropriate conjugation strategy to retain the protein biological activity.

TMV nanorods with programmed longitudinal domains of differently addressable coat proteins

NASA Astrophysics Data System (ADS)

Geiger, Fania C.; Eber, Fabian J.; Eiben, Sabine; Mueller, Anna; Jeske, Holger; Spatz, Joachim P.; Wege, Christina

2013-04-01

The spacing of functional nanoscopic elements may play a fundamental role in nanotechnological and biomedical applications, but is so far rarely achieved on this scale. In this study we show that tobacco mosaic virus (TMV) and the RNA-guided self-assembly process of its coat protein (CP) can be used to establish new nanorod scaffolds that can be loaded not only with homogeneously distributed functionalities, but with distinct molecule species grouped and ordered along the longitudinal axis. The arrangement of the resulting domains and final carrier rod length both were governed by RNA-templated two-step in vitro assembly. Two selectively addressable TMV CP mutants carrying either thiol (TMVCys) or amino (TMVLys) groups on the exposed surface were engineered and shown to retain reactivity towards maleimides or NHS esters, respectively, after acetic acid-based purification and re-assembly to novel carrier rod types. Stepwise combination of CPCys and CPLys with RNA allowed fabrication of TMV-like nanorods with a controlled total length of 300 or 330 nm, respectively, consisting of adjacent longitudinal 100-to-200 nm domains of differently addressable CP species. This technology paves the way towards rod-shaped scaffolds with pre-defined, selectively reactive barcode patterns on the nanometer scale.The spacing of functional nanoscopic elements may play a fundamental role in nanotechnological and biomedical applications, but is so far rarely achieved on this scale. In this study we show that tobacco mosaic virus (TMV) and the RNA-guided self-assembly process of its coat protein (CP) can be used to establish new nanorod scaffolds that can be loaded not only with homogeneously distributed functionalities, but with distinct molecule species grouped and ordered along the longitudinal axis. The arrangement of the resulting domains and final carrier rod length both were governed by RNA-templated two-step in vitro assembly. Two selectively addressable TMV CP mutants carrying either thiol (TMVCys) or amino (TMVLys) groups on the exposed surface were engineered and shown to retain reactivity towards maleimides or NHS esters, respectively, after acetic acid-based purification and re-assembly to novel carrier rod types. Stepwise combination of CPCys and CPLys with RNA allowed fabrication of TMV-like nanorods with a controlled total length of 300 or 330 nm, respectively, consisting of adjacent longitudinal 100-to-200 nm domains of differently addressable CP species. This technology paves the way towards rod-shaped scaffolds with pre-defined, selectively reactive barcode patterns on the nanometer scale. Electronic supplementary information (ESI) available: Two supplementary figures, showing (a) the phenotype of different Nicotiana plant species systemically infected with TMVwt, TMVCys, or TMVLys at 9 and 15 days post-inoculation, and (b) the length distribution of partially and completely assembled VLPs with a second type of RNA template (TMVwt RNA); and one supplementary table listing the sequences of primers used for site-specific mutagenesis of the TMV CP ORF and for sequencing. See DOI: 10.1039/c3nr33724c

Protein Thiol Redox Signaling in Monocytes and Macrophages.

PubMed

Short, John D; Downs, Kevin; Tavakoli, Sina; Asmis, Reto

2016-11-20

Monocyte and macrophage dysfunction plays a critical role in a wide range of inflammatory disease processes, including obesity, impaired wound healing diabetic complications, and atherosclerosis. Emerging evidence suggests that the earliest events in monocyte or macrophage dysregulation include elevated reactive oxygen species production, thiol modifications, and disruption of redox-sensitive signaling pathways. This review focuses on the current state of research in thiol redox signaling in monocytes and macrophages, including (i) the molecular mechanisms by which reversible protein-S-glutathionylation occurs, (ii) the identification of bona fide S-glutathionylated proteins that occur under physiological conditions, and (iii) how disruptions of thiol redox signaling affect monocyte and macrophage functions and contribute to atherosclerosis. Recent Advances: Recent advances in redox biochemistry and biology as well as redox proteomic techniques have led to the identification of many new thiol redox-regulated proteins and pathways. In addition, major advances have been made in expanding the list of S-glutathionylated proteins and assessing the role that protein-S-glutathionylation and S-glutathionylation-regulating enzymes play in monocyte and macrophage functions, including monocyte transmigration, macrophage polarization, foam cell formation, and macrophage cell death. Protein-S-glutathionylation/deglutathionylation in monocytes and macrophages has emerged as a new and important signaling paradigm, which provides a molecular basis for the well-established relationship between metabolic disorders, oxidative stress, and cardiovascular diseases. The identification of specific S-glutathionylated proteins as well as the mechanisms that control this post-translational protein modification in monocytes and macrophages will facilitate the development of new preventive and therapeutic strategies to combat atherosclerosis and other metabolic diseases. Antioxid. Redox Signal. 25, 816-835.

A noticeable phenomenon: thiol terminal PEG enhances the immunogenicity of PEGylated emulsions injected intravenously or subcutaneously into rats.

PubMed

Wang, Chunling; Cheng, Xiaobo; Sui, Yue; Luo, Xiang; Jiang, Gongping; Wang, Yu; Huang, Zhenjun; She, Zhennan; Deng, Yihui

2013-11-01

Repeated intravenous injection of long-circulating methoxy-polyethylene glycol (PEG)-liposomes alters the pharmacokinetics and biodistribution of the second administration, regarded as the "accelerated blood clearance (ABC) phenomenon." Nevertheless, the effect of terminal groups of distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) on the induction of the ABC phenomenon had not been reported previously. In this study, rats were injected intravenously or subcutaneously with PEG coated emulsions (DE) which were prepared using PEG terminated with either the methoxyl (OCH3), hydroxyl (OH), amino (NH2), carboxyl (COOH), or thiol (SH) group. DE-OCH3 demonstrated the longest prolonged half-life in vivo after a single intravenous injection, followed by DE-SH and DE-COOH. In contrast, DE-OH was rapidly removed from the blood circulation, as was DE-NH2. Moreover, we observed a strong positive relationship between the circulation time of initially injected PEGylated emulsions and the extent to which the ABC phenomenon was induced, but a exception of DE-SH increasing the ABC effect. Furthermore, the present study suggested that thiols might stimulate the proliferation and differentiation of B cells to induce the fastest clearance of the second intravenous administration by inducing the synthesis of the cell membrane and cytosolic proteins or reacting with follicular dendritic cells. The results strongly suggested that thiol groups played a stimulatory role in the immune response and provided a considerable implication for multiple drug therapy of thiol groups. Copyright © 2013 Elsevier B.V. All rights reserved.

Cysteine regulation of protein function--as exemplified by NMDA-receptor modulation.

PubMed

Lipton, Stuart A; Choi, Yun-Beom; Takahashi, Hiroto; Zhang, Dongxian; Li, Weizhong; Godzik, Adam; Bankston, Laurie A

2002-09-01

Until recently cysteine residues, especially those located extracellularly, were thought to be important for metal coordination, catalysis and protein structure by forming disulfide bonds - but they were not thought to regulate protein function. However, this is not the case. Crucial cysteine residues can be involved in modulation of protein activity and signaling events via other reactions of their thiol (sulfhydryl; -SH) groups. These reactions can take several forms, such as redox events (chemical reduction or oxidation), chelation of transition metals (chiefly Zn(2+), Mn(2+) and Cu(2+)) or S-nitrosylation [the catalyzed transfer of a nitric oxide (NO) group to a thiol group]. In several cases, these disparate reactions can compete with one another for the same thiol group on a single cysteine residue, forming a molecular switch composed of a latticework of possible redox, NO or Zn(2+) modifications to control protein function. Thiol-mediated regulation of protein function can also involve reactions of cysteine residues that affect ligand binding allosterically. This article reviews the basis for these molecular cysteine switches, drawing on the NMDA receptor as an exemplary protein, and proposes a molecular model for the action of S-nitrosylation based on recently derived crystal structures.

Dietary Intake of Proteins and Calories Is Inversely Associated With The Oxidation State of Plasma Thiols in End-Stage Renal Disease Patients.

PubMed

Fanti, Paolo; Giustarini, Daniela; Rossi, Ranieri; Cunningham, Sue E D; Folli, Franco; Khazim, Khaled; Cornell, John; Matteucci, Elena; Bansal, Shweta

2015-11-01

Oxidative stress contributes to the pathogenesis of protein-energy wasting in maintenance hemodialysis (MHD) patients, but knowledge of specific effectors and mechanisms remains fragmented. Aim of the study was to define whether and how food intake is involved in the causal relationship between oxidative stress and protein-energy wasting. Seventy-one adult MHD patients and 24 healthy subjects (control) were studied cross-sectionally with analyses of diet record and of oxidative stress, as measured by a battery of plasma thiols including the protein sulfhydryl (-SH) group (PSH) levels (a marker of total protein-SH reducing capacity), the protein thiolation index (PTI, the ratio between disulfide, i.e., oxidized and reduced -SH groups in proteins), low molecular mass (LMM) thiols, LMM disulfides, and mixed LMM-protein disulfides. In addition, interleukin-6 (IL-6), albumin, C-reactive protein, and neutrophil gelatinase-associated lipocalin (NGAL) were measured as markers of inflammation. The patients showed low energy (22.0 ± 8.4 kcal/kg/day) and adequate protein (1.0 ± 0.4 g/kg/day) intakes, high levels of cystine (CySS; patients vs. 113.5 [90.9-132.8] vs. 68.2 [56.2-75.7] μM), cysteinylated proteins (CySSP; 216.0 [182.8-254.0] vs. 163.5 [150.0-195.5] μM), and high PTI (0.76 [0.61-0.88] vs. 0.43 [0.40-0.54]; P < .001 in all comparisons). In patients, variation of CySSP was explained by a standard regression model (R = 0.775; P = .00001) that included significant contributions of protein intake (β = -0.361), NGAL (β = 0.387), age (β = 0.295), and albumin (β = 0.457). In the same model, variation of PTI (R = 0.624; P = .01) was explained by protein intake (β = -0.384) and age (β = 0.326) and NGAL (β = 0.311). However, when PSH was entered as dependent variable (R = 0.730; P = .0001), only serum albumin (β = 0.495) and age (β = -0.280), but not dietary intake or NGAL, contributed to the model. In MHD, markers of thiol oxidation including CySSP and PTI show independent association with dietary intake and NGAL, whereas PSH, a marker of thiol-reducing capacity, did not associate with these same variables. The mechanism(s) responsible for inverse association between oxidative stress and food intake in MHD remain undefined. Copyright © 2015 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

Determination of total antioxidant capacity of milk by CUPRAC and ABTS methods with separate characterisation of milk protein fractions.

PubMed

Çekiç, Sema Demirci; Demir, Aslı; Başkan, Kevser Sözgen; Tütem, Esma; Apak, Reşat

2015-05-01

Most milk-applied antioxidant assays in literature are based on the isolation and quantification of individual antioxidative compounds, whereas total antioxidant capacity (TAC) gives a more holistic picture due to cooperative action of antioxidants. Recently, the cupric reducing antioxidant capacity (CUPRAC) method has been modified to measure the antioxidant capacities of thiol-containing proteins, where the classical ammonium acetate buffer - that may otherwise precipitate proteins- was replaced with concentrated urea buffer (able to expose embedded thiol groups of proteins to oxidative attack) adjusted to pH 7.0. Thus, antioxidant capacity of milk was investigated with two competing TAC assays, namely CUPRAC and ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid))/persulphate, because only these assays were capable of evaluating protein contribution to the observed TAC value. As milk fat caused turbidity, experiments were carried out with skim milk or defatted milk samples. To determine TAC, modified CUPRAC method was applied to whole milk, separated and redissolved protein fractions, and the remaining liquid phase after necessary operations. Both TAC methods were investigated for their dilution sensitivity and antioxidant power assessment of separate milk fractions such as casein and whey. Proteins like β-lactoglobulin and casein (but not simple thiols) exhibited enhanced CUPRAC reactivity with surfactant (SDS) addition. Addition of milk protein fractions to whole skim milk produced significant 'negative-biased' deviations (up to -26% relative standard error) from TAC absorbance additivity in the application of the ABTS method, as opposed to that of the CUPRAC method less affected by chemical deviations from Beer's law thereby producing much smaller deviations from additivity (i.e. the property of additivity is valid when the measured TAC of a mixture is equal to the sum of individual antioxidant capacities of its constituents).

Protein disulfide isomerase and Nox: new partners in redox signaling.

PubMed

Trevelin, Silvia Cellone; Lopes, Lucia Rossetti

2015-01-01

Reactive oxygen species (ROS) contribute to the pathogenesis of cardiovascular disease, including hypertension, atherosclerosis, cardiac hypertrophy, heart failure and restenosis. Thiol proteins and thiol oxidoreductases are key players in cell signaling, and their altered expression and/or activity has been associated with a disrupture in cardiac and vascular homeostasis. Protein disulfide isomerase (PDI) is a thiol oxidoreductase member of the thioredoxin family that has multiple roles in cellular function. Originally discovered in the endoplasmic reticulum (ER), PDI is essential for protein folding. However, it can also be found in the cytosol and closely associated with the surface of platelets, smooth muscle cells, neutrophils and endothelial cells. On the cell surface, PDI is imperative for platelet aggregation and transnitrosation, which are related to thrombosis and control of vascular tone by nitric oxide, respectively. Furthermore, PDI signaling contributes to redox-dependent events such as smooth muscle cell migration induced by PDGF and TNFα-dependent angiogenesis. Studies from our group have shown that intracellular PDI regulates the expression and activity of the NADPH oxidase family of proteins (Nox), which are enzymes dedicated to ROS generation. PDI acts as a new organizer of leukocyte Nox2 by redox dependently associating with p47phox and controlling its recruitment to the plasma membrane, an essential step for assembly of the active enzyme. Such multiple effects of PDI suggest that specific targeting of this oxidoreductase could represent a new approach in the treatment of vascular disease. In this review, we present a novel role for PDI as an adaptor protein involved in redox processes and Nox signaling and propose PDI as a potential therapeutic target in the treatment of atherosclerosis, thrombosis and hypertension.

Regulation of the Cardiac Muscle Ryanodine Receptor by O2 Tension and S-Nitrosoglutathione†

PubMed Central

Sun, Junhui; Yamaguchi, Naohiro; Xu, Le; Eu, Jerry P.; Stamler, Jonathan S.; Meissner, Gerhard

2009-01-01

The cardiac and skeletal muscle sarcoplasmic reticulum ryanodine receptor Ca2+ release channels contain thiols that are potential targets of endogenously produced reactive oxygen and nitrogen intermediates. Previously, we showed that the skeletal muscle ryanodine receptor (RyR1) has O2-sensitive thiols; only when these thiols are in the reduced state (pO2 ~ 10 mmHg) can physiological concentrations of NO (nanomolar) activate RyR1. Here, we report that cardiac muscle ryanodine receptor (RyR2) activity also depends on pO2, but unlike RyR1, RyR2 was not activated or S-nitrosylated directly by NO. Rather, activation and S-nitrosylation of RyR2 required S-nitrosoglutathione. The effects of peroxynitrite were indiscriminate on RyR1 and RyR2. Our results indicate that both RyR1 and RyR2 are pO2-responsive yet point to different mechanisms by which NO and S-nitrosoglutathione influence cardiac and skeletal muscle sarcoplasmic reticulum Ca2+ release. PMID:19053230

The evolution of glutathione metabolism in phototrophic microorganisms

NASA Technical Reports Server (NTRS)

Fahey, Robert C.; Buschbacher, Ralph M.; Newton, Gerald L.

1988-01-01

The low molecular weight thiol composition of a variety of phototropic microorganisms is examined in order to ascertain how evolution of glutathione (GSH) production is related to the evolution of oxygenic photosynthesis. Cells were extracted in the presence of monobromobimane (mBBr) to convert thiols (RSH) to fluorescent derivatives (RSmB) which were analyzed by high performance liquid chromatography (HPLC). Significant levels of GSH were not found in green sulfur bacteria. Substantial levels were present in purple bacteria, cyanobacteria, and eukaryotic algae. Other thiols measured included cysteine, gamma-glutamylcysteine, thiosulfate, coenzyme A, and sulfide. Many of the organisms also exhibited a marked ability to reduce mBBr to syn-(methyl,methyl)bimane, an ability which was quenched by treatment with 2-pyridyl disulfide or 5,5 prime-bisdithio - (2-nitrobenzoic acid) prior to reaction with mBBr. These observations indicate the presence of a reducing system capable of electron transfer to mBBr and reduction of reactive disulfides. The distribution of GSH in phototropic eubacteria indicates that GSH synthesis evolved at or around the time that oxygenic photosynthesis evolved.

Facile and High-Throughput Synthesis of Functional Microparticles with Quick Response Codes.

PubMed

Ramirez, Lisa Marie S; He, Muhan; Mailloux, Shay; George, Justin; Wang, Jun

2016-06-01

Encoded microparticles are high demand in multiplexed assays and labeling. However, the current methods for the synthesis and coding of microparticles either lack robustness and reliability, or possess limited coding capacity. Here, a massive coding of dissociated elements (MiCODE) technology based on innovation of a chemically reactive off-stoichimetry thiol-allyl photocurable polymer and standard lithography to produce a large number of quick response (QR) code microparticles is introduced. The coding process is performed by photobleaching the QR code patterns on microparticles when fluorophores are incorporated into the prepolymer formulation. The fabricated encoded microparticles can be released from a substrate without changing their features. Excess thiol functionality on the microparticle surface allows for grafting of amine groups and further DNA probes. A multiplexed assay is demonstrated using the DNA-grafted QR code microparticles. The MiCODE technology is further characterized by showing the incorporation of BODIPY-maleimide (BDP-M) and Nile Red fluorophores for coding and the use of microcontact printing for immobilizing DNA probes on microparticle surfaces. This versatile technology leverages mature lithography facilities for fabrication and thus is amenable to scale-up in the future, with potential applications in bioassays and in labeling consumer products. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Diphenyl diselenide decreases serum levels of total cholesterol and tissue oxidative stress in cholesterol-fed rabbits.

PubMed

de Bem, Andreza Fabro; Portella, Rafael de Lima; Colpo, Elisângela; Duarte, Marta Maria Medeiros Frescura; Frediane, Andressa; Taube, Paulo Sergio; Nogueira, Cristina Wayne; Farina, Marcelo; da Silva, Edson Luiz; Teixeira Rocha, João Batista

2009-07-01

Hypercholesterolaemia and oxidative stress are well-known risk factors in coronary artery diseases. Diphenyl diselenide is a synthetic organoselenium compound that has been shown to have in vitro and in vivo antioxidant properties. In this study, we investigated whether diphenyl diselenide could reduce the hypercholesterolaemia and diminish the tissue oxidative stress in cholesterol-fed rabbits. Twenty-four New Zealand white male rabbits were randomly divided into four groups. Each group was fed a different diet as follows: Control group--regular chow; Cholesterol group--1% cholesterol-enriched diet; diphenyl diselenide group--regular diet supplemented with 10 ppm diphenyl diselenide; and Chol/diphenyl diselenide group--the same cholesterol-rich supplemented with 10 ppm diphenyl diselenide. After 45 days of treatment, the rabbits were killed and the blood, liver, and brain were used for laboratory analysis. The results showed that the serum levels of total cholesterol were markedly increased in cholesterol-fed rabbits and the consumption of diphenyl diselenide decreased these levels approximately twofold in Chol/diphenyl diselenide rabbits (P < 0.05). The intake of diphenyl diselenide by hypercholesterolaemic rabbits diminished the serum and hepatic thiobarbituric acid reactive substances levels as well as the production of reactive oxygen species in the blood and brain (P < 0.05) when compared to the cholesterol group. In addition, diphenyl diselenide supplementation increased hepatic and cerebral delta-aminolevulinic dehydratase activity and hepatic non-protein thiol groups levels despite hypercholesterolaemia (P < 0.05). In summary, the results showed that diphenyl diselenide reduced the hypercholesterolaemia and the oxidative stress in cholesterol-fed rabbits.

Selective chromogenic detection of thiol-containing biomolecules using carbonaceous nanospheres loaded with silver nanoparticles as carrier.

PubMed

Hu, Bo; Zhao, Yang; Zhu, Hai-Zhou; Yu, Shu-Hong

2011-04-26

Thiol-containing biomolecules show strong affinity with noble metal nanostructures and could not only stably protect them but also control the self-assembly process of these special nanostructures. A highly selective and sensitive chromogenic detection method has been designed for the low and high molecular weight thiol-containing biomolecules, including cysteine, glutathione, dithiothreitol, and bovine serum albumin, using a new type of carbonaceous nanospheres loaded with silver nanoparticles (Ag NPs) as carrier. This strategy relies upon the place-exchange process between the reporter dyes on the surface of Ag NPs and the thiol groups of thiol-containing biomolecules. The concentration of biomolecules can be determined by monitoring with the fluorescence intensity of reporter dyes dispersed in solution. This new chromogenic assay method could selectively detect these biomolecules in the presence of various other amino acids and monosaccharides and even sensitively detect the thiol-containing biomolecules with different molecular weight, even including proteins.

Heavy metal ion removal by thiol functionalized aluminum oxide hydroxide nanowhiskers

NASA Astrophysics Data System (ADS)

Xia, Zhiyong; Baird, Lance; Zimmerman, Natasha; Yeager, Matthew

2017-09-01

In this study, we developed a cost effective method of using thiol functionalized γ-aluminum oxide hydroxide (γ-AlOOH) filters for removing three key heavy metals from water: mercury, lead, and cadmium under non-concomitant conditions. Compared to non-thiol treated γ-AlOOH filters, the introduction of thiol functional groups greatly improved the heavy metal removal efficiency under both static and dynamic filtration conditions. The adsorption kinetics of thiol functionalized γ-AlOOH were investigated using the Lagergren first order and pseudo-second order kinetics models; whereas the isothermal adsorption behavior of these membranes was revealed through the Langmuir and Freundlich models. Heavy metal concentration was quantified by Inductively Coupled Plasma-Mass Spectroscopy, and the thiol level on γ-AlOOH surface was measured by a colorimetric assay using Ellman's reagent. X-ray photoelectron spectroscopy was used to further address the surface sulfur state on the membranes after heavy metal exposure. Mechanisms for heavy metal adsorption were also discussed.

Low Nourishment of Vitamin C Induces Glutathione Depletion and Oxidative Stress in Healthy Young Adults.

PubMed

Waly, Mostafa I; Al-Attabi, Zahir; Guizani, Nejib

2015-09-01

The present study was conducted to assess the status of vitamin C among healthy young adults in relation to serum antioxidant parameters [glutathione (GSH), thiols, and total antioxidant capacity, (TAC)], and oxidative stress markers [malondialdehyde (MDA), and nitrites plus nitrates (NN)]. A prospective study included 200 young adults, and their dietary intake was assessed by using food diaries. Fasting plasma vitamin C, serum levels of GSH, thiols, TAC, MDA, and NN were measured using biochemical assays. It was observed that 38% of the enrolled subjects, n=76, had an adequate dietary intake of vitamin C (ADI group). Meanwhile, 62%, n=124, had a low dietary intake of vitamin C (LDI group) as compared to the recommended dietary allowances. The fasting plasma level of vitamin C was significantly higher in the ADI group as compared to the LDI group. Oxidative stress in the sera of the LDI group was evidenced by depletion of GSH, low thiols levels, impairment of TAC, an elevation of MDA, and increased NN. In the ADI group, positive correlations were found between plasma vitamin C and serum antioxidant parameters (GSH, thiols, and TAC). Meanwhile, the plasma vitamin C was negatively correlated with serum MDA and NN levels. This study reveals a significant increase of oxidative stress status and reduced antioxidant capacity in sera from healthy young adults with low intake of the dietary antioxidant, vitamin C.

Modifying surface resistivity and liquid moisture management property of keratin fibers through thiol-ene click reactions.

PubMed

Yu, Dan; Cai, Jackie Y; Church, Jeffrey S; Wang, Lijing

2014-01-22

This paper reports on a new method for improving the antistatic and liquid moisture management properties of keratinous materials. The method involves the generation of thiols by controlled reduction of cystine disulfide bonds in keratin with tris(2-carboxyethyl) phosphine hydrochloride and subsequent grafting of hydrophilic groups onto the reduced keratin by reaction with an acrylate sulfonate or acrylamide sulfonate through thiol-ene click chemistry. The modified substrates were characterized with Raman spectroscopy and scanning electron microscopy and evaluated for their performance changes in liquid moisture management, surface resistivity, and wet burst strength. The results have revealed that the thiol-acrylate reaction is more efficient than the thiol-acrylamide reaction, and the keratinous substrate modified with an acrylate sulfonate salt exhibits significantly improved antistatic and liquid moisture management properties.

Oxidative stress, thiols, and redox profiles.

PubMed

Harris, Craig; Hansen, Jason M

2012-01-01

Oxidative stress has been recognized as a contributing factor in the toxicity of a large number of developmental toxicants. Traditional definitions of oxidative stress state that a shift in the balance between reduced and oxidized biomolecules within cells, in favor of the latter, result in changes that are deleterious to vital cell functions and can culminate in malformations and death. The glutathione (GSH)/glutathione disulfide (GSSG) redox couple has been the traditional marker of choice for characterization of oxidative stress because of its high concentrations and direct roles as antioxidant and cellular protectant. Steady state depletion of GSH through conjugation, oxidation, or export has often been reported as the sole criteria for invoking oxidative stress and a myriad of associated deleterious consequences. Numerous other, mostly qualitative, observations have also been reported to suggest oxidative stress has occurred but it is not always clear how well they reflect the state of a cell or its functions. Our emerging understanding of redox signaling and the roles of reactive oxygen species (ROS), thiols, oxidant molecules, and cellular antioxidants, all acting as second messengers, has prompted a redefinition of oxidative stress based on changes in the real posttranslational protein thiol modifications that are central to redox regulation and control. Thiol-based redox couples such as GSH/GSSG, cysteine/cystine (cys/cySS), thioredoxin-reduced/thioredoxin-oxidized (TRX(red)/TRX(ox)) form independent signaling nodes that selectively regulate developmental events and are closely linked to changes in intracellular redox potentials. Accurate assessment of the consequences of increased free radicals in developing conceptuses should best be made using a battery of measurements including the quantitative assessment of intracellular redox potential, ROS, redox status of biomolecules, and induced changes in specific redox signaling nodes. Methods are presented for a determination of ROS production, soluble thiol oxidation, redox potential, and a proteomic approach to evaluate the thiol oxidation state of specific proteins.

Ebselen, a useful tool for understanding cellular redox biology and a promising drug candidate for use in human diseases.

PubMed

Noguchi, Noriko

2016-04-01

Ebselen is an organoselenium compound with glutathione peroxidase (GPx)-like hydroperoxide reducing activity. Moreover, ebselen has its own unique reactivity, with functions that GPx does not have, since it reacts with many kinds of thiols other than glutathione. Ebselen may affect the thioredoxin systems, through which it may contribute to regulation of cell function. With high reactivity toward thiols, hydroperoxides, and peroxynitrite, ebselen has been used as a useful tool in research on cellular redox mechanisms. Unlike α-tocopherol, ebselen does not scavenge lipid peroxyl radicals, which is another advantage of ebselen for use as a research tool in comparison with radical scavenging antioxidants. Selenium is not released from the ebselen molecule, which explains the low toxicity of ebselen. To further understand the mechanism of cellular redox biology, it should be interesting to compare the effects of ebselen with that of selenoprotein P, which supplies selenium to GPx. New medical applications of ebselen as a drug candidate for human diseases such as cancer and diabetes mellitus as well as brain stroke and ischemia will be expected. Copyright © 2015 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Na; Su, Dian; Cort, John R.

Reversible disulfide oxidation between proximal cysteines in proteins represents a common regulatory control mechanism to modulate flux through metabolic pathways in response to changing environmental conditions. To enable in vivo measurements of cellular redox changes linked to disulfide bond formation, we have synthesized a cell-permeable monosubstituted cyanine dye derivatized with arsenic (i.e., TRAP_Cy3) to trap and visualize dithiols in cytosolic proteins. Alkylation of reactive thiols prior to displacement of the bound TRAP-Cy3 by ethanedithiol permits facile protein capture and mass spectrometric identification of proximal reduced dithiols to the exclusion of individual cysteines. Applying TRAP_Cy3 to evaluate cellular responses to increasesmore » in oxygen and light levels in the photosynthetic microbe Synechococcus sp. PCC 7002, we observe large decreases in the abundance of reduced dithiols in cellular proteins, which suggest redox-dependent mechanisms involving the oxidation of proximal disulfides. Under these same growth conditions that result in the oxidation of proximal thiols, there is a reduction in the abundance of post-translational oxidative modifications involving nitrotyrosine and methionine sulfoxide formation. These results suggest that the redox status of proximal cysteines respond to environmental conditions, acting to regulate metabolic flux and minimize the formation of reactive oxygen species to decrease oxidative protein damage.« less

Cysteine-independent activation/inhibition of heme oxygenase-2

PubMed Central

Vukomanovic, Dragic; Rahman, Mona N.; Maines, Mahin D.; Ozolinš, Terence RS; Szarek, Walter A.; Jia, Zongchao; Nakatsu, Kanji

2016-01-01

Reactive thiols of cysteine (cys) residues in proteins play a key role in transforming chemical reactivity into a biological response. The heme oxygenase-2 (HO-2) isozyme contains two cys residues that have been implicated in binding of heme and also the regulation of its activity. In this paper, we address the question of a role for cys residues for the HO-2 inhibitors or activators designed in our laboratory. We tested the activity of full length recombinant human heme oxygenase-2 (FL-hHO-2) and its analog in which cys265 and cys282 were both replaced by alanine to determine the effect on activation by menadione (MD) and inhibition by QC-2350. Similar inhibition by QC-2350 and almost identical activation by MD was observed for both recombinant FL-hHO-2s. Our findings are interpreted to mean that thiols of FL-hHO-2s are not involved in HO-2 activation or inhibition by the compounds that have been designed and identified by us. Activation or inhibition of HO-2 by our compounds should be attributed to a mechanism other than altering binding affinity of HO-2 for heme through cys265 and cys282. PMID:27826418

Cysteine-independent activation/inhibition of heme oxygenase-2.

PubMed

Vukomanovic, Dragic; Rahman, Mona N; Maines, Mahin D; Ozolinš, Terence Rs; Szarek, Walter A; Jia, Zongchao; Nakatsu, Kanji

2016-03-01

Reactive thiols of cysteine (cys) residues in proteins play a key role in transforming chemical reactivity into a biological response. The heme oxygenase-2 (HO-2) isozyme contains two cys residues that have been implicated in binding of heme and also the regulation of its activity. In this paper, we address the question of a role for cys residues for the HO-2 inhibitors or activators designed in our laboratory. We tested the activity of full length recombinant human heme oxygenase-2 (FL-hHO-2) and its analog in which cys265 and cys282 were both replaced by alanine to determine the effect on activation by menadione (MD) and inhibition by QC-2350. Similar inhibition by QC-2350 and almost identical activation by MD was observed for both recombinant FL-hHO-2s. Our findings are interpreted to mean that thiols of FL-hHO-2s are not involved in HO-2 activation or inhibition by the compounds that have been designed and identified by us. Activation or inhibition of HO-2 by our compounds should be attributed to a mechanism other than altering binding affinity of HO-2 for heme through cys265 and cys282.

Liquid—liquid interface-mediated Au—ZnO composite membrane using ‘thiol-ene’ click chemistry

NASA Astrophysics Data System (ADS)

Ali, Mohammed; Ghosh, Sujit Kumar

2015-07-01

A nanoparticle-decorated composite membrane has been devised at the water/CCl4 interface based on the self-assembly of ligand-stabilized gold and zinc oxide nanoparticles, exploiting the ‘thiol-ene’ click chemistry between the thiol groups of 11-mercaptoundecanoic acid-stabilized ZnO nanoparticles and the ene functionality of cinnamic acid attached to gold nanoparticles. The interfacial assembly of ultrasmall particles leads to a multilayer film that exhibits charge-dependent permeability of amino acid molecules across the membrane.

On-Chip Chemical Self-Assembly of Semiconducting Single-Walled Carbon Nanotubes (SWNTs): Toward Robust and Scale Invariant SWNTs Transistors.

PubMed

Derenskyi, Vladimir; Gomulya, Widianta; Talsma, Wytse; Salazar-Rios, Jorge Mario; Fritsch, Martin; Nirmalraj, Peter; Riel, Heike; Allard, Sybille; Scherf, Ullrich; Loi, Maria A

2017-06-01

In this paper, the fabrication of carbon nanotubes field effect transistors by chemical self-assembly of semiconducting single walled carbon nanotubes (s-SWNTs) on prepatterned substrates is demonstrated. Polyfluorenes derivatives have been demonstrated to be effective in selecting s-SWNTs from raw mixtures. In this work the authors functionalized the polymer with side chains containing thiols, to obtain chemical self-assembly of the selected s-SWNTs on substrates with prepatterned gold electrodes. The authors show that the full side functionalization of the conjugated polymer with thiol groups partially disrupts the s-SWNTs selection, with the presence of metallic tubes in the dispersion. However, the authors determine that the selectivity can be recovered either by tuning the number of thiol groups in the polymer, or by modulating the polymer/SWNTs proportions. As demonstrated by optical and electrical measurements, the polymer containing 2.5% of thiol groups gives the best s-SWNT purity. Field-effect transistors with various channel lengths, using networks of SWNTs and individual tubes, are fabricated by direct chemical self-assembly of the SWNTs/thiolated-polyfluorenes on substrates with lithographically defined electrodes. The network devices show superior performance (mobility up to 24 cm 2 V -1 s -1 ), while SWNTs devices based on individual tubes show an unprecedented (100%) yield for working devices. Importantly, the SWNTs assembled by mean of the thiol groups are stably anchored to the substrate and are resistant to external perturbation as sonication in organic solvents. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein S-nitrosylation as a therapeutic target for neurodegenerative diseases

PubMed Central

Nakamura, Tomohiro; Lipton, Stuart A.

2015-01-01

At physiological levels, nitric oxide (NO) contributes to the maintenance of normal neuronal activity and survival, thus serving as an important regulatory mechanism in the central nervous system. In contrast, accumulating evidence suggests that exposure to environmental toxins or the normal aging process can trigger excessive production of reactive oxygen/nitrogen species (such as NO), contributing to the etiology of several neurodegenerative diseases. Here we highlight protein S-nitrosylation, resulting from covalent attachment of an NO group to a cysteine thiol of the target protein, as a ubiquitous effector of NO signaling in both health and disease. We review our current understanding of this redox-dependent posttranslational modification under neurodegenerative conditions, and evaluate how targeting dysregulated protein S-nitrosylation can lead to novel therapeutics. PMID:26707925

π-Clamp-mediated cysteine conjugation

NASA Astrophysics Data System (ADS)

Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; van Voorhis, Troy; Pentelute, Bradley L.

2016-02-01

Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the ‘π-clamp’, that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

Investigations on spruce decline in the Bavarian forest.

PubMed

Osswald, W F; Elstner, E F

1987-01-01

The primary damaging reactions in spruce needles may operate as follows: 1) Trees under "stress" produce the plant hormone ethylene. 2) Ethylene and ozone react extremely fast forming hydrogen peroxide and formaldehyde, compounds which may damage the wax layer. 3) Ozone as a very aggressive oxidant will inactivate membrane bound enzymes through oxidation of their thiol groups. Thus the translocation of sugars from the chloroplast into the phloem may be inhibited or blocked. The result will be an "over-reduction" of the electron transport chain resulting in the formation of reactive oxygen species in the light. These reactive oxygen species will induce lipid peroxidation and pigment co-oxidation. 4) The visible effects are bleached needles and an impairment of structural resistance against fungal infections. 5) In addition ozone will directly reduce the content of antifungal compounds such as p-HAP. 6) Furthermore p-HAP may be involved in the bleaching reaction after its release from picein. 7) Finally, fungi may penetrate the needles and eventually grow faster in bleached needles. Infected needles will become necrotic and abscise.

Proteome-wide detection and quantitative analysis of irreversible cysteine oxidation using long column UPLC-pSRM.

PubMed

Lee, Chia-Fang; Paull, Tanya T; Person, Maria D

2013-10-04

Reactive oxygen species (ROS) play an important role in normal biological functions and pathological processes. ROS is one of the driving forces for oxidizing proteins, especially on cysteine thiols. The labile, transient, and dynamic nature of oxidative modifications poses enormous technical challenges for both accurate modification site determination and quantitation of cysteine thiols. The present study describes a mass spectrometry-based approach that allows effective discovery and quantification of irreversible cysteine modifications. The utilization of a long reverse phase column provides high-resolution chromatography to separate different forms of modified cysteine thiols from protein complexes or cell lysates. This Fourier transform mass spectrometry (FT-MS) approach enabled detection and quantitation of ataxia telangiectasia mutated (ATM) complex cysteine sulfoxidation states using Skyline MS1 filtering. When we applied the long column ultra high pressure liquid chromatography (UPLC)-MS/MS analysis, 61 and 44 peptides from cell lysates and cells were identified with cysteine modifications in response to in vitro and in vivo H2O2 oxidation, respectively. Long column ultra high pressure liquid chromatography pseudo selected reaction monitoring (UPLC-pSRM) was then developed to monitor the oxidative level of cysteine thiols in cell lysate under varying concentrations of H2O2 treatment. From UPLC-pSRM analysis, the dynamic conversion of sulfinic (S-O2H) and sulfonic acid (S-O3H) was observed within nucleoside diphosphate kinase (Nm23-H1) and heat shock 70 kDa protein 8 (Hsc70). These methods are suitable for proteome-wide studies, providing a highly sensitive, straightforward approach to identify proteins containing redox-sensitive cysteine thiols in biological systems.

Direct Nanoscale Conversion of Biomolecular Signals into Electronic Information

DTIC Science & Technology

2008-09-22

the electrode surface. In this experiment, the single free cysteine group featured in the GOx structure was exploited to demonstrate that orientation...first with GOx-ssDNA conjugates featuring a sequence complementary to the address strand, then with a non-complementary conjugate and finally with...fully-functional for an enzyme that features a free thiol group, or that can be engineered to incorporate a thiol onto its outer shell

Fully automated synthesis of 4-[18F]fluorobenzylamine based on borohydride/NiCl2 reduction.

PubMed

Way, Jenilee; Wuest, Frank

2013-04-01

4-[(18)F]Fluorobenzylamine ([(18)F]FBA) is an important building block for the synthesis of (18)F-labeled compounds. Synthesis of [(18)F]FBA usually involves application of strong reducing agents like LiAlH4 which is challenging to handle in automated synthesis units (ASUs). Therefore, alternative methods for the preparation of [(18)F]FBA compatible with remotely-controlled syntheses in ASUs are needed. (18)F]FBA was prepared in a remotely-controlled synthesis unit (GE TRACERlab™ FX) based on Ni(II)-mediated borohydride exchange resin (BER) reduction of 4-[(18)F]fluorobenzonitrile ([(18)F]FBN). [(18)F]FBA was used for the synthesis of novel thiol-reactive prosthetic group 4-[(18)F]fluorobenzyl)maleimide [(18)F]FBM and Hsp90 inhibitor 17-(4-[(18)F]fluorobenzylamino)-17-demethoxy-geldanamycin [(18)F] GA. [(18)F]FBA could be prepared in high radiochemical yield greater than 80% (decay-corrected) within 60min. In a typical experiment, 7.4GBq of [(18)F]FBA could be obtained in high radiochemical purity of greater than 95% starting from 10GBq of cyclotron-produced n.c.a. [(18)F]fluoride. [(18)F]FBA was used for the preparation of 4-[(18)F]fluorobenzyl)maleimide as a novel prosthetic group for labeling of thiol groups as demonstrated with tripeptide glutathione. [(18)F]FBA was also used as building block for the syntheses of small molecules as exemplified by the preparation of Hsp90 inhibitor 17-(4-[(18)F]fluorobenzylamino)-17-demethoxy-geldanamycin. The described remotely-controlled synthesis of [(18)F]FBA will significantly improve the availability of [(18)F]FBA as an important and versatile building block for the development of novel (18)F-labeled compounds containing a fluorobenzylamine moiety. Copyright © 2013 Elsevier Inc. All rights reserved.

Decreased plasma thiol antioxidant barrier and selenoproteins as potential biomarkers for ongoing methylmercury intoxication and an individual protective capacity.

PubMed

Usuki, Fusako; Fujimura, Masatake

2016-04-01

Manifestation of methylmercury (MeHg) toxicity depends on individual susceptibility to MeHg, as well as MeHg burden level. Therefore, biomarkers that reflect the protective capacity against MeHg are needed. The critical role of oxidative stress in the pathogenesis of MeHg cytotoxicity has been demonstrated. Because MeHg has high affinity for selenohydryl groups, sulfhydryl groups, and selenides, and causes posttranscriptional defects in selenoenzymes, proteins with selenohydryl and sulfhydryl groups should play a critical role in mediating MeHg-induced oxidative stress. Here, plasma oxidative stress markers and selenoproteins were investigated in MeHg-intoxicated rats showing neuropathological changes after 4 weeks of MeHg exposure. The thiol antioxidant barrier (-SHp) level significantly decreased 2 weeks after MeHg exposure, which is an early stage at which no systemic oxidative stress, histopathological changes, or clinical signs were detected. Diacron reactive oxidant metabolite (d-ROM) levels significantly increased 3 weeks after MeHg exposure, indicating the occurrence of systemic oxidative stress. Rats treated with lead acetate or cadmium chloride showed no changes in levels of -SHp and d-ROM. Selenoprotein P1 abundance significantly decreased in MeHg-treated rats, whereas it significantly increased in rats treated with Pb or Cd. Plasma selenium-dependent glutathione peroxidase (GPx3) activity also significantly decreased after MeHg exposure, whereas plasma non-selenoenzyme glutathione reductase activity significantly increased in MeHg-treated rats. The results suggest that decreased capacity of -SHp and selenoproteins (GPx3 and selenoprotein P) can be useful biomarkers of ongoing MeHg cytotoxicity and the individual protective capacity against the MeHg body burden.

1,4-Naphthoquinone activates the HSP90/HSF1 pathway through the S-arylation of HSP90 in A431 cells: Negative regulation of the redox signal transduction pathway by persulfides/polysulfides.

PubMed

Abiko, Yumi; Sha, Liang; Shinkai, Yasuhiro; Unoki, Takamitsu; Luong, Nho Cong; Tsuchiya, Yukihiro; Watanabe, Yasuo; Hirose, Reiko; Akaike, Takaaki; Kumagai, Yoshito

2017-03-01

The current consensus is that environmental electrophiles activate redox signal transduction pathways through covalent modification of sensor proteins with reactive thiol groups at low concentrations, while they cause cell damage at higher concentrations. We previously exposed human carcinoma A431 cells to the atmospheric electrophile 1,4-naphthoquinone (1,4-NQ) and found that heat shock protein 90 (HSP90), a negative regulator of heat shock factor 1 (HSF1), was a target of 1,4-NQ. In the study presented here, we determined whether 1,4-NQ activates HSF1. We also examined whether such redox signaling could be regulated by nucleophilic sulfur species. Exposure of A431 cells to 1,4-NQ covalently modified cellular HSP90, resulting in repression of the association between HSF1 with HSP90, thereby enhancing HSF1 translocation into the nuclei. Liquid chromatography-tandem mass spectrometry analysis with recombinant HSP90 revealed that the modifications site were Cys412 and Cys564. We found that HSF1 activation mediated by 1,4-NQ upregulated downstream genes, such as HSPA6. HSF1 knockdown accelerated 1,4-NQ-mediated cytotoxicity in the cells. While simultaneous treatment with reactive persulfide and polysulfide, Na 2 S 2 and Na 2 S 4 , blocked 1,4-NQ-dependent protein modification and HSF1 activation in A431 cells, the knockdown of Cys persulfide producing enzymes cystathionine β-synthase (CBS) and/or cystathionine γ-lyase (CSE) enhanced these phenomena. 1,4-NQ-thiol adduct and 1,4-NQ-S-1,4-NQ adduct were produced during the enzymatic reaction of recombinant CSE in the presence of 1,4-NQ. The results suggest that activation of the HSP90-HSF1 signal transduction pathway mediated by 1,4-NQ protects cells against 1,4-NQ and that per/polysulfides can diminish the reactivity of 1,4-NQ by forming sulfur adducts. Copyright © 2017 Elsevier Inc. All rights reserved.

Beneficial influence of ellagic acid on biochemical indexes associated with experimentally induced colon carcinogenesis.

PubMed

Syed, Umesalma; Ganapasam, Sudhandiran

2017-01-01

To elucidate the key biochemical indexes associated with 1, 2-dimethylhydrazine (DMH)-induced colon carcinogenesis and the modulatory efficacy of a dietary polyphenol, ellagic acid (EA). Wistar rats were chosen to study objective, and were divided into 4 groups; Group 1-control rats; Group 2-rats received EA (60 mg/kg body weight/day, orally); rats in Group 3-induced with DMH (20 mg/kg body weight) subcutaneously for 15 weeks; DMH-induced Group 4 rats were initiated with EA treatment. We examined key citric acid cycle enzymes such as isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and the activities of respiratory chain enzymes NADH dehydrogenase and Cytochrome-C-oxidase and membrane-bound enzyme profiles (Na +/K + ATPase, Ca 2+ ATPase and Mg 2+ ATPase), activities of lysosomal proteases such as β-D-glucuronidase, β-galactosidase and N-acety-β-D-glucosaminidase and cellular thiols (oxidized glutathione, protein thiols, and total thiols). It was found that administration of DMH to rats decreased both mitochondrial and membrane-bound enzymes activities, increased activities of lysosomal enzymes and further modulates cellular thiols levels. Treatment with EA significantly restored the mitochondrial and ATPases levels and further reduced lysosomal enzymes to near normalcy thereby restoring harmful effects induced by DMH. EA treatment was able to effectively restore the detrimental effects induced by DMH, which proves the chemoprotective function of EA against DMH-induced experimental colon carcinogenesis.

Dinitrogenase-Driven Photobiological Hydrogen Production Combats Oxidative Stress in Cyanothece sp. Strain ATCC 51142

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sadler, Natalie C.; Bernstein, Hans C.; Melnicki, Matthew R.

ABSTRACT Photobiologically synthesized hydrogen (H 2) gas is carbon neutral to produce and clean to combust, making it an ideal biofuel.Cyanothecesp. strain ATCC 51142 is a cyanobacterium capable of performing simultaneous oxygenic photosynthesis and H 2production, a highly perplexing phenomenon because H 2evolving enzymes are O 2sensitive. We employed a system-levelin vivochemoproteomic profiling approach to explore the cellular dynamics of protein thiol redox and how thiol redox mediates the function of the dinitrogenase NifHDK, an enzyme complex capable of aerobic hydrogenase activity. We found that NifHDK responds to intracellular redox conditions and may act as an emergency electron valve tomore » prevent harmful reactive oxygen species formation in concert with other cell strategies for maintaining redox homeostasis. These results provide new insight into cellular redox dynamics useful for advancing photolytic bioenergy technology and reveal a new understanding for the biological function of NifHDK. IMPORTANCEHere, we demonstrate that high levels of hydrogen synthesis can be induced as a protection mechanism against oxidative stress via the dinitrogenase enzyme complex inCyanothecesp. strain ATCC 51142. This is a previously unknown feature of cyanobacterial dinitrogenase, and we anticipate that it may represent a strategy to exploit cyanobacteria for efficient and scalable hydrogen production. We utilized a chemoproteomic approach to capture thein situdynamics of reductant partitioning within the cell, revealing proteins and reactive thiols that may be involved in redox sensing and signaling. Additionally, this method is widely applicable across biological systems to achieve a greater understanding of how cells navigate their environment and how redox chemistry can be utilized to alter metabolism and achieve homeostasis.« less

Comparative study of the oxidation behavior of sulfur-containing amino acids and glutathione by electrochemistry-mass spectrometry in the presence and absence of cisplatin.

PubMed

Zabel, Robert; Weber, Günther

2016-02-01

Small sulfur-containing compounds are involved in several important biochemical processes, including-but not limited to-redox regulation and drug conjugation/detoxification. While methods for stable redox pairs of such compounds (thiols/disulfides) are available, analytical data on more labile and short-lived redox intermediates are scarce, due to highly challenging analytical requirements. In this study, we employ the direct combination of reagentless electrochemical oxidation and mass spectrometric (EC-MS) identification for monitoring oxidation reactions of cysteine, N-acetylcysteine, methionine, and glutathione under simulated physiological conditions (pH 7.4, 37 °C). For the first time, all theoretically expected redox intermediates-with only one exception-are detected simultaneously and in situ, including sulfenic, sulfinic, and sulfonic acids, disulfides, thiosulfinates, thiosulfonates, and sulfoxides. By monitoring the time/potential-dependent interconversion of sulfur species, mechanistic oxidation routes are confirmed and new reactions detected, e.g., sulfenamide formation due to reaction with ammonia from the buffer. Furthermore, our results demonstrate a highly significant impact of cisplatin on the redox reactivity of sulfur species. Namely, the amount of thiol oxidation to sulfonic acid via sulfenic and sulfinic acid intermediates is diminished for glutathione in the presence of cisplatin in favor of the disulfide formation, while for N-acetylcysteine the contrary applies. N-acetylcysteine is the only ligand which displays enhanced oxidation currents upon cisplatin addition, accompanied by increased levels of thiosulfinate and thiosulfonate species. This is traced back to thiol reactivity and highlights the important role of sulfenic acid intermediates, which may function as a switch between different oxidation routes.

A lysosome-targetable turn-on fluorescent probe for the detection of thiols in living cells based on a 1,8-naphthalimide derivative

NASA Astrophysics Data System (ADS)

Liang, Beibei; Wang, Baiyan; Ma, Qiujuan; Xie, Caixia; Li, Xian; Wang, Suiping

2018-03-01

Biological thiols, like cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play crucial roles in biological systems and in lysosomal processes. Highly selective probes for detecting biological thiols in lysomes of living cells are rare. In this work, a lysosome-targetable turn-on fluorescent probe for the detection of thiols in living cells was designed and synthesized based on a 1,8-naphthalimide derivative. The probe has a 4-(2-aminoethyl)morpholine unit as a lysosome-targetable group and an acrylate group as the thiol recognition unit as well as a fluorescence quencher. In the absence of biothiols, the probe displayed weak fluorescence due to the photoinduced electron transfer (PET) process. Upon the addition of biothiols, the probe exhibited an enhanced fluorescence emission centered at 550 nm due to cleavage of the acrylate moiety. The probe had high selectivity toward biothiols. Moreover, the probe features fast response time, excitation in the visible region and ability of working in a wide pH range. The linear response range covers a concentration range of Cys from 1.5 × 10- 7 to 1.0 × 10- 5 mol·L- 1 and the detection limit is 6.9 × 10- 8 mol·L- 1 for Cys. The probe has been successfully applied to the confocal imaging of biothiols in lysosomes of A549 cells with low cell toxicity. Furthermore, the method was successfully applied to the determination of thiols in a complex multicomponent mixture such as human serum, which suggests our proposed method has great potential for diagnostic purposes. All of such good properties prove it can be used to monitor biothiols in lysosomes of living cells and to be a good fluorescent probe for the selective detection of thiols.

A Novel Tool for the Assessment Oxidative Stress in Age-Related Macular Degeneration: Thiol/Disulfide Homeostasis Revisited.

PubMed

Arıkan Yorgun, Mücella; Toklu, Yasin; Altınkaynak, Hasan; Tanrıverdi, Burak; Ergin, Merve; Biçer, Cemile

2016-12-01

To investigate thiol/disulfide status using a novel automated assay in patients with age-related macular degeneration (AMD) compared to age-matched healthy controls. A total of 64 AMD patients [51 (79%) non-exudative, 13 (21%) exudative AMD] and 21 age-matched healthy control subjects were enrolled in this study. Plasma total thiol, native thiol, disulfide levels were measured and native thiol/disulfide ratio (TDR) was calculated using a novel spectrophotometric assay. Patients with AMD had significantly lower levels of total thiol (434.8 ± 7.0 μmol/L vs. 472.2 ± 7.9 μmol/L, p < 0.001), native thiol (393.6 ± 6.5 μmol/L vs. 437.5 ± 7.1 μmol/L, p = 0.004) compared to healthy controls. However, plasma disulfide levels were higher in AMD patients (20.6 ± 0.9 μmol/L vs. 17.3 ± 1.3 μmol/L, p = 0.113) compared to healthy controls. The TDR was not statistically different between the early AMD group and healthy controls (24.2 ± 2.3 vs. 29.5 ± 3.1, p = 0.345). However, intermediate and advanced stage AMD groups had significantly lower levels of TDR compared to healthy controls (21.6 ± 2.6 vs. 29.5 ± 3.1, p = 0.023 and 20.3 ± 1.2 vs. 29.5 ± 3.1, p = 0.005, respectively). Native TDR was significantly lower in patients with exudative and non-exudative AMD (19.9 ± 2.3 vs. 29.5 ± 3.1, p = 0.024 and 21.8 ± 1.14 vs. 29.47 ± 3.1 respectively, p = 0.011). A greater extent of thiol consumption occurred in AMD patients compared to age-matched healthy controls. However, despite the similar levels of total thiol levels between several grades of AMD, the plasma native TDR value was decreased in accordance with the severity of the disease, which reflected the disease grade better.

Au36(SPh)23 nanomolecules.

PubMed

Nimmala, Praneeth Reddy; Dass, Amala

2011-06-22

A new core size protected completely by an aromatic thiol, Au(36)(SPh)(23), is synthesized and characterized by MALDI-TOF mass spectrometry and UV-visible spectroscopy. The synthesis involving core size changes is studied by MS, and the complete ligand coverage by aromatic thiol group is shown by NMR.

Mechanism of covalent modification of glyceraldehyde-3-phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents.

PubMed

Mohr, S; Stamler, J S; Brüne, B

1994-07-18

Previous studies have suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes covalent modification of an active site thiol by a NO.-induced [32P]NAD(+)-dependent mechanism. However, the efficacy of GAPDH modification induced by various NO donors was found to be independent of spontaneous rates of NO. release. To further test the validity of this mechanism, we studied the effects of nitrosonium tertrafluoroborate (BF4NO), a strong NO+ donor. BF4NO potently induces GAPDH labeling by the radioactive nucleotide. In this case, the addition of thiol significantly attenuates enzyme modification by competing for the NO moiety in the formation of RS-NO. Peroxynitrite (ONOO-) also induces GAPDH modification in the presence of thiol, consistent with the notion that this species can transfer NO+ (or NO2+) through the intermediacy of RS-NO. However, the efficiency of this reaction is limited by ONOO- -induced oxidation of protein SH groups at the active site. ONOO- generation appears to account for the modification of GAPDH by SIN-1. Thus, S-nitrosylation of the active site thiol is a prequisite for subsequent post-translational modification with NAD+, and emphasizes the role of NO+ transfer in the initial step of this pathway. Our findings thus provide a uniform mechanism by which nitric oxide and related NO donors initiate non-enzymatic ADP-ribosylation (like) reactions. In biological systems, endogenous RS-NO are likely to support the NO group transfer to thiol-containing proteins.

Patchy micelles based on coassembly of block copolymer chains and block copolymer brushes on silica particles.

PubMed

Zhu, Shuzhe; Li, Zhan-Wei; Zhao, Hanying

2015-04-14

Patchy particles are a type of colloidal particles with one or more well-defined patches on the surfaces. The patchy particles with multiple compositions and functionalities have found wide applications from the fundamental studies to practical uses. In this research patchy micelles with thiol groups in the patches were prepared based on coassembly of free block copolymer chains and block copolymer brushes on silica particles. Thiol-terminated and cyanoisopropyl-capped polystyrene-block-poly(N-isopropylacrylamide) block copolymers (PS-b-PNIPAM-SH and PS-b-PNIPAM-CIP) were synthesized by reversible addition-fragmentation chain transfer polymerization and chemical modifications. Pyridyl disulfide-functionalized silica particles (SiO2-SS-Py) were prepared by four-step surface chemical reactions. PS-b-PNIPAM brushes on silica particles were prepared by thiol-disulfide exchange reaction between PS-b-PNIPAM-SH and SiO2-SS-Py. Surface micelles on silica particles were prepared by coassembly of PS-b-PNIPAM-CIP and block copolymer brushes. Upon cleavage of the surface micelles from silica particles, patchy micelles with thiol groups in the patches were obtained. Dynamic light scattering, transmission electron microscopy, and zeta-potential measurements demonstrate the preparation of patchy micelles. Gold nanoparticles can be anchored onto the patchy micelles through S-Au bonds, and asymmetric hybrid structures are formed. The thiol groups can be oxidized to disulfides, which results in directional assembly of the patchy micelles. The self-assembly behavior of the patchy micelles was studied experimentally and by computer simulation.

Electrically conducting nanopatterns formed by chemical e-beam lithography via gold nanoparticle seeds.

PubMed

Schaal, Patrick A; Besmehn, Astrid; Maynicke, Eva; Noyong, Michael; Beschoten, Bernd; Simon, Ulrich

2012-02-07

We report the formation of thiol nanopatterns on SAM covered silicon wafers by converting sulfonic acid head groups via e-beam lithography. These thiol groups act as binding sites for gold nanoparticles, which can be enhanced to form electrically conducting nanostructures. This approach serves as a proof-of-concept for the combination of top-down and bottom-up processes for the generation of electrical devices on silicon.

Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase.

PubMed

Mohr, S; Hallak, H; de Boitte, A; Lapetina, E G; Brüne, B

1999-04-02

S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by S-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function.

Thiol-functionalization of metal-organic framework by a facile coordination-based postsynthetic strategy and enhanced removal of Hg2+ from water.

PubMed

Ke, Fei; Qiu, Ling-Guang; Yuan, Yu-Peng; Peng, Fu-Min; Jiang, Xia; Xie, An-Jian; Shen, Yu-Hua; Zhu, Jun-Fa

2011-11-30

The presence of coordinatively unsaturated metal centers in metal-organic frameworks (MOFs) provides an accessible way to selectively functionalize MOFs through coordination bonds. In this work, we describe thiol-functionalization of MOFs by choosing a well known three-dimensional (3D) Cu-based MOF, i.e. [Cu(3)(BTC)(2)(H(2)O)(3)](n) (HKUST-1, BTC=benzene-1,3,5-tricarboxylate), by a facile coordination-based postsynthetic strategy, and demonstrate their application for removal of heavy metal ion from water. A series of [Cu(3)(BTC)(2)](n) samples stoichiometrically decorated with thiol groups has been prepared through coordination bonding of coordinatively unsaturated metal centers in HKUST-1 with -SH group in dithioglycol. The obtained thiol-functionalized samples were characterized by powder X-ray diffraction, scanning electron microscope, energy dispersive X-ray spectroscopy, infrared spectroscopy, and N(2) sorption-desorption isothermal. Significantly, the thiol-functionalized [Cu(3)(BTC)(2)](n) exhibited remarkably high adsorption affinity (K(d)=4.73 × 10(5)mL g(-1)) and high adsorption capacity (714.29 mg g(-1)) for Hg(2+) adsorption from water, while the unfunctionalized HKUST-1 showed no adsorption of Hg(2+) under the same condition. Copyright © 2011 Elsevier B.V. All rights reserved.

Delta-aminolevulinate dehydratase activity and oxidative stress markers in preeclampsia.

PubMed

de Lucca, Leidiane; Rodrigues, Fabiane; Jantsch, Letícia B; Kober, Helena; Neme, Walter S; Gallarreta, Francisco M P; Gonçalves, Thissiane L

2016-12-01

Preeclampsia is an important pregnancy-specific multisystem disorder characterized by the onset of hypertension and proteinuria. It is of unknown etiology and involves serious risks for the pregnant women and fetus. One of the main factors involved in the pathophysiology of preeclampsia is oxidative stress, where excess free radicals produce harmful effects, including damage to macromolecules such as lipids, proteins and DNA. In addition, the sulfhydryl delta-aminolevulinate dehydratase enzyme (δ-ALA-D) that is part of the heme biosynthetic pathway in pro-oxidant conditions can be inhibited, which may result in the accumulation of 5-aminolevulinic acid (ALA), associated with the overproduction of free radicals, suggesting it to be an indirect marker of oxidative stress. As hypertensive pregnancy complications are a major cause of morbidity and mortality maternal and fetal where oxidative stress appears to be an important factor involved in preeclampsia, the aim of this study was to evaluate the activity of δ-ALA-D and classic oxidative stress markers in the blood of pregnant women with mild and severe preeclampsia. The analysis and quantification of the following oxidative stress markers were performed: thiobarbituric acid-reactive species (TBARS); presence of protein and non-protein thiol group; quantification of vitamin C; Catalase and δ-ALA--D activities in samples of blood of pregnant women with mild preeclampsia (n=25), with severe preeclampsia (n=30) and in a control group of healthy pregnant women (n=30). TBARS was significantly higher in women with preeclampsia, while the presence of thiol groups, levels of vitamin C, catalase and δ-ALA-D activity were significantly lower in groups of pregnant women with preeclampsia compared with healthy women. In addition, the results showed no significant difference between groups of pregnant women with mild and severe preeclampsia. The data suggest a state of increased oxidative stress in pregnant women with preeclampsia compared to healthy pregnant women, which may be related to the complications of this disease. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Dynamic asymmetry and the role of the conserved active-site thiol in rabbit muscle creatine kinase.

PubMed

Londergan, Casey H; Baskin, Rachel; Bischak, Connor G; Hoffman, Kevin W; Snead, David M; Reynoso, Christopher

2015-01-13

Symmetric and asymmetric crystal structures of the apo and transition state analogue forms, respectively, of the dimeric rabbit muscle creatine kinase have invoked an "induced fit" explanation for asymmetry between the two subunits and their active sites. However, previously reported thiol reactivity studies at the dual active-site cysteine 283 residues suggest a more latent asymmetry between the two subunits. The role of that highly conserved active-site cysteine has also not been clearly determined. In this work, the S-H vibrations of Cys283 were observed in the unmodified MM isoform enzyme via Raman scattering, and then one and both Cys283 residues in the same dimeric enzyme were modified to covalently attach a cyano group that reports on the active-site environment via its infrared CN stretching absorption band while maintaining the catalytic activity of the enzyme. Unmodified and Cys283-modified enzymes were investigated in the apo and transition state analogue forms of the enzyme. The narrow and invariant S-H vibrational bands report a homogeneous environment for the unmodified active-site cysteines, indicating that their thiols are hydrogen bonded to the same H-bond acceptor in the presence and absence of the substrate. The S-H peak persists at all physiologically relevant pH's, indicating that Cys283 is protonated at all pH's relevant to enzymatic activity. Molecular dynamics simulations identify the S-H hydrogen bond acceptor as a single, long-resident water molecule and suggest that the role of the conserved yet catalytically unnecessary thiol may be to dynamically rigidify that part of the active site through specific H-bonding to water. The asymmetric and broad CN stretching bands from the CN-modified Cys283 suggest an asymmetric structure in the apo form of the enzyme in which there is a dynamic exchange between spectral subpopulations associated with water-exposed and water-excluded probe environments. Molecular dynamics simulations indicate a homogeneous orientation of the SCN probe group in the active site and thus rule out a local conformational explanation at the residue level for the multipopulation CN stretching bands. The homogeneous simulated SCN orientation suggests strongly that a more global asymmetry between the two subunits is the cause of the CN probe's broad and asymmetric infrared line shape. Together, these spectral observations localized at the active-site cysteines indicate an intrinsic, dynamic asymmetry between the two subunits that exists already in the apo form of the dimeric creatine kinase enzyme, rather than being induced by the substrate. Biochemical and methodological consequences of these conclusions are considered.

Purification, characterization and function of dihydrolipoamide dehydrogenase from the cyanobacterium Anabaena sp. strain P.C.C. 7119.

PubMed Central

Serrano, A

1992-01-01

A dihydrolipoamide dehydrogenase (dihydrolipoamide: NAD+ oxidoreductase, EC 1.8.1.4) (DLD) has been found in the soluble fraction of cells of both unicellular (Synechococcus sp. strain P.C.C. 6301) and filamentous (Calothrix sp. strain P.C.C. 7601 and Anabaena sp. strain P.C.C. 7119) cyanobacteria. DLD from Anabaena sp. was purified 3000-fold to electrophoretic homogeneity. The purified enzyme exhibited a specific activity of 190 units/mg and was characterized as a dimeric FAD-containing protein with a native molecular mass of 104 kDa, a Stokes' radius of 4.28 nm and a very acidic pI value of about 3.7. As is the case with the same enzyme from other sources, cyanobacterial DLD showed specificity for NADH and lipoamide, or lipoic acid, as substrates. Nevertheless, the strong acidic character of the Anabaena DLD is a distinctive feature with respect to the same enzyme from other organisms. The presence of essential thiol groups was suggested by the inactivation produced by thiol-group-reactive reagents and heavy-metal ions, with lipoamide, but not NAD+, behaving as a protective agent. The function and physiological significance of Anabaena DLD are discussed in relation to the fact that 2-oxoacid dehydrogenase complexes have not been detected so far in filamentous cyanobacteria. Glycine decarboxylase activity, which might be involved in photorespiratory metabolism, has been found, however, in cell extracts of Anabaena sp. strain P.C.C. 7119 as the present study demonstrates. Images Fig. 2. PMID:1471997

Chemical Cleavage of an Asp-Cys Sequence Allows Efficient Production of Recombinant Peptides with an N-Terminal Cysteine Residue.

PubMed

Pane, Katia; Verrillo, Mariavittoria; Avitabile, Angela; Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Di Maro, Antimo; Rega, Camilla; Amoresano, Angela; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

2018-04-18

Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoB L , an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH, temperature, and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY, GTGDCHVA, GSGTDCGSR, SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures), high purity (>96%), and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.

Synthesis of quantum dots

DOEpatents

McDaniel, Hunter

2017-10-17

Common approaches to synthesizing alloyed quantum dots employ high-cost, air-sensitive phosphine complexes as the selenium precursor. Disclosed quantum dot synthesis embodiments avoid these hazardous and air-sensitive selenium precursors. Certain embodiments utilize a combination comprising a thiol and an amine that together reduce and complex the elemental selenium to form a highly reactive selenium precursor at room temperature. The same combination of thiol and amine acts as the reaction solvent, stabilizing ligand, and sulfur source in the synthesis of quantum dot cores. A non-injection approach may also be used. The optical properties of the quantum dots synthesized by this new approach can be finely tuned for a variety of applications by controlling size and/or composition of size and composition. Further, using the same approach, a shell can be grown around a quantum dot core that improves stability, luminescence efficiency, and may reduce toxicity.

End-group-functionalized poly(N,N-diethylacrylamide) via free-radical chain transfer polymerization: Influence of sulfur oxidation and cyclodextrin on self-organization and cloud points in water

PubMed Central

Reinelt, Sebastian; Steinke, Daniel

2014-01-01

Summary In this work we report the synthesis of thermo-, oxidation- and cyclodextrin- (CD) responsive end-group-functionalized polymers, based on N,N-diethylacrylamide (DEAAm). In a classical free-radical chain transfer polymerization, using thiol-functionalized 4-alkylphenols, namely 3-(4-(1,1-dimethylethan-1-yl)phenoxy)propane-1-thiol and 3-(4-(2,4,4-trimethylpentan-2-yl)phenoxy)propane-1-thiol, poly(N,N-diethylacrylamide) (PDEAAm) with well-defined hydrophobic end-groups is obtained. These end-group-functionalized polymers show different cloud point values, depending on the degree of polymerization and the presence of randomly methylated β-cyclodextrin (RAMEB-CD). Additionally, the influence of the oxidation of the incorporated thioether linkages on the cloud point is investigated. The resulting hydrophilic sulfoxides show higher cloud point values for the lower critical solution temperature (LCST). A high degree of functionalization is supported by 1H NMR-, SEC-, FTIR- and MALDI–TOF measurements. PMID:24778720

Using the Localized Surface Plasmon Resonance of Gold Nanoparticles to Monitor Lipid Membrane Assembly and Protein Binding.

PubMed

Messersmith, Reid E; Nusz, Greg J; Reed, Scott M

2013-12-19

Gold nanoparticles provide a template for preparing supported lipid layers with well-defined curvature. Here, we utilize the localized surface plasmon resonance (LSPR) of gold nanoparticles as a sensor for monitoring the preparation of lipid layers on nanoparticles. The LSPR is very sensitive to the immediate surroundings of the nanoparticle surface and it is used to monitor the coating of lipids and subsequent conversion of a supported bilayer to a hybrid membrane with an outer lipid leaflet and an inner leaflet containing hydrophobic alkanethiol. We demonstrate that both decanethiol and propanethiol are able to form hybrid membranes and that the membrane created over the shorter thiol can be stripped from the gold along with the lipid leaflet using β-mercaptoethanol. The sensitivity of the nanoparticle LSPR to the refractive index (RI) of its surroundings is greater when the shorter thiol is used (37.8 ± 1.5 nm per RI unit) than when the longer thiol is used (27.5 ± 0.5 nm per RI unit). Finally, C-reactive protein binding to the membrane is measured using this sensor allowing observation of both protein-membrane and nanoparticle-nanoparticle interactions without chemical labeling of protein or lipids.

Detection of thiol-based redox switch processes in parasites - facts and future.

PubMed

Rahbari, Mahsa; Diederich, Kathrin; Becker, Katja; Krauth-Siegel, R Luise; Jortzik, Esther

2015-05-01

Malaria and African trypanosomiasis are tropical diseases caused by the protozoa Plasmodium and Trypanosoma, respectively. The parasites undergo complex life cycles in the mammalian host and insect vector, during which they are exposed to oxidative and nitrosative challenges induced by the host immune system and endogenous processes. Attacking the parasite's redox metabolism is a target mechanism of several known antiparasitic drugs and a promising approach to novel drug development. Apart from this aspect, oxidation of cysteine residues plays a key role in protein-protein interaction, metabolic responses to redox events, and signaling. Understanding the role and dynamics of reactive oxygen species and thiol switches in regulating cellular redox homeostasis is crucial for both basic and applied biomedical approaches. Numerous techniques have therefore been established to detect redox changes in parasites including biochemical methods, fluorescent dyes, and genetically encoded probes. In this review, we aim to give an insight into the characteristics of redox networks in the pathogens Plasmodium and Trypanosoma, including a comprehensive overview of the consequences of specific deletions of redox-associated genes. Furthermore, we summarize mechanisms and detection methods of thiol switches in both parasites and discuss their specificity and sensitivity.

The effects of organosulfur compounds upon the storage stability of Jet A fuel. M.S. Thesis. Final Report

NASA Technical Reports Server (NTRS)

Heneman, F. C.

1981-01-01

This study examined the effect of sulfur-containing compounds on the storage stability of Jet A turbine fuel. It was found that alkyl sulfides and disulfides increased the fuel's stability while all thiols and thiophene derivatives tested decreased fuel stability (increased deposit formation) at temperatures and sulfur concentrations selected. Linear Arrhenius plots of sulfur-spiked fuel samples demonstrated that deposit formation decreased with increased slope for all alkyl sulfides, alkyl disulfides, thiols, and thiophene derivatives. A plot of insoluble deposit vs. concentration of added alkyl sulfide produces a negative slope. It appears that the inhibiting mechanism for alkyl sulfides is a result of the compound's reactivity with intermediate soluble precursors to deposit in the fuel. A method of approximating the relative basicity of weak organosulfur bases was developed via measurement of their resonance chemical shifts in proton NMR. Linear plots of log gm. deposit vs. change in chemical shift (shift differences between sulfur bases neat and complexed with I2) were found for alkyl sulfides and alkyl thiols. This suggests the possibility that increased deposit formation is due to base catalysis with these compound classes.

Thermodynamics of Alkanethiol Self-Assembled Monolayer Assembly on Pd Surfaces.

PubMed

Kumar, Gaurav; Van Cleve, Timothy; Park, Jiyun; van Duin, Adri; Medlin, J Will; Janik, Michael J

2018-06-05

We investigate the structure and binding energy of alkanethiolate self-assembled monolayers (SAMs) on Pd (111), Pd (100), and Pd (110) facets at different coverages. Dispersion-corrected density functional theory calculations are used to correlate the binding energy of alkanethiolates with alkyl chain length and coverage. The equilibrium coverage of thiolate layers strongly prefers 1/3 monolayer (ML) on the Pd (111) surface. The coverage of thiolates varies with chemical potential on Pd (100) and Pd (110), increasing from 1/3 to 1/2 ML on (100) and from 1/4 to 1/2 ML on (110) as the thiol chemical potential is increased. Higher coverages are driven by attractive dispersion interactions between the extended alkyl chains, such that transitions to higher coverages occur at lower thiol chemical potentials for longer chain thiolates. Stronger adsorption to the Pd (100) surface causes the equilibrium Wulff construction of Pd particles to take on a cubic shape upon saturation with thiols. The binding of H, O, and CO adsorbates is weakened as the thiolate coverage is increased, with saturation coverages causing unfavorable binding of O and CO on Pd (100) and weakened binding on other facets. Temperature-dependent CO diffuse reflectance infrared Fourier transform spectroscopy experiments are used to corroborate the weakened binding of CO in the presence of thiolate SAMs of varying surface density. Preliminary results of multiscale modeling efforts on the Pd-thiol system using a reactive force field, ReaxFF, are also discussed.

Preactivated thiolated pullulan as a versatile excipient for mucosal drug targeting.

PubMed

Leonaviciute, Gintare; Suchaoin, Wongsakorn; Matuszczak, Barbara; Lam, Hung Thanh; Mahmood, Arshad; Bernkop-Schnürch, Andreas

2016-10-20

The purpose of the present study was to generate a novel mucoadhesive thiolated pullulan with protected thiol moieties and to evaluate its suitability as mucosal drug delivery system. Two different synthetic pathways: bromination-nucleophilic substitution and reductive amination including periodate cleavage were utilized to synthesize such thiolated pullulans. The thiomer (pullulan-cysteamine) with the highest amount of free thiol groups was further enrolled in a reaction with 6-mercaptonicotinamide and its presence in pullulan structure was confirmed via NMR analysis. Furthermore, unmodified, thiolated and preactivated thiolated pullulan were investigated in terms of mucoadhesion via rotating cylinder studies and rheological synergism method as well as their toxicity potential over Caco-2 cells. Comparing both methods the reductive amination seems to be the method of choice resulting in comparatively higher coupling rates. Using this procedure pullulan-cysteamine conjugate displayed 1522±158μmol immobilized thiol groups and 280±70μmol free thiol groups per gram polymer. Furthermore, 82% of free thiol groups on this conjugate were linked with 6-mercaptonicotinamide (6-MNA). The adhesion time on the rotating cylinder was up to 46-fold prolonged in case of the thiolated polymer and up to 75-fold in case of the preactivated polymer. Rheological measurements of modified pullulan samples showed 98-fold and 160-fold increase in dynamic viscosity upon the addition of mucus within 60min, whereas unmodified pullulan did not show an increase in viscosity at all. Both conjugates had a minor effect on Caco-2 cell viability. Because of these features preactivated thiolated pullulan seems to represent a promising type of mucoadhesive polymers for the development of various mucosal drug delivery systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

Natural dendrimers: Synthesis and in vitro characterization of glycogen-cysteamine conjugates.

PubMed

Perrone, Mara; Lopedota, Angela; Liberati, Elisa; Russo, Vincenzo; Cutrignelli, Annalisa; Laquintana, Valentino; de Sousa, Irene Pereira; Franco, Massimo; Tongiani, Serena; Denora, Nunzio; Bernkop-Schnürch, Andreas

2017-06-01

The aim of this study was to synthesize, characterize and evaluate the mucoadhesive properties of the first thiolated hyperbranched natural polysaccharide with biodegradability and biocompatibility features. In detail, glycogen-cysteamine conjugates were synthesized through a first step of oxidative ring opening applying increasing concentrations of sodium periodate, to obtain polymers with different degrees of oxidation, and a second step of reductive amination with a constant amount of cysteamine. The obtained glycogen-cysteamine conjugates were characterized regarding their content of free and total thiol groups by Ellman's assay, biocompatibility, swelling/erosion behavior, rheological synergism and mucoadhesive properties in comparison to the unmodified glycogen. The higher the concentration of periodate was, the higher was the content of total thiol groups being in the range of 255.7±12-1194.5±82μmol/g, biocompatibility remained unaffected by these structural changes. On the contrary, the mucoadhesive properties, evaluated by tensile, rheological synergism and rotating cylinder studies, appear to be influenced by the thiol groups concentration on the glycogen. In particular the glycogen-cysteamine conjugate exhibiting the highest degree of thiolation showed a 79-fold increase in viscosity over a time period of 8h, as well as, remained attached on freshly excised porcine mucosa 32-fold longer than the unmodified polymer. The higher was the amount of conjugated thiol groups, the higher was the water absorption capacity of glycogen-cysteamine tablets in Simulated Intestinal Fluid pH 6.8 (SIF). The introduction of thiol moieties on polymer changed the characteristics of the polysaccharide by improving mucoadhesion properties. Therefore, this work represents the first study describing thiolated natural dendrimers as potential platform useful to realize appropriate mucoadhesive nanocarrier systems suitable to prolong mucosal residence time. Copyright © 2017 Elsevier B.V. All rights reserved.

Chemical stability and in chemico reactivity of 24 fragrance ingredients of concern for skin sensitization risk assessment.

PubMed

Avonto, Cristina; Wang, Mei; Chittiboyina, Amar G; Vukmanovic, Stanislav; Khan, Ikhlas A

2018-02-01

Twenty-four pure fragrance ingredients have been identified as potential concern for skin sensitization. Several of these compounds are chemically unstable and convert into reactive species upon exposure to air or light. In the present work, a systematic investigation of the correlation between chemical stability and reactivity has been undertaken. The compounds were subjected to forced photodegradation for three months and the chemical changes were studied with GC-MS. At the end of the stability study, two-thirds of the samples were found to be unstable. The generation of chemically reactive species was investigated using the in chemico HTS-DCYA assay. Eleven and fourteen compounds were chemically reactive before and after three months, respectively. A significant increase in reactivity upon degradation was found for isoeugenol, linalool, limonene, lyral, citronellol and geraniol; in the same conditions, the reactivity of hydroxycitronellal decreased. The non-reactive compounds α-isomethyl ionone, benzyl alcohol, amyl cinnamal and farnesol became reactive after photo-oxidative degradation. Overall, forced degradation resulted in four non-reactive fragrance compounds to display in chemico thiol reactivity, while ten out of 24 compounds remained inactive. Chemical degradation does not necessarily occur with generation of reactive species. Non-chemical activation may be involved for the 10 stable unreactive compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

Presence of closely spaced protein thiols on the surface of mammalian cells.

PubMed Central

Donoghue, N.; Yam, P. T.; Jiang, X. M.; Hogg, P. J.

2000-01-01

It has been proposed that certain cell-surface proteins undergo redox reactions, that is, transfer of hydrogens and electrons between closely spaced cysteine thiols that can lead to reduction, formation, or interchange of disulfide bonds. This concept was tested using a membrane-impermeable trivalent arsenical to identify closely spaced thiols in cell-surface proteins. We attached the trivalent arsenical, phenylarsenoxide, to the thiol of reduced glutathione to produce 4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide (GSAO). GSAO bound tightly to synthetic, peptide, and protein dithiols like thioredoxin, but not to monothiols. To identify cell-surface proteins that contain closely spaced thiols, we attached a biotin moiety through a spacer arm to the primary amino group of the gamma-glutamyl residue of GSAO (GSAO-B). Incorporation of GSAO-B into proteins was assessed by measuring the biotin using streptavidin-peroxidase. Up to 12 distinct proteins were labeled with GSAO-B on the surface of endothelial and fibrosarcoma cells. The pattern of labeled proteins differed between the different cell types. Protein disulfide isomerase was one of the proteins on the endothelial and fibrosarcoma cell surface that incorporated GSAO-B. These findings demonstrate that the cell-surface environment can support the existence of closely spaced protein thiols and suggest that at least some of these thiols are redox active. PMID:11206065

ROS-dependent signal transduction

PubMed Central

Reczek, Colleen R; Chandel, Navdeep S

2014-01-01

Reactive oxygen species (ROS) are no longer viewed as just a toxic by-product of mitochondrial respiration, but are now appreciated for their role in regulating a myriad of cellular signaling pathways. H2O2, a type of ROS, is a signaling molecule that confers target specificity through thiol oxidation. Although redox-dependent signaling has been implicated in numerous cellular processes, the mechanism by which the ROS signal is transmitted to its target protein in the face of highly reactive and abundant antioxidants is not fully understood. In this review of redox-signaling biology, we discuss the possible mechanisms for H2O2-dependent signal transduction. PMID:25305438

Cisplatin impairs rat liver mitochondrial functions by inducing changes on membrane ion permeability: prevention by thiol group protecting agents.

PubMed

Custódio, José B A; Cardoso, Carla M P; Santos, Maria S; Almeida, Leonor M; Vicente, Joaquim A F; Fernandes, Maria A S

2009-05-02

Cisplatin (CisPt) is the most important platinum anticancer drug widely used in the treatment of head, neck, ovarian and testicular cancers. However, the mechanisms by which CisPt induces cytotoxicity, namely hepatotoxicity, are not completely understood. The goal of this study was to investigate the influence of CisPt on rat liver mitochondrial functions (Ca(2+)-induced mitochondrial permeability transition (MPT), mitochondrial bioenergetics, and mitochondrial oxidative stress) to better understand the mechanism underlying its hepatotoxicity. The effect of thiol group protecting agents and some antioxidants against CisPt-induced mitochondrial damage was also investigated. Treatment of rat liver mitochondria with CisPt (20nmol/mg protein) induced Ca(2+)-dependent mitochondrial swelling, depolarization of membrane potential (DeltaPsi), Ca(2+) release, and NAD(P)H fluorescence intensity decay. These effects were prevented by cyclosporine A (CyA), a potent and specific inhibitor of the MPT. In the concentration range of up to 40nmol/mg protein, CisPt slightly inhibited state 3 and stimulated state 2 and state 4 respiration rates using succinate as respiratory substrate. The respiratory indexes, respiratory control ratio (RCR) and ADP/O ratios, the DeltaPsi, and the ADP phosphorylation rate were also depressed. CisPt induced mitochondrial inner membrane permeabilization to protons (proton leak) but did not induce significant changes on mitochondrial H(2)O(2) generation. All the effects induced by CisPt on rat liver mitochondria were prevented by thiol group protecting agents namely, glutathione (GSH), dithiothreitol (DTT), N-acetyl-L-cysteine (NAC) and cysteine (CYS), whereas superoxide-dismutase (SOD), catalase (CAT) and ascorbate (ASC) were without effect. In conclusion, the anticancer drug CisPt: (1) increases the sensitivity of mitochondria to Ca(2+)-induced MPT; (2) interferes with mitochondrial bioenergetics by increasing mitochondrial inner membrane permeabilization to H(+); (3) does not significantly affect H(2)O(2) generation by mitochondria; (4) its mitochondrial damaging effects are protected by thiol group protecting agents. Based on these conclusions, it is possible to hypothesise that small changes on the redox-status of thiol groups, affecting membrane permeability to cations (Ca(2+) and H(+)) underlie CisPt-induced liver mitochondrial damage, putatively responsible for its hepatotoxicity. Therefore, we propose that CisPt-induced mitochondrial damage and consequent hepatotoxicity could be prevented by using thiol group protecting agents as therapeutic adjuvants.

Effects of Gold Salt Speciation and Structure of Human and Bovine Serum Albumin on the Synthesis and Stability of Gold Nanostructures

NASA Astrophysics Data System (ADS)

Miranda, Érica; Tofanello, Aryane; Brito, Adrianne; Lopes, David; Giacomelli, Fernando; Albuquerque, Lindomar; Costa, Fanny; Ferreira, Fabio; Araujo-Chaves, Juliana; de Castro, Carlos; Nantes, Iseli

2016-03-01

The present study aimed to investigate the influence of albumin structure and gold speciation on the synthesis of gold nanoparticles (GNPs). The strategy of synthesis was the addition of HAuCl4 solutions at different pH values (3-12) to solutions of human and bovine serum albumins (HSA and BSA) at the same corresponding pH values. Different pH values influence the GNP synthesis due to gold speciation. Besides the inherent effect of pH on the native structure of albumins, the use N-ethylmaleimide (NEM)-treated and heat-denaturated forms of HSA and BSA provided additional insights about the influence of protein structure, net charge, and thiol group approachability on the GNP synthesis. NEM treatment, heating, and the extreme values of pH promoted loss of the native albumin structure. The formation of GNPs indicated by the appearance of surface plasmon resonance (SPR) bands became detectable from fifteen days of the synthesis processes that were carried out with native, NEM-treated and heat-denaturated forms of HSA and BSA, exclusively at pH 6 and 7. After two months of incubation, SPR band was also detected for all synthesis carried out at pH 8.0. The mean values of the hydrodynamic radius (RH) were 24 and 34 nm for GNPs synthesized with native HSA and BSA, respectively. X-ray diffraction (XRD) revealed crystallites of 13 nm. RH, XRD, and zeta potential values were consistent with GNP capping by the albumins. However, the GNPs produced with NEM-treated and heat-denaturated albumins exhibited loss of protein capping by lowering the ionic strength. This result suggests a significant contribution of non-electrostatic interactions of albumins with the GNP surface, in these conditions. The denaturation of proteins exposes hydrophobic groups to the solvent, and these groups could interact with the gold surface. In these conditions, the thiol blockage or oxidation, the latter probably favored upon heating, impaired the formation of a stable capping by thiol coordination with the gold surface. Therefore, the cysteine side chain of albumins is important for the colloidal stabilization of GNPs rather than as the reducing agent for the synthesis. Despite the presence of more reactive gold species at more acidic pH values, i.e., below

Synthesis of photolabile transcription initiators and preparation of photocleavable functional RNA by transcription.

PubMed

Huang, Faqing; Shi, Yongliang

2012-07-01

Two new photolabile adenosine-containing transcription initiators with terminal thiol and amino functionalities are chemically synthesized. Transcription in the presence of the transcription initiators under the T7 phi2.5 promoter produces 5' thiol- and amino-functionalized RNA conjugated by a photocleavable (PC) linker. Further RNA functionalization with biotin may be achieved through acyl transfer reactions from either biotinyl AMP to the RNA thiol group or biotin NHS to the RNA amino group. Photocleavage of the PC linker displays relatively fast kinetics with a half-life of 4-5 min. The availability of these transcription initiators makes new photolabile RNA accessible for affinity purification of RNA, in vitro selection of functional RNAs, and functional RNA caging. Copyright © 2012 Elsevier Ltd. All rights reserved.

Using the M13 Phage as a Biotemplate to Create Mesoporous Structures Decorated with Gold and Platinum Nanoparticles.

PubMed

Vera-Robles, L Irais; González-Gracida, Jaqueline; Hernández-Gordillo, Armin; Campero, Antonio

2015-08-25

By taking advantage of the physical and chemical properties of the M13 bacteriophage, we have used this virus to synthesize mesoporous silica structures. Major coat protein p8 was chemically modified by attaching thiol groups. As we show, the resulting thiolated phage can be used as a biotemplate able to direct the formation of mesoporous silica materials. Simultaneously, this thiol functionality acts as an anchor for binding metal ions, such as Au(3+) and Pt(4+), forming reactive M13-metal ionic complexes which evolve into metal nanoparticles (NPs) trapped in the mesoporous network. Interestingly, Au(3+) ions are reduced to Au(0) NPs by the protein residues without requiring an external reducing agent. Likewise, silica mesostructures decorated with Au and Pt NPs are prepared in a one-pot synthesis and characterized using different techniques. The obtained results allow us to propose a mechanism of formation. In addition, gold-containing mesoporous structures are tested for the reduction of 4-nitrophenol (4-NP) and methylene blue (MB) in the presence of NaBH4. Although all of the gold-containing catalysts exhibit catalytic activity, those obtained with thiolated phages present a better performance than that obtained with M13 alone. This behavior is ascribed to the position of the Au NPs, which are partially embedded in the wall of the final mesostructures.

The potential toxicological insights about the anti-HIV drug azidothymidine-derived monoselenides in human leukocytes: Toxicological insights of new selenium-azidothymidine analogs.

PubMed

Mariano, Doc; de Souza, D; Meinerz, D F; Allebrandt, J; de Bem, A F; Hassan, W; Rodrigues, Oed; da Rocha, Jbt

2017-09-01

Acquired immunodeficiency syndrome (AIDS) is a worldwide disease characterized by impairments of immune function. AIDS can be associated with oxidative stress (OS) that can be linked to selenium (Se) deficiency. Se is fundamental for the synthesis of selenoproteins, such as glutathione peroxidase and thioredoxin reductase. These enzymes catalyze the decomposition of reactive oxygen species and contribute to maintain equilibrium in cell redox status. Literature data indicate that organoselenium compounds, such as ebselen and diphenyl diselenide, have antioxidant properties in vitro and in vivo models associated with OS. Nevertheless, selenocompounds can also react and oxidize thiols groups, inducing toxicity in mammals. Here, we tested the potential cytotoxic and genotoxic properties of six analogs of the prototypal anti-HIV drug azidothymidine (AZT) containing Se (5'-Se-(phenyl)zidovudine; 5'-Se-(1,3,5-trimethylphenyl)zidovudine; 5'-Se-(1-naphtyl)zidovudine; 5'-Se-(4-chlorophenyl)zidovudine) (C4); 5'-Se-(4-methylphenyl)zidovudine (C5); and 5'-(4-methylbenzoselenoate)zidovudine). C5 increased the rate of dithiothreitol oxidation (thiol oxidase activity) and C2-C4 and C6 (at 100 µM) increased DNA damage index (DI) in human leukocytes. Moreover, C5 (200 µM) decreased human leukocyte viability to about 50%. Taken together, these results indicated the low in vitro toxicity in human leukocytes of some Se-containing analogs of AZT.

Enzymatic degradation of thiolated chitosan.

PubMed

Laffleur, Flavia; Hintzen, Fabian; Rahmat, Deni; Shahnaz, Gul; Millotti, Gioconda; Bernkop-Schnürch, Andreas

2013-10-01

The objective of this study was to evaluate the biodegradability of thiolated chitosans in comparison to unmodified chitosan. Mediated by carbodiimide, thioglycolic acid (TGA) and mercaptonicotinic acid (MNA) were covalently attached to chitosan via formation an amide bond. Applying two different concentrations of carbodiimide 50 and 100 mM, two chitosan TGA conjugates (TGA A and TGA B) were obtained. According to chitosan solution (3% m/v) thiomer solutions were prepared and chitosanolytic enzyme solutions were added. Lysozyme, pectinase and cellulase were examined in chitosan degrading activity. The enzymatic degradability of these thiomers was investigated by viscosity measurements with a plate-plate viscometer. The obtained chitosan TGA conjugate A displayed 267.7 µmol and conjugate B displayed 116.3 µmol of immobilized thiol groups. With 325.4 µmol immobilized thiol groups, chitosan MNA conjugate displayed the most content of thiol groups. In rheological studies subsequently the modification proved that chitosan TGA conjugates with a higher coupling rate of thiol groups were not only degraded to a lesser extent by 20.9-26.4% but also more slowly. Chitosan mercaptonicotinic acid was degraded by 31.4-50.1% depending the investigated enzyme and even faster than unmodified chitosan. According to these results the biodegradability can be influenced by various modifications of the polymer which showed in particular that the rate of biodegradation is increased when MNA is the ligand, whereas the degradation is hampered when TGA is used as ligand for chitosan.

Chromenoquinoline-based thiol probes: a study on the quencher position for controlling fluorescent Off-On characteristics.

PubMed

Kand, Dnyaneshwar; Kalle, Arunasree Marasanapalli; Talukdar, Pinaki

2013-02-13

The design, synthesis and thiol sensing ability of chromenoquinoline-based fluorescent probes 4, 5 and 6 and are reported here. The relative position of the maleimide moiety was varied along the chromenoquinoline fluorophore to decrease the background fluorescence. Lower background fluorescence in probes 4 and 6 was rationalized by the smaller k(r)/k(nr) values compared to that of probe 5. An intramolecular charge transfer (ICT) mechanism was proposed for quenching and the extent was dependent on the position of the maleimide quencher. Fluorescent Off-On characteristics were evaluated by theoretical calculations. All probes were selective only towards thiol containing amino acids. Thiol sensing by probes 4 and 6 were much better compared to 5. Probe 4 displayed a better fluorescence response for less hindered thiol (185-, 223- and 156-fold for Hcy, Cys and GSH, respectively), while for probe 6, a higher enhancement in fluorescence was observed with more hindered thiols (180-, 205- and 245-fold for Hcy, Cys and GSH, respectively). The better response to bulkier thiol, GSH by probe 6 was attributed to the steric crowding at the C-4 position and bulkiness of the GSH group which force the succinimide unit to be in a nearly orthogonal conformation. This spatial arrangement was important in reducing the fluorescence quenching ability of the succinimide moiety. The application of probes 4, 5 and 6 was demonstrated by naked eye detection thiols using a 96-well plate system as well as by live-cell imaging.

Molecular cloning and characterization of Bacillus alvei thiol-dependent cytolytic toxin expressed in Escherichia coli.

PubMed

Geoffroy, C; Alouf, J E

1988-07-01

A chromosomal DNA fragment from Bacillus alvei, encoding a thiol-dependent haemolytic product known as alveolysin (Mr 60,000, pI 5.0) was cloned in Escherichia coli SK1592, using pBR322 as the vector plasmid. Only a single haemolysin-positive clone was identified, by testing for haemolysis on blood agar plates. The haemolytic material was associated with the host bacterial cell. It was released by ultrasonic disruption and purified 267-fold. A 64 kDa polypeptide of pI 8.2 cofractionated with haemolytic activity during gel filtration chromatography and isoelectric focusing. It behaved identically to alveolysin in its activation by thiols, inactivation by thiol group reagents, inhibition by cholesterol, and neutralization, immunoprecipitation and immunoblotting by immune sera raised against alveolysin and streptolysin O.

Diverse Effects of Glutathione and UPF Peptides on Antioxidant Defense System in Human Erythroleukemia Cells K562.

PubMed

Kairane, Ceslava; Mahlapuu, Riina; Ehrlich, Kersti; Kilk, Kalle; Zilmer, Mihkel; Soomets, Ursel

2012-01-01

The main goal of the present paper was to examine the influence of the replacement of γ-Glu moiety to α-Glu in glutathione and in its antioxidative tetrapeptidic analogue UPF1 (Tyr(Me)-γ-Glu-Cys-Gly), resulting in α-GSH and UPF17 (Tyr(Me)-Glu-Cys-Gly), on the antioxidative defense system in K562 cells. UPF1 and GSH increased while UPF17 and α-GSH decreased the activity of CuZnSOD in K562 cells, at peptide concentration of 10 μM by 42% and 38% or 35% and 24%, respectively. After three-hour incubation, UPF1 increased and UPF17 decreased the intracellular level of total GSH. Additionally, it was shown that UPF1 is not degraded by γ-glutamyltranspeptidase, which performs glutathione breakdown. These results indicate that effective antioxidative character of peptides does not depend only on the reactivity of the thiol group, but also of the other functional groups, and on the spatial structure of peptides.

On-resin conversion of Cys(Acm)-containing peptides to their corresponding Cys(Scm) congeners.

PubMed

Mullen, Daniel G; Weigel, Benjamin; Barany, George; Distefano, Mark D

2010-05-01

The Acm protecting group for the thiol functionality of cysteine is removed under conditions (Hg(2+)) that are orthogonal to the acidic milieu used for global deprotection in Fmoc-based solid-phase peptide synthesis. This use of a toxic heavy metal for deprotection has limited the usefulness of Acm in peptide synthesis. The Acm group may be converted to the Scm derivative that can then be used as a reactive intermediate for unsymmetrical disulfide formation. It may also be removed by mild reductive conditions to generate unprotected cysteine. Conversion of Cys(Acm)-containing peptides to their corresponding Cys(Scm) derivatives in solution is often problematic because the sulfenyl chloride reagent used for this conversion may react with the sensitive amino acids tyrosine and tryptophan. In this protocol, we report a method for on-resin Acm to Scm conversion that allows the preparation of Cys(Scm)-containing peptides under conditions that do not modify other amino acids. (c) 2010 European Peptide Society and John Wiley & Sons, Ltd.

Exposure to Electrophiles Impairs Reactive Persulfide-Dependent Redox Signaling in Neuronal Cells.

PubMed

Ihara, Hideshi; Kasamatsu, Shingo; Kitamura, Atsushi; Nishimura, Akira; Tsutsuki, Hiroyasu; Ida, Tomoaki; Ishizaki, Kento; Toyama, Takashi; Yoshida, Eiko; Abdul Hamid, Hisyam; Jung, Minkyung; Matsunaga, Tetsuro; Fujii, Shigemoto; Sawa, Tomohiro; Nishida, Motohiro; Kumagai, Yoshito; Akaike, Takaaki

2017-09-18

Electrophiles such as methylmercury (MeHg) affect cellular functions by covalent modification with endogenous thiols. Reactive persulfide species were recently reported to mediate antioxidant responses and redox signaling because of their strong nucleophilicity. In this study, we used MeHg as an environmental electrophile and found that exposure of cells to the exogenous electrophile elevated intracellular concentrations of the endogenous electrophilic molecule 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), accompanied by depletion of reactive persulfide species and 8-SH-cGMP which is a metabolite of 8-nitro-cGMP. Exposure to MeHg also induced S-guanylation and activation of H-Ras followed by injury to cerebellar granule neurons. The electrophile-induced activation of redox signaling and the consequent cell damage were attenuated by pretreatment with a reactive persulfide species donor. In conclusion, exogenous electrophiles such as MeHg with strong electrophilicity impair the redox signaling regulatory mechanism, particularly of intracellular reactive persulfide species and therefore lead to cellular pathogenesis. Our results suggest that reactive persulfide species may be potential therapeutic targets for attenuating cell injury by electrophiles.

Silymarin Ameliorates Oxidative Stress and Enhances Antioxidant Defense System Capacity in Cadmium-Treated Mice.

PubMed

Farjad, Elham; Momeni, Hamid Reza

2018-10-01

Cadmium is an environmental pollutant which induces oxidative stress while silymarin as an antioxidant is able to scavenge free radicals. The aim of the present study was to investigate the effect of silymarin on oxidative stress markers and antioxidant defense system capacity in mice treated with cadmium chloride. In this experimental study, adult mice were divided into four groups as follow: i. Control, ii. Cadmium chloride (5 mg/kg b.w., s.c.), iii. Silymarin+cadmium chloride, and iv. Silymarin (100 mg/kg b.w., i.p.). Mice were treated with cadmium chloride for 24 hours and silymarin was administered 24 hours before the cadmium. Blood samples were then collected from the experimental groups and their sera were prepared. To investigate oxidative stress markers in the serum, the amount of malondialdehyde (MDA) and thiol groups (-SH) were evaluated. To measure the total antioxidant power in the serum, Ferric Reducing/ Antioxidant Power (FRAP) method was used. In addition, the activity of enzymes including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) was assessed to evaluate serum antioxidant defense power. In the cadmium-treated group, the amount of MDA significantly increased as compared to the control group. In silymarin+cadmium group, silymarin significantly ameliorated the level of MDA compared to the cadmium group. In addition, cadmium significantly reduced serum FRAP, the activity of antioxidant defense system enzymes and thiol groups compared to the control. In silymarin+cadmium group, silymarin could significantly reverse the reduction of these markers compared to the cadmium group. Administration of silymarin alone caused a significant increase in serum FRAP, the activity of antioxidant defense system enzymes and thiol groups compared to the control group. Silymarin as a powerful antioxidant reverses the toxic effect of cadmium on the serum levels of lipid peroxidation, total antioxidant power, antioxidant defense system enzymes activity and thiol groups. Copyright© by Royan Institute. All rights reserved.

Diamond surface functionalization with biomimicry - Amine surface tether and thiol moiety for electrochemical sensors

NASA Astrophysics Data System (ADS)

Sund, James B.; Causey, Corey P.; Wolter, Scott D.; Parker, Charles B.; Stoner, Brian R.; Toone, Eric J.; Glass, Jeffrey T.

2014-05-01

The surface of conducting diamond was functionalized with a terminal thiol group that is capable of binding and detecting nitrogen-oxygen species. The functionalization process employed multiple steps starting with doped diamond films grown by plasma enhanced chemical vapor deposition followed by hydrogen termination and photochemical attachment of a chemically protected amine alkene. The surface tether was deprotected to reveal the amine functionality, which enabled the tether to be extended with surface chemistry to add a terminal thiol moiety for electrochemical sensing applications. Each step of the process was validated using X-ray photoelectron spectroscopy analysis.

The nitrative and oxidative stress in blood platelets isolated from breast cancer patients: the protectory action of aronia melanocarpa extract.

PubMed

Kedzierska, Magdalena; Olas, Beata; Wachowicz, Barbara; Stochmal, Anna; Oleszek, Wieslaw; Jeziorski, Arkadiusz; Piekarski, Janusz

2010-01-01

Since mechanisms involved in the relationship between oxidative stress and breast cancer are still unclear, the aim of our present study was to evaluate oxidative/nitrative modifications of blood platelet proteins by measuring the level of biomarkers of oxidative/nitrative stress such as carbonyl groups, thiol groups and 3-nitrotyrosine in proteins in patients with benign breast diseases and in patients with invasive breast cancer, and compare with the control group. Levels of carbonyl groups and 3-nitrotyrosine residues in platelet proteins were measured by ELISA and a competition ELISA, respectively. The method with 5,5′-dithio-bis(2-nitro-benzoic acid) has been used to analyse free thiol groups in platelet proteins. Patients were hospitalized in the Department of Oncological Surgery, Medical University of Lodz, Poland. Exogenous antioxidants reduce oxidative stress, therefore we also investigated in a model system in vitro the effects of a polyphenol rich extract of Aronia melanocarpa (Rosaceae, final concentration of 50 µg/ml, 5 min, 37°C) on modified blood platelet proteins as well from patients with breast cancer and from the healthy group. We demonstrated in platelet proteins from patients with invasive breast cancer a higher level of carbonyl groups than in the control healthy group (p < 0.02). The level of 3-nitrotyrosine in platelet proteins from patients with invasive breast cancer was also significantly higher than in the healthy subject group (p < 0.001). In contrast, the amount of thiol groups in platelet proteins from patients was significantly lower (about < 50%) than in control blood platelets. In a model system in vitro we also observed that the extract from berries of A. melanocarpa (50 µg/ml, 5 min, 37°C) due to antioxidant action, significantly reduced the oxidative/nitrative stress (measured by thiol groups and 3-nitrotyrosine) in platelets, not only from the healthy group, but also from patients with benign breast diseases and in patients with invasive breast cancer.

Developing clean fuels: Novel techniques for desulfurization

NASA Astrophysics Data System (ADS)

Nehlsen, James P.

The removal of sulfur compounds from petroleum is crucial to producing clean burning fuels. Sulfur compounds poison emission control catalysts and are the source of acid rain. New federal regulations require the removal of sulfur in both gasoline and diesel to very low levels, forcing existing technologies to be pushed into inefficient operating regimes. New technology is required to efficiently produce low sulfur fuels. Two processes for the removal of sulfur compounds from petroleum have been developed: the removal of alkanethiols by heterogeneous reaction with metal oxides; and oxidative desulfurization of sulfides and thiophene by reaction with sulfuric acid. Alkanethiols, common in hydrotreated gasoline, can be selectively removed and recovered from a hydrocarbon stream by heterogeneous reaction with oxides of Pb, Hg(II), and Ba. The choice of reactive metal oxides may be predicted from simple thermodynamic considerations. The reaction is found to be autocatalytic, first order in water, and zero order in thiol in the presence of excess oxide. The thiols are recovered by reactive extraction with dilute oxidizing acid. The potential for using polymer membrane hydrogenation reactors (PEMHRs) to perform hydrogenation reactions such as hydrodesulfurization is explored by hydrogenating ketones and olefins over Pt and Au group metals. The dependence of reaction rate on current density suggests that the first hydrogen addition to the olefin is the rate limiting step, rather than the adsorption of hydrogen, for all of the metals tested. PEMHRs proved unsuccessful in hydrogenating sulfur compounds to perform HDS. For the removal of sulfides, a two-phase reactor is used in which concentrated sulfuric acid oxidizes aromatic and aliphatic sulfides present in a hydrocarbon solvent, generating sulfoxides and other sulfonated species. The polar oxidized species are extracted into the acid phase, effectively desulfurizing the hydrocarbon. A reaction scheme is proposed for this system and is justified with a thermodynamic analysis and an experimental determination of the reaction rate law.

Comparison of sensory, microbiological, and biochemical parameters of microwave versus indirect UHT fluid skim milk during storage.

PubMed

Clare, D A; Bang, W S; Cartwright, G; Drake, M A; Coronel, P; Simunovic, J

2005-12-01

Shelf-stable milk could benefit from sensory quality improvement. Current methods of heating cause flavor and nutrient degradation through exposure to overheated thermal exchange surfaces. Rapid heating with microwaves followed by sudden cooling could reduce or eliminate this problem. The objectives for this study were focused on designing and implementing continuous microwave thermal processing of skim fluid milks (white and chocolate) to compare sensory, microbiological, and biochemical parameters with conventionally prepared, indirect UHT milks. All test products were aseptically packaged and stored at ambient temperature for 12 mo. Every 3 mo, samples were taken for microbiological testing, reactive sulfhydryl determinations, active enzyme analysis, instrumental viscosity readings, color measurements, and descriptive sensory evaluation. Microbiological plate counts were negative on all milks at each time point. Enzymatic assays showed that plasmin was inactivated by both heat treatments. 5,5'-dithio-bis(2-nitrobenzoic acid) analysis, a measure of reactive sulfhydryl (-SH-) groups, showed that the initial thiol content was not significantly different between the microwave-processed and UHT-treated milks. However, both heating methods resulted in an increased thiol level compared with conventionally pasteurized milk samples due to the higher temperatures attained. Sulfhydryl oxidase, a milk enzyme that catalyzes disulfide bond formation using a variety of protein substrates, retained activity following microwave processing, and decreased during storage. Viscosity values were essentially equivalent in microwave- and UHT-heated white skim milks. Sensory analyses established that UHT-treated milks were visibly darker, and exhibited higher caramelized and stale/fatty flavors with increased astringency compared with the microwave samples. Sweet aromatic flavor and sweet taste decreased during storage in both UHT and microwave milk products, whereas stale/fatty flavors increased over time. Sensory effects were more apparent in white milks than in chocolate varieties. These studies suggest that microwave technology may provide a useful alternative processing method for delivery of aseptic milk products that retain a long shelf life.

Assessment of Mitochondrial Dysfunction Arising from Treatment with Hepatotoxicants

PubMed Central

King, Adrienne L.; Bailey, Shannon M.

2010-01-01

Studies demonstrate that mitochondrial dysfunction is a key causative factor in liver disease. Indeed, defects in mitochondrial energy metabolism, disrupted calcium handling, and increased reactive oxygen/nitrogen species production are observed in many metabolic disorders and diseases induced by toxicants. Mitochondria have emerged as a main research focus through work defining new functions of this key organelle in normal cellular physiology and pathophysiology. Specifically, studies show a critical role of mitochondrial reactive oxygen/nitrogen species production in regulating cellular signaling pathways involved in cell survival and death. Given this, along with advances made in proteomics technologies, mitochondria are recognized as top candidates for proteomics analysis. However, assessment of mitochondrial function and it’s proteome following toxicant exposure are not trivial undertakings. In this chapter a technique used to isolate mitochondria from liver tissue is presented along with methods needed to assess mitochondria functionality. The methods described include measurement of mitochondrial respiration, calcium accumulation, and reactive oxygen species production. A presentation of proteomics approaches is also included to allow researchers the basic tools needed to identify alterations in the mitochondrial proteome that contribute to toxicant-mediated diseases. Specifically, methods are presented that demonstrate how thiol labeling reagents in combination with electrophoresis and western blotting can be used to detect oxidant-mediated alterations in mitochondrial protein thiols. A few select pieces data are presented highlighting the power of proteomics to identify mitochondrial targets that contribute to mitochondrial dysfunction and hepatotoxicity in response to specific toxicant exposures and metabolic stressors such as alcohol and environmental tobacco smoke. PMID:23045017

A lysosome-targetable turn-on fluorescent probe for the detection of thiols in living cells based on a 1,8-naphthalimide derivative.

PubMed

Liang, Beibei; Wang, Baiyan; Ma, Qiujuan; Xie, Caixia; Li, Xian; Wang, Suiping

2018-03-05

Biological thiols, like cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play crucial roles in biological systems and in lysosomal processes. Highly selective probes for detecting biological thiols in lysomes of living cells are rare. In this work, a lysosome-targetable turn-on fluorescent probe for the detection of thiols in living cells was designed and synthesized based on a 1,8-naphthalimide derivative. The probe has a 4-(2-aminoethyl)morpholine unit as a lysosome-targetable group and an acrylate group as the thiol recognition unit as well as a fluorescence quencher. In the absence of biothiols, the probe displayed weak fluorescence due to the photoinduced electron transfer (PET) process. Upon the addition of biothiols, the probe exhibited an enhanced fluorescence emission centered at 550nm due to cleavage of the acrylate moiety. The probe had high selectivity toward biothiols. Moreover, the probe features fast response time, excitation in the visible region and ability of working in a wide pH range. The linear response range covers a concentration range of Cys from 1.5×10 -7 to 1.0×10 -5 mol·L -1 and the detection limit is 6.9×10 -8 mol·L -1 for Cys. The probe has been successfully applied to the confocal imaging of biothiols in lysosomes of A549 cells with low cell toxicity. Furthermore, the method was successfully applied to the determination of thiols in a complex multicomponent mixture such as human serum, which suggests our proposed method has great potential for diagnostic purposes. All of such good properties prove it can be used to monitor biothiols in lysosomes of living cells and to be a good fluorescent probe for the selective detection of thiols. Copyright © 2017 Elsevier B.V. All rights reserved.

Studies of benzamide- and thiol-based histone deacetylase inhibitors in models of oxidative-stress-induced neuronal death: identification of some HDAC3-selective inhibitors.

PubMed

Chen, Yufeng; He, Rong; Chen, Yihua; D'Annibale, Melissa A; Langley, Brett; Kozikowski, Alan P

2009-05-01

We compare three structurally different classes of histone deacetylase (HDAC) inhibitors that contain benzamide, hydroxamate, or thiol groups as the zinc binding group (ZBG) for their ability to protect cortical neurons in culture from cell death induced by oxidative stress. This study reveals that none of the benzamide-based HDAC inhibitors (HDACIs) provides any neuroprotection whatsoever, in distinct contrast to HDACIs that contain other ZBGs. Some of the sulfur-containing HDACIs, namely the thiols, thioesters, and disulfides present modest neuroprotective activity but show toxicity at higher concentrations. Taken together, these data demonstrate that the HDAC6-selective mercaptoacetamides that were reported previously provide the best protection in the homocysteic acid model of oxidative stress, thus further supporting their study in animal models of neurodegenerative diseases.

Selective chloroform sensor using thiol functionalized reduced graphene oxide at room temperature

NASA Astrophysics Data System (ADS)

Midya, Anupam; Mukherjee, Subhrajit; Roy, Shreyasee; Santra, Sumita; Manna, Nilotpal; Ray, Samit K.

2018-02-01

This paper presents a highly selective chloroform sensor using functionalised reduced graphene oxide (RGO) as a sensing layer. Thiol group is covalently attached on the basal plan of RGO film by a simple one-step aryl diazonium chemistry to improve its selectivity. Several spectroscopic techniques like X-ray photoelectron, Raman and Fourier transform infrared spectroscopy confirm successful thiol functionalization of RGO. Finally, the fabricated chemiresistor type sensor is exposed to chloroform in the concentration range 200-800 ppm (parts per million). The sensor shows a 4.3% of response towards 800 ppm chloroform. The selectivity of the sensor is analyzed using various volatile organic compounds as well. The devices show enhanced response and faster recovery attributed to the physiosorption of chloroform onto thiol functionalized graphene making them attractive for 2D materials based sensing applications.

Selective optical sensing of biothiols with Ellman's reagent: 5,5'-Dithio-bis(2-nitrobenzoic acid)-modified gold nanoparticles.

PubMed

Güçlü, Kubilay; Ozyürek, Mustafa; Güngör, Nilay; Baki, Sefa; Apak, Reşat

2013-09-10

Development of sensitive and selective methods of determination for biothiols is important because of their significant roles in biological systems. We present a new optical sensor using Ellman's reagent (DTNB)-adsorbed gold nanoparticles (Au-NPs) (DTNB-Au-NP) in a colloidal solution devised to selectively determine biologically important thiols (biothiols) from biological samples and pharmaceuticals. 5,5'-Dithio-bis(2-nitrobenzoic acid) (DTNB), a versatile water-soluble compound for quantitating free sulfhydryl groups in solution, was adsorbed through non-covalent interaction onto Au-NPs, and the absorbance changes associated with the formation of the yellow-colored 5-thio-2-nitrobenzoate (TNB(2-)) anion as a result of reaction with biothiols was measured at 410nm. The sensor gave a linear response over a wide concentration range of standard biothiols comprising cysteine, glutathione, homocysteine, cysteamine, dihydrolipoic acid and 1,4-dithioerythritol. The calibration curves of individual biothiols were constructed, and their molar absorptivities and linear concentration ranges determined. The cysteine equivalent thiol content (CETC) values of various biothiols using the DTNB-Au-NP assay were comparable to those of the conventional DTNB assay, showing that the immobilized DTNB reagent retained its reactivity toward thiols. Common biological sample ingredients like amino acids, flavonoids, vitamins, and plasma antioxidants did not interfere with the proposed sensing method. This assay was validated through linearity, additivity, precision and recovery, demonstrating that the assay is reliable and robust. DTNB-adsorbed Au-NPs probes provided higher sensitivity (i.e., lower detection limits) in biothiol determination than conventional DTNB reagent. Under optimized conditions, cysteine (Cys) was quantified by the proposed assay, with a detection limit (LOD) of 0.57μM and acceptable linearity ranging from 0.4 to 29.0μM (r=0.998). Copyright © 2013 Elsevier B.V. All rights reserved.

Regulation of Cell Physiology and Pathology by Protein S-Glutathionylation: Lessons Learned from the Cardiovascular System

PubMed Central

Pimentel, David; Haeussler, Dagmar Johanna; Matsui, Reiko; Burgoyne, Joseph Robert; Cohen, Richard Alan

2012-01-01

Abstract Significance: Reactive oxygen and nitrogen species contributing to homeostatic regulation and the pathogenesis of various cardiovascular diseases, including atherosclerosis, hypertension, endothelial dysfunction, and cardiac hypertrophy, is well established. The ability of oxidant species to mediate such effects is in part dependent on their ability to induce specific modifications on particular amino acids, which alter protein function leading to changes in cell signaling and function. The thiol containing amino acids, methionine and cysteine, are the only oxidized amino acids that undergo reduction by cellular enzymes and are, therefore, prime candidates in regulating physiological signaling. Various reports illustrate the significance of reversible oxidative modifications on cysteine thiols and their importance in modulating cardiovascular function and physiology. Recent Advances: The use of mass spectrometry, novel labeling techniques, and live cell imaging illustrate the emerging importance of reversible thiol modifications in cellular redox signaling and have advanced our analytical abilities. Critical Issues: Distinguishing redox signaling from oxidative stress remains unclear. S-nitrosylation as a precursor of S-glutathionylation is controversial and needs further clarification. Subcellular distribution of glutathione (GSH) may play an important role in local regulation, and targeted tools need to be developed. Furthermore, cellular redundancies of thiol metabolism complicate analysis and interpretation. Future Directions: The development of novel pharmacological analogs that specifically target subcellular compartments of GSH to promote or prevent local protein S-glutathionylation as well as the establishment of conditional gene ablation and transgenic animal models are needed. Antioxid. Redox Signal. 16, 524–542. PMID:22010840

Increased Cysteine Biosynthesis Capacity of Transgenic Tobacco Overexpressing an O-Acetylserine(thiol) Lyase Modifies Plant Responses to Oxidative Stress1

PubMed Central

Youssefian, Shohab; Nakamura, Michimi; Orudgev, Emin; Kondo, Noriaki

2001-01-01

O-Acetylserine(thiol) lyase (OASTL), a key enzyme of plant sulfur metabolism, catalyzes the formation of Cys from sulfide and O-acetylserine. The biosynthesis of Cys is regarded as the exclusive function of sulfur reduction in plants, and a key limiting step in the production of glutathione (GSH), a thiol implicated in various cellular functions, including sulfur transport, gene expression, scavenging of reactive oxygen species (ROS), and resistance to biotic and abiotic stresses. To examine whether an increased capacity for cysteine (Cys) biosynthesis alters cellular responses to such stresses, we studied the differential changes in thiol levels and ROS scavenging of transgenic tobacco (Nicotiana tabacum) plants expressing the wheat (Triticum aestivum) OASTL gene, cys1, to SO2 and to the ROS generator, methyl viologen. Intracellular Cys and GSH contents were generally higher in cys1 transgenics than in controls under normal growth conditions, but became especially elevated in transgenic plants after SO2 exposure. An examination of differences in the ROS scavenging system of the transgenic plants also demonstrated the specific accumulation of Cu/Zn superoxide dismutase transcripts, known to be induced by Cys or GSH, and elevated cellular superoxide dismutase activities. The transgenic plants accordingly showed dramatic reductions in the extent of both foliar and photooxidative damage in response to acute SO2, as well as reduced levels of chlorosis and membrane damage following methyl viologen treatment. Overall, our results imply that OASTL plays a pivotal role in the synthesis of Cys and GSH that are required for regulation of plant responses to oxidative stress. PMID:11457951

Properties of selected S-nitrosothiols compared to nitrosylated WR-1065.

PubMed

Whiteside, William Michael; Sears, Devin N; Young, Paul R; Rubin, David B

2002-05-01

WR-1065 ([N-mercaptoethyl]-1-3-diaminopropane), the active form of the aminothiol drug Ethyol/Amifostine, protects against toxicity caused by radiation, chemotherapy and endotoxin. Because WR-1065 and other thiols readily bind nitric oxide (NO), injurious conditions or therapies that induce the production or mobilization of NO could alter the effects of WR-1065. S-Nitrosothiols were prepared from various thiols by a standard method to compare properties and stability. Heteromolecular quantum correlation 2D nuclear magnetic resonance was used to characterize nitrosylated glutathione (GSH) and WR-1065; both S- and N-nitrosothiols were observed, depending on the experimental conditions. Three categories of S-nitrosothiol stability were observed: (1) highly stable, with t(1/2) > 8 h, N-acetyl-L-cysteine nitrosothiol (t(1/2) 15 h) > GSH nitrosothiol (t(1/2) 8 h); (2) intermediate stability, t(1/2) approximately 2 h, cysteamine nitrosothiol and WR-1065 nitrosothiol; and (3) low stability, t(1/2) < 1 h, cysteine nitrosothiol and Captopril nitrosothiol. Similar relative rates were observed for Hg(+2)-induced denitrosylation: WR-1065 reacted faster than GSH nitrosothiol, while GSH nitrosothiol reacted faster than N-acetyl-L-cysteine nitrosothiol. Mostly mediated by mixed-NPSH disulfide formation, the activity of the redox-sensitive cysteine protease, cathepsin H, was inhibited by the S-nitrosothiols, with WR-1065 nitrosothiol > cysteine nitrosothiol > N-acetyl-L-cysteine nitrosothiol and GSH nitrosothiol. These observations indicate that, relative to other nitrosylated non-protein thiols, the S-nitrosothiol of WR-1065 is an unstable non-protein S-nitrosothiols with a high reactive potential in the modification of protein thiols.

A new ABC half-transporter in Leishmania major is involved in resistance to antimony.

PubMed

Manzano, J I; García-Hernández, R; Castanys, S; Gamarro, F

2013-08-01

The characterization of ABCI4, a new intracellular ATP-binding cassette (ABC) half-transporter in Leishmania major, is described. We show that ABCI4 is involved in heavy metal export, thereby conferring resistance to Pentostam, to Sb(III), and to As(III) and Cd(II). Parasites overexpressing ABCI4 showed a lower mitochondrial toxic effect of antimony by decreasing reactive oxygen species production and maintained higher values of both the mitochondrial electrochemical potential and total ATP levels with respect to controls. The ABCI4 half-transporter forms homodimers as determined by a coimmunoprecipitation assay. A combination of subcellular localization studies under a confocal microscope and a surface biotinylation assay using parasites expressing green fluorescent protein- and FLAG-tagged ABCI4 suggests that the transporter presents a dual localization in both mitochondria and the plasma membrane. Parasites overexpressing ABCI4 present an increased replication in mouse peritoneal macrophages. We have determined that porphyrins are substrates for ABCI4. Consequently, the overexpression of ABCI4 confers resistance to some toxic porphyrins, such as zinc-protoporphyrin, due to the lower accumulation resulting from a significant efflux, as determined using the fluorescent zinc-mesoporphyrin, a validated heme analog. In addition, ABCI4 has a significant ability to efflux thiol after Sb(III) incubation, thus meaning that ABCI4 could be considered to be a potential thiol-X-pump that is able to recognize metal-conjugated thiols. In summary, we have shown that this new ABC transporter is involved in drug sensitivity to antimony and other compounds by efflux as conjugated thiol complexes.

ROS-dependent signal transduction.

PubMed

Reczek, Colleen R; Chandel, Navdeep S

2015-04-01

Reactive oxygen species (ROS) are no longer viewed as just a toxic by-product of mitochondrial respiration, but are now appreciated for their role in regulating a myriad of cellular signaling pathways. H2O2, a type of ROS, is a signaling molecule that confers target specificity through thiol oxidation. Although redox-dependent signaling has been implicated in numerous cellular processes, the mechanism by which the ROS signal is transmitted to its target protein in the face of highly reactive and abundant antioxidants is not fully understood. In this review of redox-signaling biology, we discuss the possible mechanisms for H2O2-dependent signal transduction. Copyright © 2014 Elsevier Ltd. All rights reserved.

Microbicidal activity of neutrophils is inhibited by isolates from recurrent vaginal candidiasis (RVVC) caused by Candida albicans through fungal thioredoxin reductase.

PubMed

Ratti, Bianca Altrão; Godoy, Janine Silva Ribeiro; de Souza Bonfim Mendonça, Patrícia; Bidóia, Danielle Lazarin; Nakamura, Tânia Ueda; Nakamura, Celso Vataru; Lopes Consolaro, Marcia Edilaine; Estivalet Svidzinski, Terezinha Inez; de Oliveira Silva, Sueli

2015-01-01

Vulvovaginal candidiasis (VVC) is characterized by an infection of the vulva and vagina, mainly caused by Candida albicans, a commensal microorganism that inhabits the vaginal, digestive, and respiratory mucosae. Vulvovaginal candidiasis affects approximately 75% of women, and 5% develop the recurrent form (RVVC). The aim of the present study was to evaluate whether neutrophils microbicidal response is triggered when activated with RVVC isolates caused by C. albicans. Our results showed that RVVC isolates induced neutrophil migration but significantly decrease the microbicidal activity of neutrophils, compared with VVC and ASS isolates. The microbicidal activity of neutrophils is highly dependent on the production of reactive oxygen species/reactive nitrogen species (ROS/RNS). However, this isolate induced detoxification of ROS/RNS produced by neutrophils, reflected by the high level of thiol groups and by the oxygen consumption. Therefore, RVVC isolates induced biochemical changes in the inflammatory response triggered by neutrophils, and these effects were mainly related to the detoxification of ROS/RNS through the thioredoxin reductase (TR), a key antioxidant enzyme in fungi. This might be one of the resistance mechanisms triggered by RVVC caused by C. albicans. Copyright © 2014 Elsevier Inc. All rights reserved.

The Combination of Vitamin K3 and Vitamin C Has Synergic Activity against Forms of Trypanosoma cruzi through a Redox Imbalance Process

PubMed Central

Cristina Desoti, Vânia; Lazarin-Bidóia, Danielle; Martins Ribeiro, Fabianne; Cardoso Martins, Solange; da Silva Rodrigues, Jean Henrique; Ueda-Nakamura, Tania; Vataru Nakamura, Celso; Farias Ximenes, Valdecir; de Oliveira Silva, Sueli

2015-01-01

Chagas’ disease is an infection that is caused by the protozoan Trypanosoma cruzi, affecting millions of people worldwide. Because of severe side effects and variable efficacy, the current treatments for Chagas’ disease are unsatisfactory, making the search for new chemotherapeutic agents essential. Previous studies have reported various biological activities of naphthoquinones, such as the trypanocidal and antitumor activity of vitamin K3. The combination of this vitamin with vitamin C exerted better effects against various cancer cells than when used alone. These effects have been attributed to an increase in reactive oxygen species generation. In the present study, we evaluated the activity of vitamin K3 and vitamin C, alone and in combination, against T. cruzi. The vitamin K3 + vitamin C combination exerted synergistic effects against three forms of T. cruzi, leading to morphological, ultrastructural, and functional changes by producing reactive species, decreasing reduced thiol groups, altering the cell cycle, causing lipid peroxidation, and forming autophagic vacuoles. Our hypothesis is that the vitamin K3 + vitamin C combination induces oxidative imbalance in T. cruzi, probably started by a redox cycling process that leads to parasite cell death. PMID:26641473

The Combination of Vitamin K3 and Vitamin C Has Synergic Activity against Forms of Trypanosoma cruzi through a Redox Imbalance Process.

PubMed

Cristina Desoti, Vânia; Lazarin-Bidóia, Danielle; Martins Ribeiro, Fabianne; Cardoso Martins, Solange; da Silva Rodrigues, Jean Henrique; Ueda-Nakamura, Tania; Vataru Nakamura, Celso; Farias Ximenes, Valdecir; de Oliveira Silva, Sueli

2015-01-01

Chagas' disease is an infection that is caused by the protozoan Trypanosoma cruzi, affecting millions of people worldwide. Because of severe side effects and variable efficacy, the current treatments for Chagas' disease are unsatisfactory, making the search for new chemotherapeutic agents essential. Previous studies have reported various biological activities of naphthoquinones, such as the trypanocidal and antitumor activity of vitamin K3. The combination of this vitamin with vitamin C exerted better effects against various cancer cells than when used alone. These effects have been attributed to an increase in reactive oxygen species generation. In the present study, we evaluated the activity of vitamin K3 and vitamin C, alone and in combination, against T. cruzi. The vitamin K3 + vitamin C combination exerted synergistic effects against three forms of T. cruzi, leading to morphological, ultrastructural, and functional changes by producing reactive species, decreasing reduced thiol groups, altering the cell cycle, causing lipid peroxidation, and forming autophagic vacuoles. Our hypothesis is that the vitamin K3 + vitamin C combination induces oxidative imbalance in T. cruzi, probably started by a redox cycling process that leads to parasite cell death.

Chemical fingerprints of cold physical plasmas - an experimental and computational study using cysteine as tracer compound.

PubMed

Lackmann, J-W; Wende, K; Verlackt, C; Golda, J; Volzke, J; Kogelheide, F; Held, J; Bekeschus, S; Bogaerts, A; Schulz-von der Gathen, V; Stapelmann, K

2018-05-16

Reactive oxygen and nitrogen species released by cold physical plasma are being proposed as effectors in various clinical conditions connected to inflammatory processes. As these plasmas can be tailored in a wide range, models to compare and control their biochemical footprint are desired to infer on the molecular mechanisms underlying the observed effects and to enable the discrimination between different plasma sources. Here, an improved model to trace short-lived reactive species is presented. Using FTIR, high-resolution mass spectrometry, and molecular dynamics computational simulation, covalent modifications of cysteine treated with different plasmas were deciphered and the respective product pattern used to generate a fingerprint of each plasma source. Such, our experimental model allows a fast and reliable grading of the chemical potential of plasmas used for medical purposes. Major reaction products were identified to be cysteine sulfonic acid, cystine, and cysteine fragments. Less-abundant products, such as oxidized cystine derivatives or S-nitrosylated cysteines, were unique to different plasma sources or operating conditions. The data collected point at hydroxyl radicals, atomic O, and singlet oxygen as major contributing species that enable an impact on cellular thiol groups when applying cold plasma in vitro or in vivo.

Development of a Fragment-Based in Silico Profiler for Michael Addition Thiol Reactivity.

PubMed

Ebbrell, David J; Madden, Judith C; Cronin, Mark T D; Schultz, Terry W; Enoch, Steven J

2016-06-20

The Adverse Outcome Pathway (AOP) paradigm details the existing knowledge that links the initial interaction between a chemical and a biological system, termed the molecular initiating event (MIE), through a series of intermediate events, to an adverse effect. An important example of a well-defined MIE is the formation of a covalent bond between a biological nucleophile and an electrophilic compound. This particular MIE has been associated with various toxicological end points such as acute aquatic toxicity, skin sensitization, and respiratory sensitization. This study has investigated the calculated parameters that are required to predict the rate of chemical bond formation (reactivity) of a dataset of Michael acceptors. Reactivity of these compounds toward glutathione was predicted using a combination of a calculated activation energy value (Eact, calculated using density functional theory (DFT) calculation at the B3YLP/6-31G+(d) level of theory, and solvent-accessible surface area values (SAS) at the α carbon. To further develop the method, a fragment-based algorithm was developed enabling the reactivity to be predicted for Michael acceptors without the need to perform the time-consuming DFT calculations. Results showed the developed fragment method was successful in predicting the reactivity of the Michael acceptors excluding two sets of chemicals: volatile esters with an extended substituent at the β-carbon and chemicals containing a conjugated benzene ring as part of the polarizing group. Additionally the study also demonstrated the ease with which the approach can be extended to other chemical classes by the calculation of additional fragments and their associated Eact and SAS values. The resulting method is likely to be of use in regulatory toxicology tools where an understanding of covalent bond formation as a potential MIE is important within the AOP paradigm.

Density Functional Theory Calculation of pKa's of Thiols in Aqueous Solution Using Explicit Water Molecules and the Polarizable Continuum Model.

PubMed

Thapa, Bishnu; Schlegel, H Bernhard

2016-07-21

The pKa's of substituted thiols are important for understanding their properties and reactivities in applications in chemistry, biochemistry, and material chemistry. For a collection of 175 different density functionals and the SMD implicit solvation model, the average errors in the calculated pKa's of methanethiol and ethanethiol are almost 10 pKa units higher than for imidazole. A test set of 45 substituted thiols with pKa's ranging from 4 to 12 has been used to assess the performance of 8 functionals with 3 different basis sets. As expected, the basis set needs to include polarization functions on the hydrogens and diffuse functions on the heavy atoms. Solvent cavity scaling was ineffective in correcting the errors in the calculated pKa's. Inclusion of an explicit water molecule that is hydrogen bonded with the H of the thiol group (in neutral) or S(-) (in thiolates) lowers error by an average of 3.5 pKa units. With one explicit water and the SMD solvation model, pKa's calculated with the M06-2X, PBEPBE, BP86, and LC-BLYP functionals are found to deviate from the experimental values by about 1.5-2.0 pKa units whereas pKa's with the B3LYP, ωB97XD and PBEVWN5 functionals are still in error by more than 3 pKa units. The inclusion of three explicit water molecules lowers the calculated pKa further by about 4.5 pKa units. With the B3LYP and ωB97XD functionals, the calculated pKa's are within one unit of the experimental values whereas most other functionals used in this study underestimate the pKa's. This study shows that the ωB97XD functional with the 6-31+G(d,p) and 6-311++G(d,p) basis sets, and the SMD solvation model with three explicit water molecules hydrogen bonded to the sulfur produces the best result for the test set (average error -0.11 ± 0.50 and +0.15 ± 0.58, respectively). The B3LYP functional also performs well (average error -1.11 ± 0.82 and -0.78 ± 0.79, respectively).

Serum ischemia modified albumin level and its relationship with the thiol/disulfide balance in placenta percreta patients.

PubMed

Uyanikoglu, Hacer; Sak, Muhammet Erdal; Tatli, Faik; Hilali, Nese Gul; Sak, Sibel; Incebiyik, Adnan; Barut, Mert Ulas; Erel, Ozcan; Gonel, Ataman

2018-06-08

The pathogenesis of placenta percreta (PP) is not very well known. This study was designed to analyse the oxidative stress (OS), the thiol/disulphide balance, and ischaemia-modified albumin (IMA) the women with PP. The study included 38 pregnant women with PP and 40 similarly aged healthy pregnant women in their third trimester of gestation. We measured the IMA, native and total thiols, and disulphide concentrations in the maternal sera of all of the participating women. The IMA levels were higher and the native and total thiols were lower in the PP group than in the control group. However, there was no statistical significance with respect to the thiol/disulphide balance between the two groups. The results of this study suggest that an increase in the ischaemia and OS and a decrease in the antioxidant status may contribute to the pathogenesis of PP. Impact statement What is already known on this subject? Placenta percreta (PP) is a serious complication of pregnancy. Although there are several studies investigating the pathophysiological mechanism of PP, whether the pathology results from a lack of decidua or from the over-invasiveness of trophoblasts remains controversial. The pathology of PP is poorly understood. What do the results of this study add? This prospective study has shown an increased ischaemia modified albumin (IMA) and a decreased antioxidant capacity in the patients with placenta percreta. The results from 38 women with PP suggest that the serum concentrations of IMA and the oxidative stress parameters may be able to predict PP in cases of uncertainty. What are the implications of these findings for clinical practice and/or further research? The implication of these findings shed light on understanding the pathogenesis of PP for further research.

Withaferin A induces apoptosis through the generation of thiol oxidation in human head and neck cancer cells.

PubMed

Park, Jong Won; Min, Kyoung-Jin; Kim, Dong Eun; Kwon, Taeg Kyu

2015-01-01

Withaferin A is a steroidal lactone purified from the Indian medicinal plant, Withania somnifera. Withaferin A has been shown to inhibit the proliferation, metastasis, invasion and angiogenesis of cancer cells. In the present study, we investigated whether withaferin A induces apoptosis in the human head and neck cancer cells, AMC-HN4. Withaferin A markedly increased the sub-G1 cell population and the cleavage of poly(ADP-ribose) polymerase (PARP), which are markers of apoptosis. Pan-caspase inhibitor, z-VAD-fmk (z-VAD), markedly inhibited the withaferin A-induced apoptosis. However, the withaferin A-induced increase in the expression of COX-2 was not affected by treatment with z-VAD. Furthermore, withaferin A upregulated cyclooxygenase-2 (COX-2) expression. The COX-2 inhibitor, NS-398, reduced the withaferin A-induced production of prostaglandin E2. However, treatment with NS-398 did not affect the sub-G1 population and the cleavage of PARP. In addition, the withaferin A-induced apoptosis was independent of reactive oxygen species production. Thiol donors [N-acetylcysteine (NAC) and dithiothreitol (DTT)] reversed withaferin A-induced apoptosis. Therefore, our data suggest that withaferin A induces apoptosis through the mechanism of thiol oxidation in head and neck carcinoma cells.

3D-Hydrogel Based Polymeric Nanoreactors for Silver Nano-Antimicrobial Composites Generation.

PubMed

Soto-Quintero, Albanelly; Romo-Uribe, Ángel; Bermúdez-Morales, Víctor H; Quijada-Garrido, Isabel; Guarrotxena, Nekane

2017-08-01

This study underscores the development of Ag hydrogel nanocomposites, as smart substrates for antibacterial uses, via innovative in situ reactive and reduction pathways. To this end, two different synthetic strategies were used. Firstly thiol-acrylate (PSA) based hydrogels were attained via thiol-ene and radical polymerization of polyethylene glycol (PEG) and polycaprolactone (PCL). As a second approach, polyurethane (PU) based hydrogels were achieved by condensation polymerization from diisocyanates and PCL and PEG diols. In fact, these syntheses rendered active three-dimensional (3D) hydrogel matrices which were used as nanoreactors for in situ reduction of AgNO₃ to silver nanoparticles. A redox chemistry of stannous catalyst in PU hydrogel yielded spherical AgNPs formation, even at 4 °C in the absence of external reductant; and an appropriate thiol-functionalized polymeric network promoted spherical AgNPs well dispersed through PSA hydrogel network, after heating up the swollen hydrogel at 103 °C in the presence of citrate-reductant. Optical and swelling behaviors of both series of hydrogel nanocomposites were investigated as key factors involved in their antimicrobial efficacy over time. Lastly, in vitro antibacterial activity of Ag loaded hydrogels exposed to Pseudomona aeruginosa and Escherichia coli strains indicated a noticeable sustained inhibitory effect, especially for Ag-PU hydrogel nanocomposites with bacterial inhibition growth capabilities up to 120 h cultivation.

Inactivation of thiol-dependent enzymes by hypothiocyanous acid: role of sulfenyl thiocyanate and sulfenic acid intermediates

PubMed Central

Barrett, Tessa J.; Pattison, David I.; Leonard, Stephen E.; Carroll, Kate S.; Davies, Michael J.; Hawkins, Clare L.

2012-01-01

Myeloperoxidase (MPO) forms reactive oxidants including hypochlorous and hypothiocyanous acids (HOCl and HOSCN) under inflammatory conditions. HOCl causes extensive tissue damage and plays a role in the progression of many inflammatory-based diseases. Although HOSCN is a major MPO oxidant, particularly in smokers, who have elevated plasma thiocyanate, the role of this oxidant in disease is poorly characterized. HOSCN induces cellular damage by targeting thiols. However, the specific targets and mechanisms involved in this process are not well defined. We show that exposure of macrophages to HOSCN results in the inactivation of intracellular enzymes, including creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In each case, the active-site thiol residue is particularly sensitive to oxidation, with evidence for reversible inactivation and the formation of sulfenyl thiocyanate and sulfenic acid intermediates, on treatment with HOSCN (less than fivefold molar excess). Experiments with DAz-2, a cell-permeable chemical trap for sulfenic acids, demonstrate that these intermediates are formed on many cellular proteins, including GAPDH and CK, in macrophages exposed to HOSCN. This is the first direct evidence for the formation of protein sulfenic acids in HOSCN-treated cells and highlights the potential of this oxidant to perturb redox signaling processes. PMID:22248862

Identification of novel disulfide adducts between the thiol containing leaving group of the nerve agent VX and cysteine containing tripeptides derived from human serum albumin.

PubMed

Kranawetvogl, Andreas; Küppers, Jim; Gütschow, Michael; Worek, Franz; Thiermann, Horst; Elsinghorst, Paul W; John, Harald

2017-08-01

Chemical warfare agents represent a continuous and considerable threat to military personnel and the civilian population. Such compounds are prohibited by the Chemical Weapons Convention, to which adherence by the member states is strictly controlled. Therefore, reliable analytical methods for verification of an alleged use of banned substances are required. Accordingly, current research focuses on long-term biomarkers derived from covalent adducts with biomolecules such as proteins. Recently, we have introduced a microbore liquid chromatography/electrospray ionization high-resolution tandem mass spectrometry method allowing for the investigation of two different classes of adducts of the nerve agent VX with human serum albumin (HSA). Phosphonylated tyrosine residues and novel disulfide adducts at cysteine residues of HSA were produced by enzymatic cleavage with pronase and detected simultaneously. Notably, the thiol containing leaving group of VX (2-(diisopropylamino)ethanethiol, DPAET) formed disulfide adducts that were released as cysteine and proline containing dipeptides originating from at least two different sites of HSA. Aim of this study was to identify assumed and novel adducts of DPAET with HSA using synthetic peptide reference compounds. Two novel tripeptides were identified representing disulfide adducts with DPAET (Met-Pro-Cys-DPAET, MPC-DPAET and Asp-Ile-Cys-DPAET, DIC-DPAET). MPC-DPAET was shown to undergo partial in-source decay during electrospray ionization for MS detection thereby losing the N-terminal Met residue. This results in the more stable Pro-Cys-DPAET (PC-DPAET) dipeptide detectable as protonated ion. The limit of detection for MPC-DPAET was evaluated, revealing toxicologically relevant VX plasma concentrations. The results provide novel insights into the reactivity of VX and its endogenous targets. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Local Delivery of Gene Vectors From Bare-Metal Stents by Use of a Biodegradable Synthetic Complex Inhibits In-Stent Restenosis in Rat Carotid Arteries

PubMed Central

Fishbein, Ilia; Alferiev, Ivan; Bakay, Marina; Stachelek, Stanley J.; Sobolewski, Peter; Lai, Meizan; Choi, Hoon; Chen, I.-W.; Levy, Robert J.

2012-01-01

Background Local drug delivery from polymer-coated stents has demonstrated efficacy for preventing in-stent restenosis; however, both the inflammatory effects of polymer coatings and concerns about late outcomes of drug-eluting stent use indicate the need to investigate innovative approaches, such as combining localized gene therapy with stent angioplasty. Thus, we investigated the hypothesis that adenoviral vectors (Ad) could be delivered from the bare-metal surfaces of stents with a synthetic complex for reversible vector binding. Methods and Results We synthesized the 3 components of a gene vector binding complex: (1) A polyallylamine bisphosphonate with latent thiol groups (PABT), (2) a polyethyleneimine (PEI) with pyridyldithio groups for amplification of attachment sites [PEI(PDT)], and (3) a bifunctional (amine- and thiol-reactive) cross-linker with a labile ester bond (HL). HL-modified Ad attached to PABT/PEI(PDT)-treated steel surfaces demonstrated both sustained release in vitro over 30 days and localized green fluorescent protein expression in rat arterial smooth muscle cell cultures, which were not sensitive to either inhibition by neutralizing anti-Ad antibodies or inactivation after storage at 37°C. In rat carotid studies, deployment of steel stents configured with PABT/PEI(PDT)/HL-tethered adenoviral vectors demonstrated both site-specific arterial AdGFP expression and adenovirus-luciferase transgene activity per optical imaging. Rat carotid stent delivery of adenovirus encoding inducible nitric oxide synthase resulted in significant inhibition of restenosis. Conclusions Reversible immobilization of adenovirus vectors on the bare-metal surfaces of endovascular stents via a synthetic complex represents an efficient, tunable method for sustained release of gene vectors to the vasculature. PMID:18413497

Effectiveness of γ-oryzanol in reducing neuromotor deficits, dopamine depletion and oxidative stress in a Drosophila melanogaster model of Parkinson's disease induced by rotenone.

PubMed

Araujo, Stífani Machado; de Paula, Mariane Trindade; Poetini, Marcia Rósula; Meichtry, Luana; Bortolotto, Vandreza Cardoso; Zarzecki, Micheli Stefani; Jesse, Cristiano Ricardo; Prigol, Marina

2015-12-01

The γ-orizanol present in rice bran oil contains a mix of steryl triterpenyl esters of ferulic acid, which is believed to be linked to its antioxidant potential. In this study we investigated the neuroprotective actions of γ-orizanol (ORY) against the toxicity induced by rotenone (ROT) in Drosophila melanogaster. The flies (both genders) aged between 1 and 5 days old were divided into four groups of 50 flies each: (1) control, (2) ORY 25 μM, (3) ROT 500 μM, (4) ORY 25 μM+ROT 500 μM. Flies were concomitantly exposed to a diet containing ROT and ORY for 7 days according to their respective groups. Survival and behavior analyses were carried out in vivo, and ex vivo analyses involved acetylcholinesterase activity (AChE), determination of dopaminergic levels, cellular viability and mitochondrial viability, activities of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), reactive species levels (RS), lipid peroxidation (TBARS) and contents of total thiols and non-proteic thiols (NPSH). Our results show for the first time that ORY not only acts as an endogenous activator of the cellular antioxidant defenses, but it also ameliorates rotenone induced mortality, oxidative stress and mitochondrial dysfunction. Our salient findings regarded the restoration of cholinergic deficits, dopamine levels and improved motor function provided by ORY. These results demonstrate the neuroprotective potential of ORY and that this effect can be potentially due to its antioxidant action. In conclusion, the present results show that ORY is effective in reducing the ROT induced toxicity in D. melanogaster, which showed a neuroprotective action, possibly due to the presence of the antioxidant constituents such as the ferulic acid. Copyright © 2015. Published by Elsevier B.V.

Identification of a protein with antioxidant activity that is important for the protection against beer ageing.

PubMed

Wu, Ming J; Clarke, Frank M; Rogers, Peter J; Young, Paul; Sales, Narelle; O'Doherty, Patrick J; Higgins, Vincent J

2011-01-01

This study was carried out with fresh Australian lager beer which was sampled directly off the production line, the same samples aged for 12 weeks at 30 °C, and the vintage beer which was kept at 20 °C for 5 years. Characteristic Australian lager flavour was maintained in the fresh and vintage beers but was lost in the aged beer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and free thiol group labelling analyses of beer proteins found that this flavour stability correlated with the presence of an unknown 10 kilodaltons (kDa) protein with a higher level of free thiols. The protein was purified by size-exclusion chromatography, then peptide sequencing and database matching identified it as the barley lipid transfer protein (LTP1). Further characterisation using diphenylpicrylhydrazyl (DPPH) free radical scavenging and a Saccharomyces cerevisiae-based antioxidant screening assay demonstrated that the LTP1 protein was active in DPPH reduction and antioxidant activity. The absence of free thiol in the aged beer indicates that the thiol functional groups within the LTP1 protein were saturated and suggests that it is important in the flavour stability of beer by maintaining reduction capacity during the ageing process.

The chemical foundations of nitroalkene fatty acid signaling through addition reactions with thiols.

PubMed

Turell, Lucía; Steglich, Martina; Alvarez, Beatriz

2018-03-22

Nitroalkene fatty acids can be formed in vivo and administered exogenously. They exert pleiotropic signaling actions with cytoprotective and antiinflammatory effects. The presence of the potent electron withdrawing nitro group confers electrophilicity to the adjacent β-carbon. Thiols (precisely, thiolates) are strong nucleophiles and can react with nitroalkene fatty acids through reversible Michael addition reactions. In addition, nitroalkene fatty acids can undergo several other processes including metabolic oxidation, reduction, esterification, nitric oxide release and partition into hydrophobic compartments. The signaling actions of nitroalkenes are mainly mediated by reactions with critical thiols in regulatory proteins. Thus, the thio-Michael addition reaction provides a framework for understanding the molecular basis of the biological effects of nitroalkene fatty acids at the crossroads of thiol signaling and electrophilic lipid signaling. In this review, we describe the reactions of nitroalkene fatty acids in biological contexts. We focus on the Michael addition-elimination reaction with thiols and its mechanism, and extrapolate kinetic and thermodynamic considerations to in vivo settings. Copyright © 2018 Elsevier Inc. All rights reserved.

Striped gold nanoparticles: New insights from molecular dynamics simulations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Velachi, Vasumathi, E-mail: vasuphy@gmail.com; Cordeiro, M. Natália D. S., E-mail: ncordeir@fc.up.pt; Bhandary, Debdip

Recent simulations have improved our knowledge of the molecular-level structure and hydration properties of mixed self-assembled monolayers (SAMs) with equal and unequal alkyl thiols at three different arrangements, namely, random, patchy, and Janus. In our previous work [V. Vasumathi et al., J. Phys. Chem. C 119, 3199–3209 (2015)], we showed that the bending of longer thiols over shorter ones clearly depends on the thiols’ arrangements and chemical nature of their terminal groups. In addition, such a thiol bending revealed to have a strong impact on the structural and hydration properties of SAMs coated on gold nanoparticles (AuNPs). In this paper,more » we extend our previous atomistic simulation study to investigate the bending of longer thiols by increasing the stripe thickness of mixed SAMs of equal and unequal lengths coated on AuNPs. We study also the effect of stripe thickness on the structural morphology and hydration of the coated SAMs. Our results show that the structural and hydration properties of SAMs are affected by the stripe thickness for mixtures of alkyl thiols with unequal chain length but not for equal length. Hence, the stability of the stripe configuration depends on the alkyl’s chain length, the length difference between the thiol mixtures, and solvent properties.« less

Formation of mercury sulfide from Hg(II)−thiolate complexes in natural organic matter

USGS Publications Warehouse

Alain Manceau,; Cyprien Lemouchi,; Mironel Enescu,; Anne-Claire Gaillot,; Martine Lanson,; Valerie Magnin,; Pieter Glatzel,; Poulin, Brett; Ryan, Joseph N.; Aiken, George R.; Isabelle Gautier-Lunea,; Kathryn L. Nagy,

2015-01-01

Methylmercury is the environmental form of neurotoxic mercury that is biomagnified in the food chain. Methylation rates are reduced when the metal is sequestered in crystalline mercury sulfides or bound to thiol groups in macromolecular natural organic matter. Mercury sulfide minerals are known to nucleate in anoxic zones, by reaction of the thiol-bound mercury with biogenic sulfide, but not in oxic environments. We present experimental evidence that mercury sulfide forms from thiol-bound mercury alone in aqueous dark systems in contact with air. The maximum amount of nanoparticulate mercury sulfide relative to thiol-bound mercury obtained by reacting dissolved mercury and soil organic matter matches that detected in the organic horizon of a contaminated soil situated downstream from Oak Ridge, TN, in the United States. The nearly identical ratios of the two forms of mercury in field and experimental systems suggest a common reaction mechanism for nucleating the mineral. We identified a chemical reaction mechanism that is thermodynamically favorable in which thiol-bound mercury polymerizes to mercury–sulfur clusters. The clusters form by elimination of sulfur from the thiol complexes via breaking of mercury–sulfur bonds as in an alkylation reaction. Addition of sulfide is not required. This nucleation mechanism provides one explanation for how mercury may be immobilized, and eventually sequestered, in oxygenated surface environments.

Fluorimetric determination of some thiol compounds in their dosage forms.

PubMed

Al-Ghannam, Sh M; El-Brashy, A M; Al-Farhan, B S

2002-08-01

A simple fluorimetric procedure was adopted for determination of three pharmaceutical compounds containing thiol groups namely, captopril, D-penicillamine and N-acetylcysteine. In this method, the drugs are treated with 1,2-naphthoquinone-4-sulfonic acid. The latter is reduced to 1,2-dihydroxynaphthalene-4-sulfonic acid which has a maximum fluorescence intensity at 480/318 nm (lambdaEm/Ex). The method is sensitive to 0.5-4.5 pg ml(- 1) with minimum detectability 0.05 microg ml(-1) (S/N = 2), and has been applied to determine these three thiols in their dosage forms. The results obtained are compared favourably with those obtained by their pharmacopeial methods.

Structure- and reactivity-based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor (EGFR).

PubMed

Ward, Richard A; Anderton, Mark J; Ashton, Susan; Bethel, Paul A; Box, Matthew; Butterworth, Sam; Colclough, Nicola; Chorley, Christopher G; Chuaqui, Claudio; Cross, Darren A E; Dakin, Les A; Debreczeni, Judit É; Eberlein, Cath; Finlay, M Raymond V; Hill, George B; Grist, Matthew; Klinowska, Teresa C M; Lane, Clare; Martin, Scott; Orme, Jonathon P; Smith, Peter; Wang, Fengjiang; Waring, Michael J

2013-09-12

A novel series of small-molecule inhibitors has been developed to target the double mutant form of the epidermal growth factor receptor (EGFR) tyrosine kinase, which is resistant to treatment with gefitinib and erlotinib. Our reported compounds also show selectivity over wild-type EGFR. Guided by molecular modeling, this series was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and demonstrates high levels of activity in cellular models of the double mutant form of EGFR. In addition, these compounds show significant activity against the activating mutations, which gefitinib and erlotinib target and inhibition of which gives rise to their observed clinical efficacy. A glutathione (GSH)-based assay was used to measure thiol reactivity toward the electrophilic functionality of the inhibitor series, enabling both the identification of a suitable reactivity window for their potency and the development of a reactivity quantitative structure-property relationship (QSPR) to support design.

The basics of thiols and cysteines in redox biology and chemistry.

PubMed

Poole, Leslie B

2015-03-01

Cysteine is one of the least abundant amino acids, yet it is frequently found as a highly conserved residue within functional (regulatory, catalytic, or binding) sites in proteins. It is the unique chemistry of the thiol or thiolate group of cysteine that imparts to functional sites their specialized properties (e.g., nucleophilicity, high-affinity metal binding, and/or ability to form disulfide bonds). Highlighted in this review are some of the basic biophysical and biochemical properties of cysteine groups and the equations that apply to them, particularly with respect to pKa and redox potential. Also summarized are the types of low-molecular-weight thiols present in high concentrations in most cells, as well as the ways in which modifications of cysteinyl residues can impart or regulate molecular functions important to cellular processes, including signal transduction. Copyright © 2014 Elsevier Inc. All rights reserved.

Thiol Versus Hydroxamate as Zinc Binding Group In HDAC Inhibition: An Ab Initio QM/MM Molecular Dynamics Study

PubMed Central

Gong, Wenjing; Wu, Ruibo; Zhang, Yingkai

2015-01-01

Zinc-dependent histone deacetylases (HDACs) play a critical role in transcriptional repression and gene silencing, and are among the most attractive targets for the development of new therapeutics against cancer and various other diseases. Two HDAC inhibitors have been approved by FDA as anti-cancer drugs: one is SAHA whose hydroxamate is directly bound to zinc, the other is FK228 whose active form may use thiol as the zinc binding group. In spite of extensive studies, it remains to be ambiguous regarding how thiol and hydroxamate are bound to the zinc active site of HDACs. In this work, our computational approaches center on Born-Oppenheimer ab initio quantum mechanical/molecular mechanical (QM/MM) molecular dynamics with umbrella sampling, which allow for modeling of the zinc active site with reasonable accuracy while properly including dynamics and effects of protein environment. Meanwhile, an improved short-long effective function (SLEF2) to describe non-bonded interactions between zinc and other atoms has been employed in initial MM equilibrations. Our ab initio QM/MM MD simulations have confirmed that hydroxamate is neutral when it is bound to HDAC8, and found that thiol is deprotonated when directly bound to zinc in the HDAC active site. By comparing thiol and hydroxamate, our results elucidated the differences in their binding environment in the HDAC active sites, and emphasized the importance of the linker design to achieve more specific binding towards class IIa HDACs. PMID:26452222

Thiol versus hydroxamate as zinc binding group in HDAC inhibition: An ab initio QM/MM molecular dynamics study.

PubMed

Gong, Wenjing; Wu, Ruibo; Zhang, Yingkai

2015-11-15

Zinc-dependent histone deacetylases (HDACs) play a critical role in transcriptional repression and gene silencing, and are among the most attractive targets for the development of new therapeutics against cancer and various other diseases. Two HDAC inhibitors have been approved by FDA as anti-cancer drugs: one is SAHA whose hydroxamate is directly bound to zinc, the other is FK228 whose active form may use thiol as the zinc binding group. In spite of extensive studies, it remains to be ambiguous regarding how thiol and hydroxamate are bound to the zinc active site of HDACs. In this work, our computational approaches center on Born-Oppenheimer ab initio quantum mechanical/molecular mechanical (QM/MM) molecular dynamics with umbrella sampling, which allow for modeling of the zinc active site with reasonable accuracy while properly including dynamics and effects of protein environment. Meanwhile, an improved short-long effective function (SLEF2) to describe non-bonded interactions between zinc and other atoms has been employed in initial MM equilibrations. Our ab initio QM/MM MD simulations have confirmed that hydroxamate is neutral when it is bound to HDAC8, and found that thiol is deprotonated when directly bound to zinc in the HDAC active site. By comparing thiol and hydroxamate, our results elucidated the differences in their binding environment in the HDAC active sites, and emphasized the importance of the linker design to achieve more specific binding toward class IIa HDACs. © 2015 Wiley Periodicals, Inc.

Ester-free cross-linker molecules for ultraviolet-light-cured polysilsesquioxane gate dielectric layers of organic thin-film transistors

NASA Astrophysics Data System (ADS)

Okada, Shuichi; Nakahara, Yoshio; Uno, Kazuyuki; Tanaka, Ichiro

2018-04-01

Pentacene thin-film transistors (TFTs) were fabricated with ultraviolet-light (UV)-cured polysilsesquioxane (PSQ) gate dielectric layers using cross-linker molecules with or without ester groups. To polymerize PSQ without ester groups, thiol-ene reaction was adopted. The TFTs fabricated with PSQ layers comprising ester-free cross-linkers showed a higher carrier mobility than the TFTs with PSQ layers cross-linked with ester groups, which had large electric dipole moments that limited the carrier mobility. It was demonstrated that the thiol-ene reaction is more suitable than the conventional radical reaction for UV-cured PSQ with small dielectric constant.

Comparative Study of Protein Immobilization Properties on Calixarene Monolayers

PubMed Central

Chen, Hongxia; Lee, Minsu; Choi, Sungwook; Kim, Jae-Ho; Choi, Heung-Jin; Kim, Sung-Hoon; Lee, Jeabeom; Koh, Kwangnak

2007-01-01

Three calix[4]arene (Cal-4) derivatives of which contain ethylester (1), carboxylic acid (2), and crownether (3) at the lower rim with a common reactive thiol at the upper rim were synthesized and constructed to self-assembled monolayers (SAMs) on Au films. After spectroscopic characterization of monolayers, the interaction between Cal-4 and surface confined bovine serum albumin (BSA) in the SAMs was analyzed by surface plasmon resonance (SPR). The estimated surface concentration of BSA on the Cal-4 SAM with crownether group was the highest among the three Cal-4 derivatives. Anti-hIgG and hIgG pair was employed for the investigation of protein-protein interaction. Molecular interaction between anti-hIgG and hIgG can be detected in a concentration range of 10 pg/mL to 200 pg/mL on the Cal-4 derivative 3 SAM modified SPR chip.

Dual-Responsive Molecular Probe for Tumor Targeted Imaging and Photodynamic Therapy

PubMed Central

Meng, Xiaoqing; Yang, Yueting; Zhou, Lihua; Zhang, li; Lv, Yalin; Li, Sanpeng; Wu, Yayun; Zheng, Mingbin; Li, Wenjun; Gao, Guanhui; Deng, Guanjun; Jiang, Tao; Ni, Dapeng; Gong, Ping; Cai, Lintao

2017-01-01

The precision oncology significantly relies on the development of multifunctional agents to integrate tumor targeting, imaging and therapeutics. In this study, a first small-molecule theranostic probe, RhoSSCy is constructed by conjugating 5′-carboxyrhodamines (Rho) and heptamethine cyanine IR765 (Cy) using a reducible disulfide linker and pH tunable amino-group to realize thiols/pH dual sensing. In vitro experiments verify that RhoSSCy is highly sensitive for quantitative analysis and imaging intracellular pH gradient and biothiols. Furthermore, RhoSSCy shows superb tumor targeted dual-modal imaging via near-infrared fluorescence (NIRF) and photoacoustic (PA). Importantly, RhoSSCy also induces strongly reactive oxygen species for tumor photodynamic therapy (PDT) with robust antitumor activity both in vitro and in vivo. Such versatile small-molecule theranostic probe may be promising for tumor targeted imaging and precision therapy. PMID:28638467

A validated HPLC-MS/MS assay for quantifying unstable pharmacologically active metabolites of clopidogrel in human plasma: application to a clinical pharmacokinetic study.

PubMed

Furlong, Michael T; Savant, Ishani; Yuan, Moucun; Scott, Laura; Mylott, William; Mariannino, Thomas; Kadiyala, Pathanjali; Roongta, Vikram; Arnold, Mark E

2013-05-01

Clopidogrel is prescribed for the treatment of Acute Coronary Syndrome and recent myocardial infarction, recent stroke, or established peripheral arterial disease. A sensitive and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay was developed and validated to enable reliable quantification of four diastereomeric and chemically reactive thiol metabolites, two of which are pharmacologically active, in human plasma. The metabolites were stabilized by alkylation of their reactive thiol moieties with 2-bromo-3'-methoxyacetophenone (MPB). Following organic solvent mediated-protein precipitation in a 96-well plate format, chromatographic separation was achieved by gradient elution on an Ascentis Express RP-amide column. Chromatographic conditions were optimized to ensure separation of the four derivatized active metabolites. Derivatized metabolites and stable isotope-labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The HPLC-MS/MS assay was validated over concentration ranges of 0.125-125 ng/mL for metabolites H1-H3 and 0.101-101 ng/mL for H4. Intra- and inter-assay precision values for replicate quality control samples were within 14.3% for all analytes during the assay validation. Mean quality control accuracy values were within ±6.3% of nominal values for all analytes. Assay recoveries were high (>79%). The four derivatized analytes were stable in human blood for at least 2 h at room temperature and on ice. The analytes were also stable in human plasma for at least 25 h at room temperature, 372 days at -20 °C and -70 °C, and following at least five freeze-thaw cycles. The validated assay was successfully applied to the quantification of all four thiol metabolites in human plasma in support of a human pharmacokinetic study. Copyright © 2013 Elsevier B.V. All rights reserved.

Site-directed mutagenesis of mouse glutathione transferase P1-1 unlocks masked cooperativity, introduces a novel mechanism for 'ping pong' kinetic behaviour, and provides further structural evidence for participation of a water molecule in proton abstraction from glutathione.

PubMed

McManus, Gavin; Costa, Marta; Canals, Albert; Coll, Miquel; Mantle, Timothy J

2011-01-01

Mouse liver glutathione transferase P1-1 has three cysteine residues at positions 14, 47 and 169. We have constructed the single, double and triple cysteine to alanine mutants to define the behaviour of all three thiols. We confirm that C47 is the 'fast' thiol (pK 7.4), and define C169 as the alkaline reactive residue with a pK(a) of 8.6. Only a small proportion of C14 is reactive with 5,5'-dithiobis-(2-nitrobenoic acid) (DTNB) at pH 9 in the C47A/C169A double mutant. The native enzyme and the C169A mutant exhibited Michaelis-Menten kinetics, but all other thiol to alanine mutants exhibited sigmoidal kinetics to varying degrees. The C169A mutant exhibited 'ping pong' kinetics, consistent with a mechanism whereby liberation of a proton from a reduced enzyme-glutathione (GSH) complex to form an enzyme-GS(-) (unprotonated) complex is essentially irreversible. Intriguingly, similar behaviour has recently been reported for a mutant of the yeast prion Ure2p. This cooperative behaviour is 'mirrored' in the crystal structure of the C47A mutant, which binds the p-nitrobenzyl moiety of p-nitrobenzyglutathione in distinct orientations in the two crystallographic subunits. The asymmetry seen in this structure for product binding is associated with absence of a water molecule W0 in the standard wild-type conformation of product binding that is clearly identifiable in the new structure, which may represent a structural model for binding of incoming GSH prior to displacement of W0. Elimination of W0 as a hydroxonium ion may be the mechanism for the initial proton extrusion from the active site. © 2010 The Authors Journal compilation © 2010 FEBS.

The Intracellular Redox Stress Caused by Hexavalent Chromium is Selective for Proteins that Have Key Roles in Cell Survival and Thiol Redox Control

PubMed Central

Myers, Judith M.; Antholine, William E.; Myers, Charles R.

2011-01-01

Hexavalent chromium [Cr(VI)] compounds (e.g. chromates) are strong oxidants that readily enter cells where they are reduced to reactive Cr intermediates that can directly oxidize some cell components and can promote the generation of reactive oxygen and nitrogen species. Inhalation is a major route of exposure which directly exposes the bronchial epithelium. Previous studies with non-cancerous human bronchial epithelial cells (BEAS-2B) demonstrated that Cr(VI) treatment results in the irreversible inhibition of thioredoxin reductase (TrxR) and the oxidation of thioredoxins (Trx) and peroxiredoxins (Prx). The mitochondrial Trx/Prx system is somewhat more sensitive to Cr(VI) than the cytosolic Trx/Prx system, and other redox-sensitive mitochondrial functions are subsequently affected including electron transport complexes I and II. Studies reported here show that Cr(VI) does not cause indiscriminant thiol oxidation, and that the Trx/Prx system is among the most sensitive of cellular protein thiols. Trx/Prx oxidation is not unique to BEAS-2B cells, as it was also observed in primary human bronchial epithelial cells. Increasing the intracellular levels of ascorbate, an endogenous Cr(VI) reductant, did not alter the effects on TrxR, Trx, or Prx. The peroxynitrite scavenger MnTBAP did not protect TrxR, Trx, Prx, or the electron transport chain from the effects of Cr(VI), implying that peroxynitrite is not required for these effects. Nitration of tyrosine residues of TrxR was not observed following Cr(VI) treatment, further ruling out peroxynitrite as a significant contributor to the irreversible inhibition of TrxR. Cr(VI) treatments that disrupt the TrxR/Trx/Prx system did not cause detectable mitochondrial DNA damage. Overall, the redox stress that results from Cr(VI) exposure shows selectivity for key proteins which are known to be important for redox signaling, antioxidant defense, and cell survival. PMID:21237240

Thiol/disulfide homeostasis in pregnant women with obstructive sleep apnea syndrome.

PubMed

Üstündağ, Yasemin; Demirci, Hakan; Balık, Rifat; Erel, Ozcan; Özaydın, Fahri; Kücük, Bilgen; Ertaş, Dilber; Ustunyurt, Emin

2017-11-27

Repetitive episodes of hypoxia and reoxygenation during sleep in patients with obstructive sleep apnea syndrome (OSAS) resemble an ischemia-reperfusion injury. We aimed to test the hypothesis that oxidative stress occurs in pregnant women with OSAS. We also aimed to compare thiol/disulfide homeostasis with ischemia-modified albumin (IMA) and total antioxidant capacity (TAC) as markers of ischemia-reperfusion injury in pregnant women with and without OSAS and healthy control. This study included 29 pregnant women with OSAS, 30 women without OSAS in the third trimester applying for periodic examinations, and 30 healthy women. Serum IMA and TAC (using the ferric reducing power of plasma method) were measured. Serum thiol/disulfide homeostasis was determined by a novel automated method. The mean age of the pregnant women with OSAS was 31.0 ± 4.7 years with a mean gestational age of 36.5 ± 3.0 weeks. The mean age of pregnant women without OSAS was 29.8 ± 4.9 years with a mean gestational age of 36.9 ± 2.7 weeks. The mean age of the nonpregnant control group was 29.7 ± 6.4 years. Both native thiol (291 ± 29 μmol/L versus 314 ± 30 μmol/L; p = .018) and total thiol (325 ± 32 versus 350 ± 32, p = .025) levels were lower in pregnant women with OSAS compared to pregnant women without OSAS, respectively (p < .01). This is the first study demonstrating the thiol/disulfide homeostasis in pregnant women with OSAS. Native thiol and total thiol levels were lower in pregnant women with OSAS compared to those without OSAS. However, dynamic thiol/disulfide homeostasis parameters cannot provide valuable information to discriminate OSAS in pregnant women.

Click-PEGylation - A mobility shift approach to assess the redox state of cysteines in candidate proteins.

PubMed

van Leeuwen, Lucie A G; Hinchy, Elizabeth C; Murphy, Michael P; Robb, Ellen L; Cochemé, Helena M

2017-07-01

The redox state of cysteine thiols is critical for protein function. Whereas cysteines play an important role in the maintenance of protein structure through the formation of internal disulfides, their nucleophilic thiol groups can become oxidatively modified in response to diverse redox challenges and thereby function in signalling and antioxidant defences. These oxidative modifications occur in response to a range of agents and stimuli, and can lead to the existence of multiple redox states for a given protein. To assess the role(s) of a protein in redox signalling and antioxidant defence, it is thus vital to be able to assess which of the multiple thiol redox states are present and to investigate how these alter under different conditions. While this can be done by a range of mass spectrometric-based methods, these are time-consuming, costly, and best suited to study abundant proteins or to perform an unbiased proteomic screen. One approach that can facilitate a targeted assessment of candidate proteins, as well as proteins that are low in abundance or proteomically challenging, is by electrophoretic mobility shift assays. Redox-modified cysteine residues are selectively tagged with a large group, such as a polyethylene glycol (PEG) polymer, and then the proteins are separated by electrophoresis followed by immunoblotting, which allows the inference of redox changes based on band shifts. However, the applicability of this method has been impaired by the difficulty of cleanly modifying protein thiols by large PEG reagents. To establish a more robust method for redox-selective PEGylation, we have utilised a Click chemistry approach, where free thiol groups are first labelled with a reagent modified to contain an alkyne moiety, which is subsequently Click-reacted with a PEG molecule containing a complementary azide function. This strategy can be adapted to study reversibly reduced or oxidised cysteines. Separation of the thiol labelling step from the PEG conjugation greatly facilitates the fidelity and flexibility of this approach. Here we show how the Click-PEGylation technique can be used to interrogate the redox state of proteins. Copyright © 2017. Published by Elsevier Inc.

Metal-chelating polymers by anionic ring-opening polymerization and their use in quantitative mass cytometry.

PubMed

Illy, Nicolas; Majonis, Daniel; Herrera, Isaac; Ornatsky, Olga; Winnik, Mitchell A

2012-08-13

Metal-chelating polymers (MCPs) are important reagents for multiplexed immunoassays based on mass cytometry. The role of the polymer is to carry multiple copies of individual metal isotopes, typically as lanthanide ions, and to provide a reactive functionality for convenient attachment to a monoclonal antibody (mAb). For this application, the optimum combination of chain length, backbone structure, end group, pendant groups, and synthesis strategy has yet to be determined. Here we describe the synthesis of a new type of MCP based on anionic ring-opening polymerization of an activated cyclopropane (the diallyl ester of 1,1-cyclopropane dicarboxylic acid) using a combination of 2-furanmethanethiol and a phosphazene base as the initiator. This reaction takes place with rigorous control over molecular weight, yielding a polymer with a narrow molecular weight distribution, reactive pendant groups for introducing a metal chelator, and a functional end group with orthogonal reactivity for attaching the polymer to the mAbs. Following the ring-opening polymerization, a two-step transformation introduced diethylenetriaminepentaacetic acid (DTPA) chelating groups on each pendant group. The polymers were characterized by NMR, size exclusion chromatography (SEC), and thermogravimetric analysis (TGA). The binding properties toward Gd(3+) as a prototypical lanthanide (Ln) ion were also studied by isothermal titration calorimetry (ITC). Attachment to a mAb involves a Diels-Alder reaction of the terminal furan with a bismaleimide, followed by a Michael addition of a thiol on the mAb, generated by mild reduction of a disulfide bond in the hinge region. Polymer samples with a number average degree of polymerization of 35, with a binding capacity of 49.5 ± 6 Ln(3+) ions per chain, were loaded with 10 different types of Ln ions and conjugated to 10 different mAbs. A suite of metal-tagged Abs was tested by mass cytometry in a 10-plex single cell analysis of human adult peripheral blood, allowing us to quantify the antibody binding capacity of 10 different cell surface antigens associated with specific cell types.

Synthesis of a novel poly-thiolated magnetic nano-platform for heavy metal adsorption. Role of thiol and carboxyl functions

NASA Astrophysics Data System (ADS)

Odio, Oscar F.; Lartundo-Rojas, Luis; Palacios, Elia Guadalupe; Martínez, Ricardo; Reguera, Edilso

2016-11-01

We report a novel strategy for the synthesis of magnetic nano-platforms containing free thiol groups. It first involves the synthesis of a poly(acrylic acid) copolymer containing disulfide bridges between the linear chains through di-ester linkages, followed by the anchoring of this new ligand to magnetite nanoparticles using a ligand exchange reaction. Finally, free sbnd SH groups are obtained by treating the resulting disulfide-functionalized magnetic nano-system with tributyl phosphine as reducing agent. The characterization of the resulting 17 nm nanoparticles (Fe3O4@PAA-HEDred) by FTIR and TGA confirms the attachment of the copolymer through iron carboxylates. XRD, TEM and magnetic measurements indicate an increase in the inorganic core diameter and the occurrence of strong magnetic inter-particle interactions during the exchange reaction, although coercitivity and remanence drop to near zero at room temperature. Afterwards, Fe3O4@PAA-HEDred nanoparticles were tested as sorbent for Pb2+ and Cd2+ cations in aqueous media. XPS measurements were performed in order to unravel the role of both carboxyl and thiol functions in the adsorption process. For the sake of comparison, the same study was performed using bare Fe3O4 nanoparticles and a nanosystem with disulfide groups (Fe3O4@DMSA). The joint analysis of the Pb 4f, Cd 3d, Fe 2p and S 2p high resolution spectra for the nanostructured materials indicates that metal-sulfur interactions are dominant if free sbnd SH groups are present, but if not, the main adsorption route entails metal-carboxyl interactions. Even in presence of unbound thiol moieties, carboxyl groups participate due to favoured steric availability.

Spatial proximity and sequence localization of the reactive sulfhydryls of porphobilinogen synthase.

PubMed Central

Markham, G. D.; Myers, C. B.; Harris, K. A.; Volin, M.; Jaffe, E. K.

1993-01-01

The zinc metalloenzyme porphobilinogen synthase (PBGS) contains several functionally important, but previously unidentified, reactive sulfhydryl groups. The enzyme has been modified with the reversible sulfhydryl-specific nitroxide spin label derivative of methyl methanethiosulfonate (MMTS), (1-oxyl-2,2,5,5-tetramethyl-delta 3-pyrroline-3-methyl)methanethiosulfonate (SL-MMTS) (Berliner, L. J., Grunwald, J., Hankovszky, H. O., & Hideg, K., 1982, Anal. Biochem. 119, 450-455). EPR spectra show that SL-MMTS labels three groups per PBGS subunit (24 per octamer), as does MMTS. EPR signals reflecting nitroxides of different mobilities are observed. Two of the three modified cysteines have been identified as Cys-119 and Cys-223 by sequencing peptides produced by an Asp-N protease digest of the modified protein. Because MMTS-reactive thiols have been implicated as ligands to the required Zn(II), EPR spectroscopy has been used to determine the spatial proximity of the modified cysteine residues. A forbidden (delta m = 2) EPR transition is observed indicating a through-space dipolar interaction between at least two of the nitroxides. The relative intensity of the forbidden and allowed transitions show that at least two of the unpaired electrons are within at most 7.6 A of each other. SL-MMTS-modified PBGS loses all Zn(II) and cannot catalyze product formation. The modified enzyme retains the ability to bind one of the two substrates at each active site. Binding of this substrate has no influence on the EPR spectral properties of the spin-labeled enzyme, or on the rate of release of the nitroxides when 2-mercaptoethanol is added.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8382991

An Alternative Thiol-Reactive Dye to Analyze Ligand Interactions with the Chemokine Receptor CXCR2 Using a New Thermal Shift Assay Format.

PubMed

Bergsdorf, Christian; Fiez-Vandal, Cédric; Sykes, David A; Bernet, Pascal; Aussenac, Sonia; Charlton, Steven J; Schopfer, Ulrich; Ottl, Johannes; Duckely, Myriam

2016-03-01

Integral membrane proteins (IMPs) play an important role in many cellular events and are involved in numerous pathological processes. Therefore, understanding the structure and function of IMPs is a crucial prerequisite to enable successful targeting of these proteins with low molecular weight (LMW) ligands early on in the discovery process. To optimize IMP purification/crystallization and to identify/characterize LMW ligand-target interactions, robust, reliable, high-throughput, and sensitive biophysical methods are needed. Here, we describe a differential scanning fluorimetry (DSF) screening method using the thiol-reactive BODIPY FL-cystine dye to monitor thermal unfolding of the G-protein-coupled receptor (GPCR), CXCR2. To validate this method, the seven-transmembrane protein CXCR2 was analyzed with a set of well-characterized antagonists. This study showed that the new DSF assay assessed reliably the stability of CXCR2 in a 384-well format. The analysis of 14 ligands with a potency range over 4 log units demonstrated the detection/characterization of LMW ligands binding to the membrane protein target. Furthermore, DSF results cross-validated with the label-free differential static light scattering (DSLS) thermal denaturation method. These results underline the potential of the BODIPY assay format as a general tool to investigate membrane proteins and their interaction partners. © 2015 Society for Laboratory Automation and Screening.

Nitrosative damage to free and zinc-bound cysteine thiols underlies nitric oxide toxicity in wild-type Borrelia burgdorferi

PubMed Central

Bourret, Travis J; Boylan, Julie A; Lawrence, Kevin A; Gherardini, Frank C

2011-01-01

Borrelia burgdorferi encounters potentially harmful reactive nitrogen species (RNS) throughout its infective cycle. In this study, diethylamine NONOate (DEA/NO) was used to characterize the lethal effects of RNS on B. burgdorferi. RNS produce a variety of DNA lesions in a broad spectrum of microbial pathogens; however, levels of the DNA deamination product, deoxyinosine, and the numbers of apurinic/apyrimidinic (AP) sites were identical in DNA isolated from untreated and DEA/NO-treated B. burgdorferi cells. Strains with mutations in the nucleotide excision repair (NER) pathway genes uvrC or uvrB treated with DEA/NO had significantly higher spontaneous mutation frequencies, increased numbers of AP sites in DNA and reduced survival compared with wild-type controls. Polyunsaturated fatty acids in B. burgdorferi cell membranes, which are susceptible to peroxidation by reactive oxygen species (ROS), were not sensitive to RNS-mediated lipid peroxidation. However, treatment of B. burgdorferi cells with DEA/NO resulted in nitrosative damage to several proteins, including the zinc-dependent glycolytic enzyme fructose-1,6-bisphosphate aldolase (BB0445), the Borrelia oxidative stress regulator (BosR) and neutrophil-activating protein (NapA). Collectively, these data suggested that nitrosative damage to proteins harbouring free or zinc-bound cysteine thiols, rather than DNA or membrane lipids underlies RNS toxicity in wild-type B. burgdorferi. PMID:21564333

Sulfur Denitrosylation by an Engineered Trx-like DsbG Enzyme Identifies Nucleophilic Cysteine Hydrogen Bonds as Key Functional Determinant.

PubMed

Lafaye, Céline; Van Molle, Inge; Tamu Dufe, Veronica; Wahni, Khadija; Boudier, Ariane; Leroy, Pierre; Collet, Jean-François; Messens, Joris

2016-07-15

Exposure of bacteria to NO results in the nitrosylation of cysteine thiols in proteins and low molecular weight thiols such as GSH. The cells possess enzymatic systems that catalyze the denitrosylation of these modified sulfurs. An important player in these systems is thioredoxin (Trx), a ubiquitous, cytoplasmic oxidoreductase that can denitrosylate proteins in vivo and S-nitrosoglutathione (GSNO) in vitro However, a periplasmic or extracellular denitrosylase has not been identified, raising the question of how extracytoplasmic proteins are repaired after nitrosative damage. In this study, we tested whether DsbG and DsbC, two Trx family proteins that function in reducing pathways in the Escherichia coli periplasm, also possess denitrosylating activity. Both DsbG and DsbC are poorly reactive toward GSNO. Moreover, DsbG is unable to denitrosylate its specific substrate protein, YbiS. Remarkably, by borrowing the CGPC active site of E. coli Trx-1 in combination with a T200M point mutation, we transformed DsbG into an enzyme highly reactive toward GSNO and YbiS. The pKa of the nucleophilic cysteine, as well as the redox and thermodynamic properties of the engineered DsbG are dramatically changed and become similar to those of E. coli Trx-1. X-ray structural insights suggest that this results from a loss of two direct hydrogen bonds to the nucleophilic cysteine sulfur in the DsbG mutant. Our results highlight the plasticity of the Trx structural fold and reveal that the subtle change of the number of hydrogen bonds in the active site of Trx-like proteins is the key factor that thermodynamically controls reactivity toward nitrosylated compounds. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

In vitro BMP-2 peptide release from thiolated chitosan based hydrogel.

PubMed

Liu, Xujie; Yu, Bo; Huang, Qianli; Liu, Rui; Feng, Qingling; Cai, Qiang; Mi, Shengli

2016-12-01

Thiolated chitosan based thermo-sensitive hydrogel is a water soluble system and the existing thiol groups are beneficial for the delivery of cysteine-rich peptides. In the present study, a kind of thiolated chitosan, i.e. chitosan-4-thio-butylamidine (CS-TBA) conjugate was characterized and used to prepare CS-TBA/hydroxyapatite (HA)/beta-glycerophosphate disodium (β-GP) thermo-sensitive hydrogel. The cysteine terminated peptide 24 (P24) containing residues 73-92 of the knuckle epitope of BMP-2 (N→C: KIPKASSVPTELSAISTLYLSGGC) was synthesized and characterized. The release behavior of P24 from CS-TBA based hydrogel was investigated in vitro. The thiol groups in CS-TBA may react with thiol groups in P24, thus decreases the P24 release rate and maintains the peptide release for a longer time compared with unmodified chitosan based hydrogel. Moreover, the bioactivity of P24 is preserved during release process. These results indicate that P24 loaded CS-TBA based thermosensitive hydrogel is a potential material for minimally invasive surgery of bone repair. Copyright © 2016 Elsevier B.V. All rights reserved.

Functionalization of gold and graphene electrodes by p-maleimido-phenyl towards thiol-sensing systems investigated by EQCM and IR ellipsometric spectroscopy

NASA Astrophysics Data System (ADS)

Neubert, Tilmann J.; Rösicke, Felix; Sun, Guoguang; Janietz, Silvia; Gluba, Marc A.; Hinrichs, Karsten; Nickel, Norbert H.; Rappich, Jörg

2017-11-01

Electrografting of gold and graphene surfaces by functional p-(N-maleimido)phenyl groups was performed by reduction of p-(N-maleimido)phenyldiazonium tetrafluoroborate. The reduction was carried out using cyclic voltammetry coupled with micro-gravimetric measurements by means of electrochemical quartz crystal microbalance (EQCM). The overall deposited mass on gold was higher than on graphene. However, the Faradaic efficiency was lower on Au (14%) compared to graphene (22%) after the first potential scan. Subsequently, the maleimide functional groups have been tested for immobilization of terminal thiols using (4-nitrobenzyl)mercaptan for the functionalized graphene surface and a cysteine-modified peptide for the functionalized gold surface. The functionalization by p-(N-maleimido)phenyl groups and the following thiol coupling of the particular surface was proven by infrared spectroscopic ellipsometry (IRSE). In addition, the interaction of the tetrabutylammonium and tetrafluoroborate ions present in the electrolyte with the Au and graphene electrodes was investigated by EQCM and revealed less electrostatic interaction of graphene with these ions in solution compared to the metal (Au) surface.

Hydrogen bonding as the origin of the switching behavior in dithiolated phenylene-vinylene oligomers

NASA Astrophysics Data System (ADS)

Obodo, J. T.; Gkionis, K.; Rungger, I.; Sanvito, S.; Schwingenschlögl, U.

2013-08-01

We investigate theoretically the switching behavior of a dithiolated phenylene-vinylene oligomer sandwiched between Au(111) electrodes using self-interaction corrected density-functional theory combined with the nonequilibrium Green's-function method for quantum transport. The molecule presents a configurational bistability, which can be exploited in constructing molecular memories, switches, and sensors. We find that protonation of the terminating thiol groups is at the origin of the change in conductance. H bonding at the thiol group weakens the S-Au bond and reduces by about one order of magnitude the transmission coefficient at the Fermi level, and thus the linear response conductance. Furthermore, protonation downshifts in energy the position of the highest occupied molecular orbital, so that the current of the protonated species is lower than that of the unprotonated one along the entire bias range investigated, from -1.5 to 1.5 V. A second protonation at the opposite thiol group has only minor effects and no further drastic reduction in transmission takes place. Our results allow us to re-interpret the experimental data originally attributing the conductance reduction to H dissociation.

Photomodulation of the nucleating activity of a photocleavable crosslinked actin dimer.

PubMed

Marriott, G; Miyata, H; Kinosita, K

1992-04-01

The ability to generate substrate concentration jumps through photo-deprotection of amine, carboxyl and phosphate groups has been an important development for investigations of protein activity in complex systems. To broaden the versatility and applications of photo-deprotection techniques for the photomodulation of protein activity we describe the synthesis and characterisation of a reagent for generating free thiol from thioether groups and a related photocleavable, heterobifunctional crosslinking reagent. Chemical and spectroscopic studies of a model thiol protected derivative were used to show some features of thiol group photodeprotection. To demonstrate how the photocleavable crosslinking reagent may be used to modulate the activity of proteins we investigated the effect of light on the nucleating activity of crosslinked actin dimer; thus following near-ultraviolet irradiation of the actin dimer the crosslink was cleaved, presumeably at the thioether bond, resulting in the concomitant dissociation of dimer, loss of nucleating activity and creation of a concentration jump of polymerisable G-actin monomer. On the basis of this initial study we discuss applications and limitations of these reagents for the photomodulation of protein activity in vitro and in vivo.

Ethanol Attenuates Histiotrophic Nutrition Pathways and Alters the Intracellular Redox Environment and Thiol Proteome during Rat Organogenesis

PubMed Central

Jilek, Joseph L.; Sant, Karilyn E.; Cho, Katherine H.; Reed, Matthew S.; Pohl, Jan; Hansen, Jason M.; Harris, Craig

2015-01-01

Ethanol (EtOH) is a reactive oxygen-generating teratogen involved in the etiology of structural and functional developmental defects. Embryonic nutrition, redox environment, and changes in the thiol proteome following EtOH exposures (1.56.0 mg/ml) were studied in rat whole embryo culture. Glutathione (GSH) and cysteine (Cys) concentrations with their respective intracellular redox potentials (Eh) were determined using high-performance liquid chromatography. EtOH reduced GSH and Cys concentrations in embryo (EMB) and visceral yolk sac (VYS) tissues, and also in yolk sac and amniotic fluids. These changes produced greater oxidation as indicated by increasingly positive Eh values. EtOH reduced histiotrophic nutrition pathway activities as measured by the clearance of fluorescin isothiocyanate (FITC)-albumin from culture media. A significant decrease in total FITC clearance was observed at all concentrations, reaching approximately 50% at the highest dose. EtOH-induced changes to the thiol proteome were measured in EMBs and VYSs using isotope-coded affinity tags. Decreased concentrations for specific proteins from cytoskeletal dynamics and endocytosis pathways (α-actinin, α-tubulin, cubilin, and actin-related protein 2); nuclear translocation (Ran and RanBP1); and maintenance of receptor-mediated endocytosis (cubilin) were observed. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis also identified a decrease in ribosomal proteins in both EMB and VYS. Results show that EtOH interferes with nutrient uptake to reduce availability of amino acids and micronutrients required by the conceptus. Intracellular antioxidants such as GSH and Cys are depleted following EtOH and Eh values increase. Thiol proteome analysis in the EMB and VYS show selectively altered actin/cytoskeleton, endocytosis, ribosome biogenesis and function, nuclear transport, and stress-related responses. PMID:26185205

Phospholipase A2 activation regulates cytotoxicity of methylmercury in vascular endothelial cells.

PubMed

Mazerik, Jessica N; Hagele, Thomas; Sherwani, Shariq; Ciapala, Valorie; Butler, Susan; Kuppusamy, M Lakshmi; Hunter, Melissa; Kuppusamy, Periannan; Marsh, Clay B; Parinandi, Narasimham L

2007-01-01

Mercury has been identified as a risk factor for cardiovascular disease among humans. Through diet, mainly fish consumption, humans are exposed to methylmercury, the biomethylated organic form of environmental mercury. As the endothelium is an important player in homeostasis of the cardiovascular system, here, the authors tested their hypothesis that methylmercury activates the lipid signaling enzyme phospholipase A(2) (PLA(2)) in vascular endothelial cells (ECs), causing upstream regulation of cytotoxicity. To test this hypothesis, the authors used bovine pulmonary artery ECs (BPAECs) cultured in monolayers, following labeling of their membrane phospholipids with [(3)H]arachidonic acid (AA). The cells were exposed to methylmercury chloride (MMC) and then the release of free AA (index of PLA(2) activity) and lactate dehydrogenase (LDH; index of cytotoxicity) were determined by liquid scintillation counting and spectrophotometry, respectively. MMC significantly activated PLA(2) in a dose-dependent (5 to 15 microM) and time-dependent (0 to 60 min) fashion. Sulfhydryl (thiol-protective) agents, calcium chelators, antioxidants, and PLA(2)-specific inhibitors attenuated the MMC-induced PLA(2) activation, suggesting the role of thiols, reactive oxygen species (ROS), and calcium in the activation of PLA(2) in BPAECs. MMC also induced the loss of thiols and increase of lipid peroxidation in BPAECs. MMC induced cytotoxicity in BPAECs as observed by the altered cell morphology and LDH leak, which was significantly attenuated by PLA(2) inhibitors. This study established that PLA(2) activation through thiols, calcium, and oxidative stress was associated with the cytotoxicity of MMC in BPAECs, drawing attention to the involvement of PLA(2) signaling in the methylmercury-induced vascular endothelial dysfunctions.

Synthesis of aryl thioethers through the N-chlorosuccinimide-promoted cross-coupling reaction of thiols with Grignard reagents.

PubMed

Cheng, Jun-Hao; Ramesh, Chintakunta; Kao, Hsin-Lun; Wang, Yu-Jen; Chan, Chien-Ching; Lee, Chin-Fa

2012-11-16

A convenient one-pot approach for the synthesis of aryl sulfides through the coupling of thiols with Grignard reagents in the presence of N-chlorosuccinimide is described. The sulfenylchlorides were formed when thiols were treated with N-chlorosuccinimide, and the resulting sulfenylchlorides were then directly reacted with Grignard reagents to provide aryl sulfides in good to excellent yields under mild reaction conditions. Functional groups including ester, fluoro, and chloro are tolerated by the reaction conditions employed. It is important to note that this method has a short reaction time (30 min in total) and represents an alternative approach for the synthesis of aryl sulfides over the existing protocols.

Functional Group Analysis.

ERIC Educational Resources Information Center

Smith, Walter T., Jr.; Patterson, John M.

1980-01-01

Discusses analytical methods selected from current research articles. Groups information by topics of general interest, including acids, aldehydes and ketones, nitro compounds, phenols, and thiols. Cites 97 references. (CS)

Rapid, Efficient and Versatile Strategies for Functionally Sophisticated Polymers and Nanoparticles: Degradable Polyphosphoesters and Anisotropic Distribution of Chemical Functionalities

NASA Astrophysics Data System (ADS)

Zhang, Shiyi

The overall emphasis of this dissertation research included two kinds of asymmetrically-functionalized nanoparticles with anisotropic distributions of chemical functionalities, three degradable polymers synthesized by organocatalyzed ring-opening polymerizations, and two polyphosphoester-based nanoparticle systems for various biomedical applications. Inspired by the many hierarchical assembly processes that afford complex materials in Nature, the construction of asymmetrically-functionalized nanoparticles with efficient surface chemistries and the directional organization of those building blocks into complex structures have attracted much attention. The first method generated a Janus-faced polymer nanoparticle that presented two orthogonally click-reactive surface chemistries, thiol and azido. This robust method involved reactive functional group transfer by templating against gold nanoparticle substrates. The second method produced nanoparticles with sandwich-like distribution of crown ether functionalities through a stepwise self-assembly process that utilized crown ether-ammonium supramolecular interactions to mediate inter-particle association and the local intra-particle phase separation of unlike hydrophobic polymers. With the goal to improve the efficiency of the production of degradable polymers with tunable chemical and physical properties, a new type of reactive polyphosphoester was synthesized bearing alkynyl groups by an organocatalyzed ring-opening polymerization, the chemical availability of the alkyne groups was investigated by employing "click" type azide-alkyne Huisgen cycloaddition and thiol-yne radical-mediated reactions. Based on this alkyne-functionalized polyphosphoester polymer and its two available "click" type reactions, two degradable nanoparticle systems were developed. To develop the first system, the well defined poly(ethylene oxide)-block-polyphosphester diblock copolymer was transformed into a multifunctional Paclitaxel drug conjugate by densely attaching the polyphosphoester block with azide-functionalized Paclitaxel by azide-alkyne Huisgen cycloaddition. This Paclitaxel drug conjugate provides a powerful platform for combinational cancer therapy and bioimaging due to its ultra-high Paclitaxel loading (> 65 wt%), high water solubility (>6.2 mg/mL for PTX) and easy functionalization. Another polyphosphoester-based nanoparticle system has been developed by a programmable process for the rapid and facile preparation of a family of nanoparticles with different surface charges and functionalities. The non-ionic, anionic, cationic and zwitterionic nanoparticles with hydrodynamic diameters between 13 nm to 21 nm and great size uniformity could be rapidly prepared from small molecules in 6 h or 2 days. The anionic and zwitterionic nanoparticles were designed to load silver ions to treat pulmonary infections, while the cationic nanoparticles are being applied to regulate lung injuries by serving as a degradable iNOS inhibitor conjugates. In addition, a direct synthesis of acid-labile polyphosphoramidate by organobase-catalyzed ring-opening polymerization and an improved two-step preparation of polyphosphoester ionomer by acid-assisted cleavage of phosphoramidate bonds on polyphosphoramidate were developed. Polyphosphoramidate and polyphosphoester ionomers may be applied to many applications, due to their unique chemical and physical properties.

Heterofunctional Glycopolypeptides by Combination of Thiol-Ene Chemistry and NCA Polymerization.

PubMed

Krannig, Kai-Steffen; Schlaad, Helmut

2016-01-01

Glycopolypeptides are prepared either by the polymerization of glycosylated amino acid N-carboxyanhydrides (NCAs) or by the post-polymerization functionalization of polypeptides with suitable functional groups. Here we present a method for the in-situ functionalization and (co-) polymerization of allylglycine N-carboxyanhydride in a facile one-pot procedure, combining radical thiol-ene photochemistry and nucleophilic ring-opening polymerization techniques, to yield well-defined heterofunctional glycopolypeptides.

DNA biosensor for detection of Salmonella typhi from blood sample of typhoid fever patient using gold electrode modified by self-assembled monolayers of thiols

NASA Astrophysics Data System (ADS)

Suryapratiwi, Windha Novita; Paat, Vlagia Indira; Gaffar, Shabarni; Hartati, Yeni Wahyuni

2017-05-01

Electrochemical biosensors are currently being developed in order to handle various clinical problems in diagnosing infectious diseases caused by pathogenic bacteria, or viruses. On this research, voltammetric DNA biosensor using gold electrode modified by thiols with self-assembled monolayers had been developed to detect a certain sequence of Salmonella typhi DNA from blood sample of typhoid fever patient. Thiol groups of cysteamines (Cys) and aldehyde groups from glutaraldehydes (Glu) were used as a link to increase the performance of gold electrode in detecting guanine oxidation signal of hybridized S. typhi DNA and ssDNA probe. Standard calibration method was used to determine analytical parameters from the measurements. The result shown that, the detection of S. typhi DNA from blood sample of typhoid fever patient can be carried out by voltammetry using gold electrode modified by self-assembled monolayers of thiols. A characteristic oxidation potential of guanine using Au/Cys/Gluwas obtained at +0.17 until +0.20 V. Limit of detection and limit of quantification from this measurements were 1.91μg mL-1 and 6.35 μg mL-1. The concentration of complement DNA from sample was 6.96 μg mL-1.

Exposure of the blue mussel, Mytilus edulis, to gold nanoparticles and the pro-oxidant menadione.

PubMed

Tedesco, Sara; Doyle, Hugh; Blasco, Julian; Redmond, Gareth; Sheehan, David

2010-03-01

Relatively little is known about how gold nanoparticles (GNP) might interact in vivo with marine organisms. Mytilus edulis was exposed (24h) to approximately 15 nm GNP, menadione and both compounds simultaneously (GNP/menadione). GNP was detected by inductively coupled plasma-optical emission spectroscopy mainly in digestive gland of samples exposed to GNP though not GNP/menadione, perhaps due to impaired feeding. Thioredoxin reductase activity and malondialdehyde levels were determined in all tissues. Thioredoxin reductase inhibition was detected only in digestive gland exposed to menadione whilst malondialdehyde levels did not vary in response to treatment in all tissues. GNP caused a decrease in the reduced/oxidized glutathione ratio in digestive gland, but no difference was found in other tissues or for other treatments. One dimensional electrophoresis of proteins containing thiol groups was performed in all tissues and revealed a reduction in protein thiols for all treatments in digestive gland. Two dimensional electrophoresis of digestive gland extracts, from GNP and control groups, showed decreased levels of thiol proteins in response to GNP which we attribute to oxidation. Our results suggest that GNP causes a modest level of oxidative stress sufficient to oxidize thiols in glutathione and proteins but without causing lipid peroxidation or induction of thioredoxin reductase activity.

Identification of a Protein with Antioxidant Activity that is Important for the Protection against Beer Ageing

PubMed Central

Wu, Ming J.; Clarke, Frank M.; Rogers, Peter J.; Young, Paul; Sales, Narelle; O’Doherty, Patrick J.; Higgins, Vincent J.

2011-01-01

This study was carried out with fresh Australian lager beer which was sampled directly off the production line, the same samples aged for 12 weeks at 30 °C, and the vintage beer which was kept at 20 °C for 5 years. Characteristic Australian lager flavour was maintained in the fresh and vintage beers but was lost in the aged beer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and free thiol group labelling analyses of beer proteins found that this flavour stability correlated with the presence of an unknown 10 kilodaltons (kDa) protein with a higher level of free thiols. The protein was purified by size-exclusion chromatography, then peptide sequencing and database matching identified it as the barley lipid transfer protein (LTP1). Further characterisation using diphenylpicrylhydrazyl (DPPH) free radical scavenging and a Saccharomyces cerevisiae-based antioxidant screening assay demonstrated that the LTP1 protein was active in DPPH reduction and antioxidant activity. The absence of free thiol in the aged beer indicates that the thiol functional groups within the LTP1 protein were saturated and suggests that it is important in the flavour stability of beer by maintaining reduction capacity during the ageing process. PMID:22016646

Hesperidin, a citrus bioflavonoid, alleviates trichloroethylene-induced oxidative stress in Drosophila melanogaster.

PubMed

Abolaji, Amos Olalekan; Babalola, Oluwatoyin Victoria; Adegoke, Abimbola Kehinde; Farombi, Ebenezer Olatunde

2017-10-01

Trichloroethylene (TCE) is a chlorinated organic pollutant of groundwater with diverse toxic effects in animals and humans. Here, we investigated the ameliorative role of hesperidin, a citrus bioflavonoid on TCE-induced toxicity in Drosophila melanogaster. Four groups of D. melanogaster (50 flies/vial, with 5 vials/group) were exposed to ethanol (2.5%, control), HSP (400mg/10g diet), TCE (10μM/10g diet) and TCE (10μM/10g diet)+HSP (400mg/10g diet) respectively in the diet for 5days. Then, selected oxidative stress and antioxidant markers were evaluated. The results showed that TCE significantly increased the level of reactive oxygen species (ROS) and inhibited catalase, glutathione S-transferase and acetylcholinesterase (AChE) activities with concurrent depletion of total thiol level. However, co-administration of TCE and hesperidin mitigated TCE-induced depletion of antioxidants, and restored ROS level and AChE activity in the flies (p<0.05). Overall, hesperidin offered protective potency on TCE-induced oxidative stress in the flies via anti-oxidative mechanism. Copyright © 2017 Elsevier B.V. All rights reserved.

INACTIVATION OF E. COLI PYRUVATE FORMATE-LYASE: ROLE OF AdhE AND SMALL MOLECULES

PubMed Central

Nnyepi, Mbako R.; Peng, Yi; Broderick, Joan B.

2007-01-01

E. coli AdhE has been reported to harbor three distinct enzymatic activities: alcohol dehydrogenase, acetaldehyde-CoA dehydrogenase, and pyruvate formate-lyase (PFL) deactivase. Herein we report on the cloning, expression, and purification of E. coli AdhE, and the re-investigation of its purported enzymatic activities. While both the alcohol dehydrogenase and acetaldehyde-CoA dehydrogenase activities were readily detectible, we were unable to obtain any evidence for catalytic deactivation of PFL by AdhE, regardless of whether the reported cofactors for deactivation (Fe(II), NAD, and CoA) were present. Our results demonstrate that AdhE is not a PFL deactivating enzyme. We have also examined the potential for deactivation of active PFL by small-molecule thiols. Both β-mercaptoethanol and dithiothreitol deactivate PFL efficiently, with the former providing quite rapid deactivation. PFL deactivated by these thiols can be reactivated, suggesting that this deactivation is non-destructive transfer of an H atom equivalent to quench the glycyl radical. PMID:17280641

Measuring protection of aromatic wine thiols from oxidation by competitive reactions vs wine preservatives with ortho-quinones.

PubMed

Nikolantonaki, Maria; Magiatis, Prokopios; Waterhouse, Andrew L

2014-11-15

Quinones are central intermediates in wine oxidation that can degrade the quality of wine by reactions with varietal thiols, such as 3-sulfanylhexanol, decreasing desirable aroma. Protection by wine preservatives (sulphur dioxide, glutathione, ascorbic acid and model tannin, phloroglucinol) was assessed by competitive sacrificial reactions with 4-methyl-1,2-benzoquinone, quantifying products and ratios by HPLC-UV-MS. Regioselectivity was assessed by product isolation and identification by NMR spectroscopy. Nucleophilic addition reactions compete with two electron reduction of quinones by sulphur dioxide or ascorbic acid, and both routes serve as effective quenching pathways, but minor secondary products from coupled redox reactions between the products and reactants are also observed. The wine preservatives were all highly reactive and thus all very protective against 3-sulfanylhexanol loss to the quinone, but showed only additive antioxidant effects. Confirmation of these reaction rates and pathways in wine is needed to assess the actual protective action of each tested preservative. Copyright © 2014 Elsevier Ltd. All rights reserved.

3D-Hydrogel Based Polymeric Nanoreactors for Silver Nano-Antimicrobial Composites Generation

PubMed Central

Soto-Quintero, Albanelly; Romo-Uribe, Ángel; Bermúdez-Morales, Víctor H.; Quijada-Garrido, Isabel

2017-01-01

This study underscores the development of Ag hydrogel nanocomposites, as smart substrates for antibacterial uses, via innovative in situ reactive and reduction pathways. To this end, two different synthetic strategies were used. Firstly thiol-acrylate (PSA) based hydrogels were attained via thiol-ene and radical polymerization of polyethylene glycol (PEG) and polycaprolactone (PCL). As a second approach, polyurethane (PU) based hydrogels were achieved by condensation polymerization from diisocyanates and PCL and PEG diols. In fact, these syntheses rendered active three-dimensional (3D) hydrogel matrices which were used as nanoreactors for in situ reduction of AgNO3 to silver nanoparticles. A redox chemistry of stannous catalyst in PU hydrogel yielded spherical AgNPs formation, even at 4 °C in the absence of external reductant; and an appropriate thiol-functionalized polymeric network promoted spherical AgNPs well dispersed through PSA hydrogel network, after heating up the swollen hydrogel at 103 °C in the presence of citrate-reductant. Optical and swelling behaviors of both series of hydrogel nanocomposites were investigated as key factors involved in their antimicrobial efficacy over time. Lastly, in vitro antibacterial activity of Ag loaded hydrogels exposed to Pseudomona aeruginosa and Escherichia coli strains indicated a noticeable sustained inhibitory effect, especially for Ag–PU hydrogel nanocomposites with bacterial inhibition growth capabilities up to 120 h cultivation. PMID:28763050

A crucial role for thiol antioxidants in estrogen-deficiency bone loss

PubMed Central

Lean, Jenny M.; Davies, Julie T.; Fuller, Karen; Jagger, Christopher J.; Kirstein, Barrie; Partington, Geoffrey A.; Urry, Zoë L.; Chambers, Timothy J.

2003-01-01

The mechanisms through which estrogen prevents bone loss are uncertain. Elsewhere, estrogen exerts beneficial actions by suppression of reactive oxygen species (ROS). ROS stimulate osteoclasts, the cells that resorb bone. Thus, estrogen might prevent bone loss by enhancing oxidant defenses in bone. We found that glutathione and thioredoxin, the major thiol antioxidants, and glutathione and thioredoxin reductases, the enzymes responsible for maintaining them in a reduced state, fell substantially in rodent bone marrow after ovariectomy and were rapidly normalized by exogenous 17-β estradiol. Moreover, administration of N-acetyl cysteine (NAC) or ascorbate, antioxidants that increase tissue glutathione levels, abolished ovariectomy-induced bone loss, while L-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of glutathione synthesis, caused substantial bone loss. The 17-β estradiol increased glutathione and glutathione and thioredoxin reductases in osteoclast-like cells in vitro. Furthermore, in vitro NAC prevented osteoclast formation and NF-κB activation. BSO and hydrogen peroxide did the opposite. Expression of TNF-α, a target for NF-κB and a cytokine strongly implicated in estrogen-deficiency bone loss, was suppressed in osteoclasts by 17-β estradiol and NAC. These observations strongly suggest that estrogen deficiency causes bone loss by lowering thiol antioxidants in osteoclasts. This directly sensitizes osteoclasts to osteoclastogenic signals and entrains ROS-enhanced expression of cytokines that promote osteoclastic bone resorption. PMID:12975476

Controlled free radical attack in the apoplast: A hypothesis for roles of O, N and S species in regulatory and polysaccharide cleavage events during rapid abscission by Azolla

PubMed Central

Cohen, Michael F.; Gurung, Sushma; Fukuto, Jon M.; Yamasaki, Hideo

2014-01-01

Shedding of organs by abscission is a key terminal step in plant development and stress responses. Cell wall (CW) loosening at the abscission zone can occur through a combination chain breakage of apoplastic polysaccharides and tension release of cellulose microfibrils. Two distinctly regulated abscission cleavage events are amenable to study in small water ferns of the genus Azolla; one is a rapid abscission induced by environmental stimuli such as heat or chemicals, and the other is an ethylene-induced process occurring more slowly through the action of hydrolytic enzymes. Although free radicals are suggested to be involved in the induction of rapid root abscission, its mechanism is not fully understood. The apoplast contains peroxidases, metal-binding proteins and phenolic compounds that potentially generate free radicals from H2O2 to cleave polysaccharides in the CW and middle lamella. Effects of various thiol-reactive agents implicate the action of apoplastic peroxidases having accessible cysteine thiols in rapid abscission. The Ca2+ dependency of rapid abscission may reflect the stabilization Ca2+ confers to peroxidase structure and binding to pectin. To spur further investigation, we present a hypothetical model for small signaling molecules H2O2 and NO and their derivatives in regulating, via modification of putative protein thiols, free radical attack of apoplastic polysaccharides. PMID:24467903

Bacillithiol, a New Player in Bacterial Redox Homeostasis

PubMed Central

2011-01-01

Abstract Bacillithiol (BSH), the α-anomeric glycoside of l-cysteinyl-d-glucosamine with l-malic acid, plays a dominant role in the cytosolic thiol redox chemistry of the low guanine and cytosine (GC) Gram-positive bacteria (phylum Firmicutes). BSH is functionally analogous to glutathione (GSH) but differs sufficiently in chemical structure that cells have evolved a distinct set of enzymes that use BSH as cofactor. BSH was discovered in Bacillus subtilis as a mixed disulfide with the redox-sensing repressor OhrR and in B. anthracis by biochemical analysis of pools of labeled thiols. The structure of BSH was determined after purification from Deinococcus radiodurans. Similarities in structure between BSH and mycothiol (MSH) facilitated the identification of biosynthetic genes for BSH in the model organism B. subtilis. Phylogenomic analyses have identified several candidate BSH-using or associated proteins, including a BSH reductase, glutaredoxin-like thiol-dependent oxidoreductases (bacilliredoxins), and a BSH-S-transferase (FosB) involved in resistance to the epoxide antibiotic fosfomycin. Preliminary results implicate BSH in cellular processes to maintain cytosolic redox balance and for adaptation to reactive oxygen, nitrogen, and electrophilic species. BSH also is predicted to chelate metals avidly, in part due to the appended malate moiety, although the implications of BSH for metal ion homeostasis have yet to be explored in detail. Antioxid. Redox Signal. 15, 123–133. PMID:20712413

Graphene Oxide-Polymer Composite Langmuir Films Constructed by Interfacial Thiol-Ene Photopolymerization

NASA Astrophysics Data System (ADS)

Luo, Xiaona; Ma, Kai; Jiao, Tifeng; Xing, Ruirui; Zhang, Lexin; Zhou, Jingxin; Li, Bingbing

2017-02-01

The effective synthesis and self-assembly of graphene oxide (GO) nanocomposites are of key importance for a broad range of nanomaterial applications. In this work, a one-step chemical strategy is presented to synthesize stable GO-polymer Langmuir composite films by interfacial thiol-ene photopolymerization at room temperature, without use of any crosslinking agents and stabilizing agents. It is discovered that photopolymerization reaction between thiol groups modified GO sheets and ene in polymer molecules is critically responsible for the formation of the composite Langmuir films. The film formed by Langmuir assembly of such GO-polymer composite films shows potential to improve the mechanical and chemical properties and promotes the design of various GO-based nanocomposites. Thus, the GO-polymer composite Langmuir films synthesized by interfacial thiol-ene photopolymerization with such a straightforward and clean manner, provide new alternatives for developing chemically modified GO-based hybrid self-assembled films and nanomaterials towards a range of soft matter and graphene applications.

Photoinduced Cross-Linking of Dynamic Poly(disulfide) Films via Thiol Oxidative Coupling.

PubMed

Feillée, Noémi; Chemtob, Abraham; Ley, Christian; Croutxé-Barghorn, Céline; Allonas, Xavier; Ponche, Arnaud; Le Nouen, Didier; Majjad, Hicham; Jacomine, Léandro

2016-01-01

Initially developed as an elastomer with an excellent record of barrier and chemical resistance properties, poly(disulfide) has experienced a revival linked to the dynamic nature of the S-S covalent bond. A novel photobase-catalyzed oxidative polymerization of multifunctional thiols to poly(disulfide) network is reported. Based solely on air oxidation, the single-step process is triggered by the photodecarboxylation of a xanthone acetic acid liberating a strong bicyclic guanidine base. Starting with a 1 μm thick film based on trithiol poly(ethylene oxide) oligomer, the UV-mediated oxidation of thiols to disulfides occurs in a matter of minutes both selectively, i.e., without overoxidation, and quantitatively as assessed by a range of spectroscopic techniques. Thiolate formation and film thickness determine the reaction rates and yield. Spatial control of the photopolymerization serves to generate robust micropatterns, while the reductive cleavage of S-S bridges allows the recycling of 40% of the initial thiol groups. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemical addressability of potato virus X for its applications in bio/nanotechnology.

PubMed

Le, Duc H T; Hu, He; Commandeur, Ulrich; Steinmetz, Nicole F

2017-12-01

Potato virus X (PVX), a type member of the plant virus potexvirus group, offers a unique nanotechnology platform based on its high aspect ratio and flexible filamentous shape. The PVX platform has already been engineered and studied for its uses in imaging, drug delivery, and immunotherapies. While genetic engineering procedures are well established for PVX, there is limited information about chemical conjugation strategies for functionalizing PVX, partly due to the lack of structural information of PVX at high resolution. To overcome these challenges, we built a structural model of the PVX particle based on the available structures from pepino mosaic virus (PepMV), a close cousin of PVX. Using the model and a series of chemical conjugation experiments, we identified and probed the addressability of cysteine side chains. Chemical reactivity of cysteines was confirmed using Michael-addition and thiol-selective probes, including fluorescent dyes and biotin tags. LC/MS/MS was used to map Cys 121 as having the highest selectivity for modification. Finally, building on the availability of two reactive groups, the newly identified Cys and previously established Lys side chains, we prepared multifunctional PVX nanoparticles by conjugating Gd-DOTA for magnetic resonance imaging (MRI) to lysines and fluorescent dyes for optical imaging to cysteines. The resulting functionalized nanofilament could have applications in dual-modal optical-MRI imaging applications. These results further extend the understanding of the chemical properties of PVX and enable development of novel multifunctional platforms in bio/nanotechnology. Copyright © 2017 Elsevier Inc. All rights reserved.

Homocysteinemia control by cysteine in cerebral vascular patients after methionine loading test: evidences in physiological and pathological conditions in cerebro-vascular and multiple sclerosis patients.

PubMed

Ulivelli, Monica; Priora, Raffaella; Di Giuseppe, Danila; Coppo, Lucia; Summa, Domenico; Margaritis, Antonios; Frosali, Simona; Bartalini, Sabina; Martini, Giuseppe; Cerase, Alfonso; Di Simplicio, Paolo

2016-06-01

The toxicity risk of hyperhomocysteinemia is prevented through thiol drug administration which reduces plasma total homocysteine (tHcy) concentrations by activating thiol exchange reactions. Assuming that cysteine (Cys) is a homocysteinemia regulator, the hypothesis was verified in healthy and pathological individuals after the methionine loading test (MLT). The plasma variations of redox species of Cys, Hcy, cysteinylglycine, glutathione and albumin (reduced, HS-ALB, and at mixed disulfide, XSS-ALB) were compared in patients with cerebral small vessels disease (CSVD) (n = 11), multiple sclerosis (MS) (n = 12) and healthy controls (n = 11) at 2-4-6 h after MLT. In MLT-treated subjects, the activation of thiol exchange reactions provoked significant changes over time in redox species concentrations of Cys, Hcy, and albumin. Significant differences between controls and pathological groups were also observed. In non-methionine-treated subjects, total Cys concentrations, tHcy and thiol-protein mixed disulfides (CSS-ALB, HSS-ALB) of CSVD patients were higher than controls. After MLT, all groups displayed significant cystine (CSSC) increases and CSS-ALB decreases, that in pathological groups were significantly higher than controls. These data would confirm the Cys regulatory role on the homocysteinemia; they also explain that the Cys-Hcy mixed disulfide excretion is an important point of hyperhomocysteinemia control. Moreover, in all groups after MLT, significant increases in albumin concentrations, named total albumin (tALB) and measured as sum of HS-ALB (spectrophometric), and XSS-ALB (assayed at HPLC) were observed. tALB increases, more pronounced in healthy than in the pathological subjects, could indicate alterations of albumin equilibria between plasma and other extracellular spaces, whose toxicological consequences deserve further studies.

A comparison of thiolated and disulfide-crosslinked polyethylenimine for nonviral gene delivery.

PubMed

Aravindan, Latha; Bicknell, Katrina A; Brooks, Gavin; Khutoryanskiy, Vitaliy V; Williams, Adrian C

2013-09-01

Branched polyethylenimine (25 kDa) is thiolated and compared with redox-sensitive crosslinked derivatives. Both polymers thiol contents are assessed; the thiolated polymers have 390-2300 mmol SH groups/mol, whereas the crosslinked polymers have lower thiol contents. Cytotoxicity assays show that both modified polymers give lower hemolysis than unmodified PEI. Increased thiol content increases gene transfer efficiency but also elevates cytotoxicity. Crosslinking improves plasmid DNA condensation and enhances transfection efficiency, but extensive crosslinking overstabilizes the polyplexes and decreases transfection, emphasizing the need to balance polyplex stabilization and unpacking. Thus, at low levels of crosslinking, 25 kDa PEI can be an efficient redox-sensitive carrier system. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamic Thiol/Disulphide Homeostasis in Children and Adolescents with Non-Autoimmune Subclinical Hypothyroidism

PubMed Central

Uçaktürk, Seyit Ahmet; Alışık, Murat; Uğur, Çağatay; Elmaoğulları, Selin; Mengen, Eda; Erel, Özcan

2018-01-01

Objective To evaluate the thiol/disulphide homeostasis in children with non-autoimmune subclinical hypothyroidism (SHT). Subjects and Methods Thiol/disulphide homeosta sis, involving native thiol (SH), disulphide (SS), and total thiol (SS + SH), was evaluated in 60 children and adolescents who were negative for thyroid auto-antibodies (anti-thyroid peroxidase, anti-thyroglobulin) and had a thyroid-stimulating hormone (TSH) value of > 5 mIU/L, and in 40 sex- and age-matched healthy control subjects who were negative for thyroid autoantibodies and had normal TSH levels. Lipid profiles and urine iodine levels were also determined. Results SH (466 ± 32.8 vs. 462 ± 32.1 μmol/L p = 0.59), SH + SS (508 ± 34.0 vs. 506 ± 32.7 μmol/L, p = 0.81), SS (21 ± 5.5 vs. 22 ± 5.8 μmol/L, p = 0.41), SS/SH (4.5 ± 1.2 vs. 4.8 ± 1.3%, p = 0.36), SS/SH + SS (4.1 ± 1.0 vs. 4.3 ± 1.1%, p = 0.36) and SH/SH + SS (91 ± 2.1 vs. 91 ± 2.1%, p = 0.31) levels were similar in children with SHT and control subjects (p > 0.05). There was no difference between total cholesterol, triglyceride, and low-density lipoprotein levels in SHT patients and controls. No difference was detected between the patients with or without iodine deficiency in the SHT group in terms of thiol/disulphide homeostasis parameters. Conclusion The status of dynamic thiol/disulphide homeostasis did not change in children and adolescents with non-autoimmune SHT. Future studies are needed for the evaluation of oxidative stress in patients with long-standing non-autoimmune SHT. PMID:29402856

Biotin Switch Assays for Quantitation of Reversible Cysteine Oxidation.

PubMed

Li, R; Kast, J

2017-01-01

Thiol groups in protein cysteine residues can be subjected to different oxidative modifications by reactive oxygen/nitrogen species. Reversible cysteine oxidation, including S-nitrosylation, S-sulfenylation, S-glutathionylation, and disulfide formation, modulate multiple biological functions, such as enzyme catalysis, antioxidant, and other signaling pathways. However, the biological relevance of reversible cysteine oxidation is typically underestimated, in part due to the low abundance and high reactivity of some of these modifications, and the lack of methods to enrich and quantify them. To facilitate future research efforts, this chapter describes detailed procedures to target the different modifications using mass spectrometry-based biotin switch assays. By switching the modification of interest to a biotin moiety, these assays leverage the high affinity between biotin and avidin to enrich the modification. The use of stable isotope labeling and a range of selective reducing agents facilitate the quantitation of individual as well as total reversible cysteine oxidation. The biotin switch assay has been widely applied to the quantitative analysis of S-nitrosylation in different disease models and is now also emerging as a valuable research tool for other oxidative cysteine modifications, highlighting its relevance as a versatile, robust strategy for carrying out in-depth studies in redox proteomics. © 2017 Elsevier Inc. All rights reserved.

Assessing the issue of instability due to Michael adduct formation in novel chemical entities possessing a carbon-carbon double bond during early drug development--applicability of common laboratory analytical protocols.

PubMed

Polepally, Akshanth Reddy; Kumar, Venkata V Pavan; Bhamidipati, Ravikanth; Kota, Jagannath; Naveed, Shaik Abdul; Reddy, Karnati Harinder; Mamidi, Rao N V S; Selvakumar, N; Mullangi, Ramesh; Srinivas, Nuggehally R

2008-09-01

The discovery of small-molecule novel chemical entities (NCEs) is often a complex play between appropriate structural requirements and optimization of the desired efficacy, safety and pharmacokinetic properties. One of the typical structural variants such as having an active carbon-carbon double bond (alpha, beta-unsaturated carbonyl group) in xenobiotics may lead to stability issues. Such functionalities are extremely reactive, paving way to nucleophilic attack by endogenously occurring and ubiquitous nucleophiles like thiols. While it is easy to make a unilateral decision to not pursue the development of xenobiotics with such functionalities, we question the wisdom of such a decision. In this report, we present in vitro methodologies with appropriate examples to illustrate the ease of assessing the reactivity of the xenobiotics containing double bonds with a known nucleophile. The protocols involve simple reaction procedures followed by measurements using standard laboratory equipments (UV spectrophotometer, HPLC and LC-MS). Our data suggests that not all xenobiotics with carbon-carbon double bonds readily form a Michael's adduct product with glutathione. Hence, the criterion for dropping discovery compounds because of alpha,beta-unsaturated double bonds needs to be reconsidered.

Antioxidant, antihyperglycemic, and antidyslipidemic effects of Brazilian-native fruit extracts in an animal model of insulin resistance.

PubMed

de Souza Cardoso, Juliane; Oliveira, Pathise Souto; Bona, Natália Pontes; Vasconcellos, Flávia Aleixo; Baldissarelli, Jucimara; Vizzotto, Marcia; Soares, Mayara Sandrielly Pereira; Ramos, Vanessa Plasse; Spanevello, Roselia Maria; Lencina, Claiton Leoneti; Tavares, Rejane Giacomelli; Stefanello, Francieli Moro

2018-12-01

Insulin resistance (IR) plays an important role in the development of many diseases, such as diabetes mellitus. Therefore, the aim of the present study was to evaluate the effects of the extracts from fruits native to Brazil on metabolic parameters and hepatic oxidative markers in an animal model of insulin resistance induced by dexamethasone (DEX). Wistar rats received water or extracts of Eugenia uniflora or Psidium cattleianum, once a day for 21 days. For the last 5 days, the rats received an intraperitoneal injection of saline or DEX. DEX caused a reduction in body weight gain and relative pancreatic weight, as well as glucose intolerance, and an increase in serum glucose and triacylglycerol levels. The extracts were found to prevent hyperglycemia and hypertriglyceridemia. DEX caused an increase in the levels of thiobarbituric acid-reactive substances and reactive oxygen species production in the liver of rats, and both extracts prevented these changes. In addition, hepatic glutathione peroxidase activity was reduced by DEX. However, total thiol content and activities of catalase, superoxide dismutase, and delta-aminolevulinate dehydratase were not altered in any of the tested groups. Fruit extracts of E. uniflora and P. cattleianum exhibited considerable antihyperglycemic, antidyslipidemic, and antioxidant effects, and may be useful in the therapeutic management of alterations due to IR.

Synthesis, chemical reactivity as Michael acceptors, and biological potency of monocyclic cyanoenones, novel and highly potent anti-inflammatory and cytoprotective agents.

PubMed

Zheng, Suqing; Santosh Laxmi, Y R; David, Emilie; Dinkova-Kostova, Albena T; Shiavoni, Katherine H; Ren, Yanqing; Zheng, Ying; Trevino, Isaac; Bumeister, Ronald; Ojima, Iwao; Wigley, W Christian; Bliska, James B; Mierke, Dale F; Honda, Tadashi

2012-05-24

Novel monocyclic cyanoenones examined to date display unique features regarding chemical reactivity as Michael acceptors and biological potency. Remarkably, in some biological assays, the simple structure is more potent than pentacyclic triterpenoids (e.g., CDDO and bardoxolone methyl) and tricycles (e.g., TBE-31). Among monocyclic cyanoenones, 1 is a highly reactive Michael acceptor with thiol nucleophiles. Furthermore, an important feature of 1 is that its Michael addition is reversible. For the inhibition of NO production, 1 shows the highest potency. Notably, its potency is about three times higher than CDDO, whose methyl ester (bardoxolone methyl) is presently in phase III clinical trials. For the induction of NQO1, 1 also demonstrated the highest potency. These results suggest that the reactivity of these Michael acceptors is closely related to their biological potency. Interestingly, in LPS-stimulated macrophages, 1 causes apoptosis and inhibits secretion of TNF-α and IL-1β with potencies that are higher than those of bardoxolone methyl and TBE-31.

Mechanical Characterization of Polydopamine-Assisted Silver Deposition on Polymer Substrates

NASA Astrophysics Data System (ADS)

Cordes, Amanda Laurence

Inspired by the adhesive proteins in marine mussels, polydopamine has become a popular adhesive ad-layer for surface functionalization of a variety of substrates. Based on the chemistry of the dopamine monomer, amine and thiol functional groups are hypothesized to increase adhesion between polymer substrates and polydopamine thin films. This hypothesis was the central motivation for development of a tailorable thiol-ene system in order to study the effects of substrate chemistry on polydopamine adhesion. While polydopamine-adhered silver has been studied on a variety of substrates, no in depth mechanical characterization has been performed and to date, no research has been published on thiol-enes coated in polydopamine-adhered silver. The purpose of this study was to characterize the mechanical durability and adhesion properties of a polydopamine-adhered silver film on commercial substrates and a tailorable thiol-ene system. Polydopamine and silver coatings were deposited on a variety of polymer substrates through a simple dip-coat process. The polydopamine forms a thin uniform adhesive layer and the silver deposits in a discontinuous manner with a nanoparticle sized base layer covering the full surface and micron-sized clusters adhered sporadically on top. Mechanical tensile testing was performed to characterize the durability of the silver coating on commercial polymers. Coated nylon and HDPE showed no signs of degradation or delamination of the polydopamine-adhered silver coating up to 30% strain although both substrates showed large plastic deformation. Peel tests were performed on both commercial polymers as well as a tailorable thiol-ene system. Results support the hypothesis that polydopamine adhesion is increased with the presence of functional groups. Parts of the HDPE sample were cleanly peeled, but silver patches were left sporadically across the surface pointing to weaker adhesion between polyethylene and polydopamine. A high adhesive strength tape was used on nylon and the thiol-ene polymers and removed some of the large clusters but was ineffective at removing the particle base layer. The silver base layer remained firmly attached on the surface after multiple rounds of peel testing. With the addition of functional groups in the polymer makeup, the adhesion strength of polydopamine-adhered silver coatings can be increased to create a mechanically durable and adhesively robust silver coating.

Surface Functionalization of Diamond Films by Photoreaction of Elemental Sulfur and Their Surface Properties

NASA Astrophysics Data System (ADS)

Nakamura, Takako; Ohana, Tsuguyori

2012-08-01

A useful method for direct sulfurization of diamond film surfaces by photoreaction of elemental sulfur was developed. The introduction of thiol groups onto the diamond films was confirmed by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, and scanning electron microscopy (SEM) analyses. The sulfur-modified diamond films attached to gold nanoparticles by self-assembly. The degrees of thiol group introduction and gold attachment were found to depend on photoirradiation time by monitoring by XPS. The gold-modified diamond film was observed to act as a surface-enhanced Raman scattering substrate for measurement of picric acid.

Synthesis of Programmable Main-chain Liquid-crystalline Elastomers Using a Two-stage Thiol-acrylate Reaction.

PubMed

Saed, Mohand O; Torbati, Amir H; Nair, Devatha P; Yakacki, Christopher M

2016-01-19

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline elastomers (LCEs) with facile control over network structure and programming of an aligned monodomain. Tailored LCE networks were synthesized using routine mixing of commercially available starting materials and pouring monomer solutions into molds to cure. An initial polydomain LCE network is formed via a self-limiting thiol-acrylate Michael-addition reaction. Strain-to-failure and glass transition behavior were investigated as a function of crosslinking monomer, pentaerythritol tetrakis(3-mercaptopropionate) (PETMP). An example non-stoichiometric system of 15 mol% PETMP thiol groups and an excess of 15 mol% acrylate groups was used to demonstrate the robust nature of the material. The LCE formed an aligned and transparent monodomain when stretched, with a maximum failure strain over 600%. Stretched LCE samples were able to demonstrate both stress-driven thermal actuation when held under a constant bias stress or the shape-memory effect when stretched and unloaded. A permanently programmed monodomain was achieved via a second-stage photopolymerization reaction of the excess acrylate groups when the sample was in the stretched state. LCE samples were photo-cured and programmed at 100%, 200%, 300%, and 400% strain, with all samples demonstrating over 90% shape fixity when unloaded. The magnitude of total stress-free actuation increased from 35% to 115% with increased programming strain. Overall, the two-stage TAMAP methodology is presented as a powerful tool to prepare main-chain LCE systems and explore structure-property-performance relationships in these fascinating stimuli-sensitive materials.

Use of an Anaerobic Chamber Environment for the Assay of Endogenous Cellular Protein-Tyrosine Phosphatase Activities

PubMed Central

Zhu, Li

2002-01-01

Protein-tyrosine phosphatases (PTPases) have a catalytic cysteine residue whose reduced state is integral to the reaction mechanism. Since exposure to air can artifactually oxidize this highly reactive thiol, PTPase assays have typically used potent reducing agents to reactivate the enzymes present; however, this approach does not allow for the measurement of the endogenous PTPase activity directly isolated from the in vivo cellular environment. Here we provide a method for using an anaerobic chamber to preserve the activity of the total PTPase complement in a tissue lysate or of an immunoprecipitated PTPase homolog to characterize their endogenous activation state. Comparison with a sample treated with biochemical reducing agents allows the determination of the activatable (reducible) fraction of the endogenous PTPase pool. PMID:12734574

The role of labile sulfur compounds in thermochemical sulfate reduction

USGS Publications Warehouse

Amrani, A.; Zhang, T.; Ma, Q.; Ellis, G.S.; Tang, Y.

2008-01-01

The reduction of sulfate to sulfide coupled with the oxidation of hydrocarbons to carbon dioxide, commonly referred to as thermochemical sulfate reduction (TSR), is an important abiotic alteration process that most commonly occurs in hot carbonate petroleum reservoirs. In the present study we focus on the role that organic labile sulfur compounds play in increasing the rate of TSR. A series of gold-tube hydrous pyrolysis experiments were conducted with n-octane and CaSO4 in the presence of reduced sulfur (e.g. H2S, S??, organic S) at temperatures of 330 and 356 ??C under a constant confining pressure. The in-situ pH was buffered to 3.5 (???6.3 at room temperature) with talc and silica. For comparison, three types of oil with different total S and labile S contents were reacted under similar conditions. The results show that the initial presence of organic or inorganic sulfur compounds increases the rate of TSR. However, organic sulfur compounds, such as 1-pentanethiol or diethyldisulfide, were significantly more effective in increasing the rate of TSR than H2S or elemental sulfur (on a mole S basis). The increase in rate is achieved at relatively low concentrations of 1-pentanethiol, less than 1 wt% of the total n-octane, which is comparable to the concentration of organic S that is common in many oils (???0.3 wt%). We examined several potential reaction mechanisms to explain the observed reactivity of organic LSC. First, the release of H2S from the thermal degradation of thiols was discounted as an important mechanism due to the significantly greater reactivity of thiol compared to an equivalent amount of H2S. Second, we considered the generation of olefines in association with the elimination of H2S during thermal degradation of thiols because olefines are much more reactive than n-alkanes during TSR. In our experiments, olefines increased the rate of TSR, but were less effective than 1-pentanethiol and other organic LSC. Third, the thermal decomposition of organic LSC creates free-radicals that in turn might initiate a radical chain-reaction that creates more reactive species. Experiments involving radical initiators, such as diethyldisulfide and benzyldisulfide, did not show an increase in reactivity compared to 1-pentanethiol. Therefore, we conclude that none of these can sufficiently explain our observations of the initial stages of TSR; they may, however, be important in the later stages. In order to gain greater insight into the potential mechanism for the observed reactivity of these organic sulfur compounds during TSR, we applied density functional theory-based molecular modeling techniques to our system. The results of these calculations indicate that 1-pentanethiol or its thermal degradation products may directly react with sulfate and reduce the activation energy required to rupture the first S-O bond through the formation of a sulfate ester. This study demonstrates the importance of labile sulfur compounds in reducing the onset timing and temperature of TSR. It is therefore essential that labile sulfur concentrations are taken into consideration when trying to make accurate predictions of TSR kinetics and the potential for H2S accumulation in petroleum reservoirs. ?? 2008.

The role of labile sulfur compounds in thermochemical sulfate reduction

NASA Astrophysics Data System (ADS)

Amrani, Alon; Zhang, Tongwei; Ma, Qisheng; Ellis, Geoffrey S.; Tang, Yongchun

2008-06-01

The reduction of sulfate to sulfide coupled with the oxidation of hydrocarbons to carbon dioxide, commonly referred to as thermochemical sulfate reduction (TSR), is an important abiotic alteration process that most commonly occurs in hot carbonate petroleum reservoirs. In the present study we focus on the role that organic labile sulfur compounds play in increasing the rate of TSR. A series of gold-tube hydrous pyrolysis experiments were conducted with n-octane and CaSO4 in the presence of reduced sulfur (e.g. H2S, S°, organic S) at temperatures of 330 and 356 °C under a constant confining pressure. The in-situ pH was buffered to 3.5 (∼6.3 at room temperature) with talc and silica. For comparison, three types of oil with different total S and labile S contents were reacted under similar conditions. The results show that the initial presence of organic or inorganic sulfur compounds increases the rate of TSR. However, organic sulfur compounds, such as 1-pentanethiol or diethyldisulfide, were significantly more effective in increasing the rate of TSR than H2S or elemental sulfur (on a mole S basis). The increase in rate is achieved at relatively low concentrations of 1-pentanethiol, less than 1 wt% of the total n-octane, which is comparable to the concentration of organic S that is common in many oils (∼0.3 wt%). We examined several potential reaction mechanisms to explain the observed reactivity of organic LSC. First, the release of H2S from the thermal degradation of thiols was discounted as an important mechanism due to the significantly greater reactivity of thiol compared to an equivalent amount of H2S. Second, we considered the generation of olefines in association with the elimination of H2S during thermal degradation of thiols because olefines are much more reactive than n-alkanes during TSR. In our experiments, olefines increased the rate of TSR, but were less effective than 1-pentanethiol and other organic LSC. Third, the thermal decomposition of organic LSC creates free-radicals that in turn might initiate a radical chain-reaction that creates more reactive species. Experiments involving radical initiators, such as diethyldisulfide and benzyldisulfide, did not show an increase in reactivity compared to 1-pentanethiol. Therefore, we conclude that none of these can sufficiently explain our observations of the initial stages of TSR; they may, however, be important in the later stages. In order to gain greater insight into the potential mechanism for the observed reactivity of these organic sulfur compounds during TSR, we applied density functional theory-based molecular modeling techniques to our system. The results of these calculations indicate that 1-pentanethiol or its thermal degradation products may directly react with sulfate and reduce the activation energy required to rupture the first S-O bond through the formation of a sulfate ester. This study demonstrates the importance of labile sulfur compounds in reducing the onset timing and temperature of TSR. It is therefore essential that labile sulfur concentrations are taken into consideration when trying to make accurate predictions of TSR kinetics and the potential for H2S accumulation in petroleum reservoirs.

A Study of Functional Polymer Colloids Prepared Using Thiol-Ene/Yne Click Chemistry

NASA Astrophysics Data System (ADS)

Durham, Olivia Z.

This project demonstrates the first instance of thiol-ene chemistry as the polymerization method for the production of polymer colloids in two-phase heterogeneous suspensions, miniemulsions, and emulsions. This work was also expanded to thiol-yne chemistry for the production of polymer particles containing increased crosslinking density. The utility of thiol-ene and thiol-yne chemistries for polymerization and polymer modification is well established in bulk systems. These reactions are considered 'click' reactions, which can be defined as processes that are both facile and simple, offering high yields with nearly 100% conversion, no side products, easy product separation, compatibility with a diverse variety of commercially available starting materials, and orthogonality with other chemistries. In addition, thiol-ene and thiol-yne chemistry follow a step-growth mechanism for the development of highly uniform polymer networks, where polymer growth is dependent on the coupling of functional groups. These step-growth polymerization systems are in stark contrast to the chain-growth mechanisms of acrylic and styrenic monomers that have dominated the field of conventional heterogeneous polymerizations. Preliminary studies evaluated the mechanism of particle production in suspension and miniemulsion systems. Monomer droplets were compared to the final polymer particles to confirm that particle growth occurred through the polymerization of monomer droplets. Additional parameters examined include homogenization energy (mechanical mixing), diluent species and concentration, and monomer content. These reactions were conducted using photoinitiation to yield particles in a matter of minutes with diameters in the size range of several microns to hundreds of microns in suspensions or submicron particles in miniemulsions. Improved control over the particle size and size distribution was examined through variation of reaction parameters. In addition, a method of seeded suspension polymerization was attempted. This project was further expanded through an extensive evaluation of stabilizers in thiol-ene suspension polymerizations. The scope of stabilizers used included synthetic surfactants (ionic and nonionic), natural gums, and colloidal silica (Pickering stabilization). Suspension polymerizations were further expanded to include thiol-yne chemistry for the evaluation of polymer composition and thermal properties. In addition, polymer particles with excess ene, yne, or thiol functionality were successfully developed to demonstrate the potential for further functionalization. The self-limiting behavior of thiol-ene/yne reactions allows for successful synthesis of functional polymer colloids using off-stoichiometric amounts of monomers. This capacity to control functionality is illustrated through the creation of fluorescent polymer particles using both an in situ thiol-ene polymerization reaction with a vinyl chromophore as well as through post-polymerization modification of thiol-ene and thiol-yne polymers with excess thiol functionality via thiol-isocyanate chemistry. To produce smaller polymer particles without the need for intense homogenization energy or high stabilizer concentrations, an emulsion polymerization system was implemented using a water soluble-thermal initiator. It was found that unlike thiol-ene suspensions, which are limited to crosslinked systems, thiol-ene emulsion polymerizations allowed for the production of polymer particles comprised of either crosslinked or linear polymer networks. For the crosslinked systems, various anionic SDS surfactant concentrations were examined to observe the influence on particle size. In linear polymer systems, variations in polymer composition were examined. Preliminary studies performed with a monomer with an ethylene glycol-like structure indicated that the synthesis of polymer particles with narrower size distributions compared to any of the other emulsion compositions was possible. Finally, thiol-ene chemistry was also employed toward the synthesis of degradable polyanhydride polymer particles. Unlike the aforementioned studies, the approach to particle synthesis was conducted by using a premade thiol-ene polymer. Various linear thiol-ene polyanhydrides were emulsified in water or buffered solutions via sonication. Polymer latex was obtained upon solvent evaporation of the dichloromethane (DCM) solvent used to solubilize the polymer. In this work, variation of polymer composition as well as degradation was examined. Additional experiments included a study of the release of Rhodamine B dye, functionalization of the linear polymers, and studies involving the delay of degradation through the incorporation of crosslinking in the polymer particles. The projects presented herein provide an innovative approach to the synthesis of polymer colloids using thiol-ene and thiol-yne 'click' chemistry in both heterogeneous polymerizations as well as through solvent evaporation of premade polymer solutions. Polymer colloids prove to be an area of great interest for numerous applications that encompass various areas involving biomedical and industrial technologies including paints and coatings, cosmetics, diagnostics, and drug delivery. Improvements in methods of chemical synthesis as well as advances in the tailoring of material properties are of utmost importance for the ever increasing demands of new technologies and educational enlightenment.

Generator-specific targets of mitochondrial reactive oxygen species.

PubMed

Bleier, Lea; Wittig, Ilka; Heide, Heinrich; Steger, Mirco; Brandt, Ulrich; Dröse, Stefan

2015-01-01

To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for "signaling" and "damaging" ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase; cytochrome bc1 complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H2O2) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress. Copyright © 2014 Elsevier Inc. All rights reserved.

Role of Heme Oxygenase-1 in Polymyxin B-Induced Nephrotoxicity in Rats

PubMed Central

Watanabe, Mirian

2012-01-01

Polymyxin B (PMB) is a cationic polypeptide antibiotic with activity against multidrug-resistant Gram-negative bacteria. PMB-induced nephrotoxicity consists of direct toxicity to the renal tubules and the release of reactive oxygen species (ROS) with oxidative damage. This study evaluated the nephroprotective effect of heme oxygenase-1 (HO-1) against PMB-induced nephrotoxicity in rats. Adult male Wistar rats, weighing 286 ± 12 g, were treated intraperitoneally once a day for 5 days with saline, hemin (HO-1 inducer; 10 mg/kg), zinc protoporphyrin (ZnPP) (HO-1 inhibitor; 50 μmol/kg, administered before PMB on day 5), PMB (4 mg/kg), PMB plus hemin, and PMB plus ZnPP. Renal function (creatinine clearance, Jaffe method), urinary peroxides (ferrous oxidation of xylenol orange version 2 [FOX-2]), urinary thiobarbituric acid-reactive substances (TBARS), renal tissue thiols, catalase activity, and renal tissue histology were analyzed. The results showed that PMB reduced creatinine clearance (P < 0.05), with an increase in urinary peroxides and TBARS. The PMB toxicity caused a reduction in catalase activity and thiols (P < 0.05). Hemin attenuated PMB nephrotoxicity by increasing the catalase antioxidant activity (P < 0.05). The combination of PMB and ZnPP incremented the fractional interstitial area of renal tissue (P < 0.05), and acute tubular necrosis in the cortex area was also observed. This is the first study demonstrating the protective effect of HO-1 against PMB-induced nephrotoxicity. PMID:22802257

Claudin-2-mediated cation and water transport share a common pore

PubMed Central

Rosenthal, Rita; Günzel, Dorothee; Krug, Susanne M.; Schulzke, Jörg-Dieter; Fromm, Michael; Yu, Alan S.L.

2016-01-01

Aim Claudin-2 is a tight junction protein typically located in “leaky” epithelia exhibiting large paracellular permeabilities like small intestine and proximal kidney tubule. Former studies revealed that claudin-2 forms paracellular channels for small cations like sodium and potassium and also paracellular channels for water. This study analyzes whether the diffusive transport of sodium and water occurs through a common pore of the claudin-2 channel. Methods Wild-type claudin-2 and different claudin-2 mutants were expressed in MDCK I kidney tubule cells using an inducible system. Ion and water permeability and the effect of blocking reagents on both were investigated on different clones of the mutants. Results Neutralization of a negatively charged cation interaction site in the pore with the mutation, D65N, decreased both, sodium permeability and water permeability. Claudin-2 mutants (I66C and S68C) with substitution of the pore-lining amino acids with cysteine were used to test the effect of steric blocking of the claudin-2 pore by thiol-reactive reagents. Addition of thiol-reactive reagents to these mutants simultaneously decreased conductance and water permeability. Remarkably, all experimental perturbations caused parallel changes in ion conductance and water permeability, disproving different or independent passage pathways. Conclusion Our results indicate that claudin-2-mediated cation and water transport are frictionally coupled and share a common pore. This pore is lined and determined in permeability by amino acid residues of the first extracellular loop of claudin-2. PMID:27359349

Glutathione Redox Control of Asthma: From Molecular Mechanisms to Therapeutic Opportunities

PubMed Central

Jones, Dean P.; Brown, Lou Ann S.

2012-01-01

Abstract Asthma is a chronic inflammatory disorder of the airways associated with airway hyper-responsiveness and airflow limitation in response to specific triggers. Whereas inflammation is important for tissue regeneration and wound healing, the profound and sustained inflammatory response associated with asthma may result in airway remodeling that involves smooth muscle hypertrophy, epithelial goblet-cell hyperplasia, and permanent deposition of airway extracellular matrix proteins. Although the specific mechanisms responsible for asthma are still being unraveled, free radicals such as reactive oxygen species and reactive nitrogen species are important mediators of airway tissue damage that are increased in subjects with asthma. There is also a growing body of literature implicating disturbances in oxidation/reduction (redox) reactions and impaired antioxidant defenses as a risk factor for asthma development and asthma severity. Ultimately, these redox-related perturbations result in a vicious cycle of airway inflammation and injury that is not always amenable to current asthma therapy, particularly in cases of severe asthma. This review will discuss disruptions of redox signaling and control in asthma with a focus on the thiol, glutathione, and reduced (thiol) form (GSH). First, GSH synthesis, GSH distribution, and GSH function and homeostasis are discussed. We then review the literature related to GSH redox balance in health and asthma, with an emphasis on human studies. Finally, therapeutic opportunities to restore the GSH redox balance in subjects with asthma are discussed. Antioxid. Redox Signal. 17, 375–408. PMID:22304503

Iodoacetyl-functionalized pullulan: A supplemental enhancer for single-domain antibody-polyclonal antibody sandwich enzyme-linked immunosorbent assay for detection of survivin.

PubMed

Matsushita, Takahiko; Arai, Hidenao; Koyama, Tetsuo; Hatano, Ken; Nemoto, Naoto; Matsuoka, Koji

2017-11-01

Survivin, an inhibitor of the apoptosis protein family, is a potent tumor marker for diagnosis and prognosis. The enzyme-linked immunosorbent assay (ELISA) is one of the methods that has been used for detection of survivin. However, ELISA has several disadvantages caused by the use of conventional antibodies, and we have therefore been trying to develop a novel ELISA system using camelid single-domain antibodies (VHHs) as advantageous replacements. Here we report a supplemental approach to improve the VHH-polyclonal antibody sandwich ELISA for survivin detection. Iodoacetyl-functionalized pullulan was synthesized, and its thiol reactivity was characterized by a model reaction with l-cysteine. The thiophilic pullulan was applied to an immunoassay asan additive upon coating of standard assay plates with an anti-survivin VHH fusion protein with C-terminal cysteine. The results showed that the mole ratio of the additive to VHH had a significant effect on the consequent response. Mole ratios of 0.07, 0.7, and 7 led to 90% lower, 15% higher, and 69% lower responses, respectively, than the response of a positive control in which no additive was used. The background levels observed in any additive conditions were as low as that of a negative control lacking both VHH and the additive. These results indicate the applicability of the thiol-reactive pullulan as a response enhancer to VHH-based ELISA. Copyright © 2017 Elsevier Ltd. All rights reserved.

Implementation of molecularly imprinted polymer beads for surface enhanced Raman detection.

PubMed

Kamra, Tripta; Zhou, Tongchang; Montelius, Lars; Schnadt, Joachim; Ye, Lei

2015-01-01

Molecularly imprinted polymers (MIPs) have a predesigned molecular recognition capability that can be used to build robust chemical sensors. MIP-based chemical sensors allow label-free detection and are particularly interesting due to their simple operation. In this work we report the use of thiol-terminated MIP microspheres to construct surfaces for detection of a model organic analyte, nicotine, by surface enhanced Raman scattering (SERS). The nicotine-imprinted microspheres are synthesized by RAFT precipitation polymerization and converted into thiol-terminated microspheres through aminolysis. The thiol groups on the MIP surface allow the microspheres to be immobilized on a gold-coated substrate. Three different strategies are investigated to achieve surface enhanced Raman scattering in the vicinity of the imprinted sites: (1) direct sputtering of gold nanoparticles, (2) immobilization of gold colloids through the MIP's thiol groups, and (3) trapping of the MIP microspheres in a patterned SERS substrate. For the first time we show that large MIP microspheres can be turned into selective SERS surfaces through the three different approaches of assembly. The MIP-based sensing surfaces are used to detect nicotine to demonstrate the proof of concept. As synthesis and surface functionalization of MIP microspheres and nanoparticles are well established, the methods reported in this work are handy and efficient for constructing label-free chemical sensors, in particular for those based on SERS detection.

Water-soluble polymers and compositions thereof

DOEpatents

Smith, B.F.; Robison, T.W.; Gohdes, J.W.

1999-04-06

Water-soluble polymers including functionalization from the group of amino groups, carboxylic acid groups, phosphonic acid groups, phosphonic ester groups, acylpyrazolone groups, hydroxamic acid groups, aza crown ether groups, oxy crown ethers groups, guanidinium groups, amide groups, ester groups, aminodicarboxylic groups, permethylated polyvinylpyridine groups, permethylated amine groups, mercaptosuccinic acid groups, alkyl thiol groups, and N-alkylthiourea groups are disclosed.

Water-soluble polymers and compositions thereof

DOEpatents

Smith, Barbara F.; Robison, Thomas W.; Gohdes, Joel W.

2002-01-01

Water-soluble polymers including functionalization from the group of amino groups, carboxylic acid groups, phosphonic acid groups, phosphonic ester groups, acylpyrazolone groups, hydroxamic acid groups, aza crown ether groups, oxy crown ethers groups, guanidinium groups, amide groups, ester groups, aminodicarboxylic groups, permethylated polvinylpyridine groups, permethylated amine groups, mercaptosuccinic acid groups, alkyl thiol groups, and N-alkylthiourea groups are disclosed.

Water-soluble polymers and compositions thereof

DOEpatents

Smith, Barbara F.; Robison, Thomas W.; Gohdes, Joel W.

1999-01-01

Water-soluble polymers including functionalization from the group of amino groups, carboxylic acid groups, phosphonic acid groups, phosphonic ester groups, acylpyrazolone groups, hydroxamic acid groups, aza crown ether groups, oxy crown ethers groups, guanidinium groups, amide groups, ester groups, aminodicarboxylic groups, permethylated polyvinylpyridine groups, permethylated amine groups, mercaptosuccinic acid groups, alkyl thiol groups, and N-alkylthiourea groups are disclosed.

Silver Coordination Polymers for Prevention of Implant Infection: Thiol Interaction, Impact on Respiratory Chain Enzymes, and Hydroxyl Radical Induction▿ †

PubMed Central

Gordon, Oliver; Vig Slenters, Tünde; Brunetto, Priscilla S.; Villaruz, Amer E.; Sturdevant, Daniel E.; Otto, Michael; Landmann, Regine; Fromm, Katharina M.

2010-01-01

Prosthetic joint replacements are used increasingly to alleviate pain and improve mobility of the progressively older and more obese population. Implant infection occurs in about 5% of patients and entails significant morbidity and high social costs. It is most often caused by staphylococci, which are introduced perioperatively. They are a source of prolonged seeding and difficult to treat due to antibiotic resistance; therefore, infection prevention by prosthesis coating with nonantibiotic-type anti-infective substances is indicated. A renewed interest in topically used silver has fostered development of silver nanoparticles, which, however, present a potential health hazard. Here we present new silver coordination polymer networks with tailored physical and chemical properties as nanostructured coatings on metallic implant substrates. These compounds exhibited strong biofilm sugar-independent bactericidal activity on in vitro-grown biofilms and prevented murine Staphylococcus epidermidis implant infection in vivo with slow release of silver ions and limited transient leukocyte cytotoxicity. Furthermore, we describe the biochemical and molecular mechanisms of silver ion action by gene screening and by targeting cell metabolism of S. epidermidis at different levels. We demonstrate that silver ions inactivate enzymes by binding sulfhydryl (thiol) groups in amino acids and promote the release of iron with subsequent hydroxyl radical formation by an indirect mechanism likely mediated by reactive oxygen species. This is the first report investigating the global metabolic effects of silver in the context of a therapeutic application. We anticipate that the compounds presented here open a new treatment field with a high medical impact. PMID:20660682

Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5′-protected thiol function and a 3′-amino group

PubMed Central

Aubert, Yves; Bourgerie, Sylvain; Meunier, Laurent; Mayer, Roger; Roche, Annie-Claude; Monsigny, Michel; Thuong, Nguyen T.; Asseline, Ulysse

2000-01-01

A new deprotection procedure enables a medium scale preparation of phosphodiester and phosphorothioate oligonucleotides substituted with a protected thiol function at their 5′-ends and an amino group at their 3′-ends in good yield (up to 72 OD units/µmol for a 19mer phosphorothioate). Syntheses of 3′-amino-substituted oligonucleotides were carried out on a modified support. A linker containing the thioacetyl moiety was manually coupled in two steps by first adding its phosphoramidite derivative in the presence of tetrazole followed by either oxidation or sulfurization to afford the bis-derivatized oligonucleotide bound to the support. Deprotection was achieved by treating the fully protected oligonucleotide with a mixture of 2,2′-dithiodipyridine and concentrated aqueous ammonia in the presence of phenol and methanol. This procedure enables (i) cleavage of the oligonucleotide from the support, releasing the oligonucleotide with a free amino group at its 3′-end, (ii) deprotection of the phosphate groups and the amino functions of the nucleic bases, as well as (iii) transformation of the 5′-terminal S-acetyl function into a dithiopyridyl group. The bis-derivatized phosphorothioate oligomer was further substituted through a two-step procedure: first, the 3′-amino group was reacted with fluorescein isothiocyanate to yield a fluoresceinylated oligonucleotide; the 5′-dithiopyridyl group was then quantitatively reduced to give a free thiol group which was then substituted by reaction with an Nα-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide–oligonucleotide conjugate. PMID:10637335

Atmospheric reactivity of alcohols, thiols and fluoroalcohols with chlorine atoms

NASA Astrophysics Data System (ADS)

Garzon Ruiz, Andres

Alcohols, thiols and fluoroalcohols are volatile organic compounds (VOCs) which are emitted to the atmosphere from both natural (vegetation, oceans, volcanoes, etc.) and anthropogenic sources (fuels, solvents, wastewater, incinerators, refrigerants, etc.). These pollutants can be eliminated from the troposphere by deposition on the terrestrial surface, direct photolysis or reaction with different tropospheric oxidants. Reactions of VOCs with tropospheric oxidants are involved in the well-known atmospheric phenomenon of photochemical smog or the production of tropospheric ozone. The oxidation of these VOCs in the troposphere is mainly initiated by reaction with OH radicals during the daytime and with NO radicals at night. However, in recent years, the oxidation by chlorine atoms (Cl) has gained great importance in the study of atmospheric reactions because they may exert some influence in the boundary layer, particularly in marine and coastal environments. In general, Cl atoms are much more reactive species than OH and NO; radicals and therefore low concentrations of Cl may compete with OH and NO3 in hydrocarbon oxidation processes. The main source of tropospheric Cl atoms is believed to be the photolysis of chlorine-containing molecules generated by heterogeneous reactions of sea salt aerosols. It has also been proposed that Cl atoms, produced in the photolysis of Cl2 emitted from industrial processes, may enhance hydrocarbon oxidation rates and ozone production in urban environments. In this work, a kinetic, theoretical and mechanistic study of the reaction of several alcohols, thiols, and fluoroalcohols with Cl atoms has been carried out. Pulsed laser photolysis-fluorescence resonance (PLP-RF) technique was used for the kinetic study as a function of temperature and pressure. An environmental chamber-Fourier transform infrared (FTIR) system was also employed in the kinetic studies. Tropospheric lifetimes of these pollutants were estimated using obtained kinetic data. Products of these reactions were determined by FTIR and derivatization with 2,4-dinitrophenylhydrazine and HPLC analysis. Finally, in order to determine the main reaction pathways a theoretical study at QCISD(T)/6-311G**//MP2(Full)(6-311C** level was performed for each reaction.

Recent advances in self-assembled monolayers based biomolecular electronic devices.

PubMed

Arya, Sunil K; Solanki, Pratima R; Datta, Monika; Malhotra, Bansi D

2009-05-15

Self-assembled monolayers (SAMs) have aroused much interest due to their potential applications in biosensors, biomolecular electronics and nanotechnology. This has been largely attributed to their inherent ordered arrangement and controllable properties. SAMs can be formed by chemisorption of organic molecules containing groups like thiols, disulphides, amines, acids or silanes, on desired surfaces and can be used to fabricate biomolecular electronic devices. We focus on recent applications of organosulphur compounds (thiols) based SAMs to biomolecular electronic devices in the last about 3 years.

Application of thiol-olefin co-oxygenation methodology to a new synthesis of the 1,2,4-trioxane pharmacophore.

PubMed

O'Neill, Paul M; Mukhtar, Amira; Ward, Stephen A; Bickley, Jamie F; Davies, Jill; Bachi, Mario D; Stocks, Paul A

2004-09-02

[reaction: see text] Thiol-olefin co-oxygenation (TOCO) of substituted allylic alcohols generates alpha-hydroxyperoxides that can be condensed in situ with various ketones to afford a series of functionalized 1,2,4-trioxanes in good yields. Manipulation of the phenylsulfenyl group in 4a allows for convenient modification to the spiro-trioxane substituents, and we describe, for the first time, the preparation of a new class of antimalarial prodrug.

Colloidal Stability of Gold Nanoparticles Coated with Multithiol-Poly(ethylene glycol) Ligands: Importance of Structural Constraints of the Sulfur Anchoring Groups

DTIC Science & Technology

2013-08-13

21-45 Citric acid has been the most commonly used ligand when negatively charged surfaces or a rather loose ligand shell is required for further...biomolecules in biological serum (e.g., proteins, peptides, nucleic acids ); (5) resistance to heat treatments (e.g., >80 °C for DNA hybridization).6-8, 16...demonstrated by a variety of thiol-based ligands such as mercaptopropionic acid ,36 glutathione,37 thiol-terminated poly(ethylene glycol) (PEG-SH), 19

Highly ordered gold nanotubes using thiols at a cleavable block copolymer interface.

PubMed

Ryu, Ja-Hyoung; Park, Soojin; Kim, Bokyung; Klaikherd, Akamol; Russell, Thomas P; Thayumanavan, S

2009-07-29

We have prepared functionalized nanoporous thin films from a polystyrene-block-polyethylene oxide block copolymer, which was made cleavable due to the intervening disulfide bond. The cleavage reaction of the disulfide bond leaves behind free thiol groups inside the nanopores of polystyrene thin film. This nanoporous thin film can be used as a template for generating gold nanoring structures. This strategy can provide a facile method to form a highly ordered array of biopolymer or metal-polymer composite structures.

Covalent Incorporation of Ionic Liquid into Ion-Conductive Networks via Thiol-Ene Photopolymerization.

PubMed

Tibbits, Andrew C; Yan, Yushan S; Kloxin, Christopher J

2017-07-01

Ene-functionalized ionic liquids with a range of different cationic groups and counteranions react stoichiometrically within a tetrathiol-divinyl ether formulation within 20 minutes to form thiol-ene polymers with measurable ionic conductivities via a photoinitiated polymerization and crosslinking reaction. Dynamic mechanical analysis indicates that these networks are more spatially heterogeneous and possess higher glass transition temperatures (T g ) compared with thiol-ene formulations without charge. While tuning the molar content of ionic liquid monomer is one method for adjusting the crosslink and charge densities of the thiol-ene polymeric ionic liquid networks, the presence of cation-anion interactions also plays a critical role in dictating the thermomechanical and conductive properties. Particularly, while cationic structure effects are not significant on the polymer properties, the use of a weakly coordinating hydrophobic anion (bistriflimide) instead of bromide-based networks results in an apparent decrease in hydrated ion conductivity (7.4 to 1.5 mS cm -1 ) and T g (-9.6 to -17.8 °C). © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thiol derivatization of Xanthan gum and its evaluation as a mucoadhesive polymer.

PubMed

Bhatia, Meenakshi; Ahuja, Munish; Mehta, Heena

2015-10-20

Thiol-derivatization of xanthan gum polysaccharide was carried out by esterification with mercaptopropionic acid and thioglycolic acid. Thiol-derivatization was confirmed by Fourier-transformed infra-red spectroscopy. Xanthan-mercaptopropionic acid conjugate and xanthan-thioglycolic acid conjugate were found to possess 432.68mM and 465.02mM of thiol groups as determined by Ellman's method respectively. Comparative evaluation of mucoadhesive property of metronidazole loaded buccal pellets of xanthan and thiolated xanthan gum using chicken buccal pouch membrane revealed higher ex vivo bioadhesion time of thiolated xanthan gum as compared to xanthan gum. Improved mucoadhesive property of thiolated xanthan gum over the xanthan gum can be attributed to the formation of disulfide bond between mucus and thiolated xanthan gum. In vitro release study conducted using phosphate buffer (pH 6.8) revealed a sustained release profile of metronidazole from thiolated xanthan pellets as compared to xanthan pellets. In conclusion, thiolation of xanthan improves its mucoadhesive property and sustained the release of metronidazole over a prolonged period. Copyright © 2015 Elsevier Ltd. All rights reserved.

Experimental and theoretical study of the absorption properties of thiolated diamondoids

NASA Astrophysics Data System (ADS)

Landt, Lasse; Bostedt, Christoph; Wolter, David; Möller, Thomas; Dahl, Jeremy E. P.; Carlson, Robert M. K.; Tkachenko, Boryslav A.; Fokin, Andrey A.; Schreiner, Peter R.; Kulesza, Alexander; Mitrić, Roland; Bonačić-Koutecký, Vlasta

2010-04-01

Nanoscale hybrid systems are a new class of molecular aggregates that offer numerous new possibilities in materials design. Diamondoid thiols are promising nanoscale building blocks for such hybrid systems. They allow the incorporation of functional groups and the investigation of their effects on the unique materials' properties of diamondoids. Here we combine experimental data with ab initio theory to explore the optical properties of diamondoid thiols and their dependence on size and shape. Agreement between theoretically and experimentally obtained absorption spectra allows the identification of the nature of the optical transitions that are responsible for some photophysical and photochemical processes. We show that the optical properties of diamondoid thiols in the deep UV regime depend on the functionalization site but are largely size independent. Our findings provide an explanation for the disappearance of diamondoid UV photoluminescence upon thiolation for smaller diamondoids. However, our theoretical results indicate that for larger diamondoid thiols beyond the critical size of six diamondoid cages the lowest energy transitions are characterized by diamondoidlike states suggesting that UV luminescence may be regained.

Thiolated citrus low-methoxyl pectin: Synthesis, characterization and rheological and oxidation-responsive gelling properties.

PubMed

Chen, Jinfeng; Ye, Fayin; Zhou, Yun; Zhao, Guohua

2018-02-01

In the present study, citrus low-methoxyl pectin was modified by conjugating cysteine via amide bonds, and the resultant polymer (CYS-PEC) was characterized. CYS-PEC conjugates with thiol contents varying from 77.8μmol/g to 296μmol/g were synthesized, and the successful conjugation was evidenced by elemental, and FT-IR analyses. The sulfur in CYS-PEC is predominately in the thiol form, with a minor fraction forming disulfide bonds (∼15%), which occur when thiol/disulfide interchange interrupts the intended thiolation. Both native and modified pectin dispersions exhibited strong pseudoplastic properties, and the frequency sweeps revealed them to be dispersions containing microgel particles. Dynamic viscoelastic analysis was used to determine the oxidation-response gelling capacities of polymer dispersions containing H 2 O 2 , especially those that are highly thiolated and have cross-linked gel properties. For oxidation-induced CYS-PEC gels, their gelation time, hardness, viscosity and elastic moduli and swelling-disintegration ratio are dependent on the thiol group content, H 2 O 2 concentration and polymer concentration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Crystal structure of bis-[μ-(4-meth-oxy-phen-yl)methane-thiol-ato-κ(2) S:S]bis-[chlorido-(η(6)-1-isopropyl-4-methyl-benzene)-ruthenium(II)] chloro-form disolvate.

PubMed

Stíbal, David; Süss-Fink, Georg; Therrien, Bruno

2015-10-01

The mol-ecular structure of the title complex, [Ru2(C8H9OS)2Cl2(C10H14)2]·2CHCl3 or (p-MeC6H4Pr (i) )2Ru2(SCH2-p-C6H5-OCH3)2Cl2·2CHCl3, shows inversion symmetry. The two symmetry-related Ru(II) atoms are bridged by two 4-meth-oxy-α-toluene-thiol-ato [(4-meth-oxy-phen-yl)methane-thiol-ato] units. One chlorido ligand and the p-cymene ligand complete the typical piano-stool coordination environment of the Ru(II) atom. In the crystal, the CH moiety of the chloro-form mol-ecule inter-acts with the chlorido ligand of the dinuclear complex, while one Cl atom of the solvent inter-acts more weakly with the methyl group of the bridging 4-meth-oxy-α-toluene-thiol-ato unit. This assembly leads to the formation of supra-molecular chains extending parallel to [021].

Differentiation of Sclerotinia minor depends on thiol redox state and oxidative stress.

PubMed

Patsoukis, Nikolaos; Georgiou, Christos D

2008-01-01

Sclerotial differentiation in Sclerotinia minor is associated with oxidative stress and thiol redox state. The significance of oxidative stress to sclerotial differentiation was revealed by the higher oxidative stress of S. minor compared with a nonsclerotiogenic counterpart. The effect of thiol redox state on sclerotial differentiation was shown by the antioxidant action of the thiol (-SH) group of N-acetylcysteine and cysteine and by an unknown (not antioxidant) role of glutathione (GSH) on S. minor. The nonantioxidant role of GSH was indicated by the differentiation-inhibiting and differentiation-noninhibiting actions of the GSH biosynthesis inhibitor L-buthionine-S,R-sulfoximine and the GSH biosynthesis inducer L-2-oxo-thiazolidine-4-carboxylate, respectively, and by the increase of oxidative stress they caused during the transition from the undifferentiated to differentiated state of S. minor. Moreover, N-acetylcysteine can be used as a potent nontoxic fungicide against this phytopathogenic fungus by acting as a growth-inhibiting cytotoxic oxidant and by sustaining the fungus in the undifferentiated hyphal stage, which is vulnerable to degradation by soil microorganisms.

Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound.

PubMed

Yang, Yang; Guan, Xiangming

2017-05-01

Thiols (-SH) play various roles in biological systems. They are divided into protein thiols (PSH) and non-protein thiols (NPSH). Due to the significant roles thiols play in various physiological/pathological functions, numerous analytical methods have been developed for thiol assays. Most of these methods are developed for glutathione, the major form of NPSH. Majority of these methods require tissue/cell homogenization before analysis. Due to a lack of effective thiol-specific fluorescent/fluorogenic reagents, methods for imaging and quantifying thiols in live cells are limited. Determination of an analyte in live cells can reveal information that cannot be revealed by analysis of cell homogenates. Previously, we reported a thiol-specific thiol-sulfide exchange reaction. Based on this reaction, a benzofurazan sulfide thiol-specific fluorogenic reagent was developed. The reagent was able to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. The reagent was later named as GUALY's reagent. Here we would like to report an extension of the work by synthesizing a novel benzofurazan sulfide triphenylphosphonium derivative [(((7,7'-thiobis(benzo[c][1,2,5]oxadiazole-4,4'-sulfonyl))bis(methylazanediyl))bis(butane-4,1-diyl))bis(triphenylphosphonium) (TBOP)]. Like GUALY's reagent, TBOP is a thiol-specific fluorogenic agent that is non-fluorescent but forms fluorescent thiol adducts in a thiol-specific fashion. Different than GUALY's reagent, TBOP reacts only with NPSH but not with PSH. TBOP was effectively used to image and quantify NPSH in live cells using fluorescence microscopy. TBOP is a complementary reagent to GUALY's reagent in determining the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions in live cells through fluorescence microscopy. Graphical Abstract Live cell imaging and quantification of non-protein thiols by TBOP.

Controlled free radical attack in the apoplast: a hypothesis for roles of O, N and S species in regulatory and polysaccharide cleavage events during rapid abscission by Azolla.

PubMed

Cohen, Michael F; Gurung, Sushma; Fukuto, Jon M; Yamasaki, Hideo

2014-03-01

Shedding of organs by abscission is a key terminal step in plant development and stress responses. Cell wall (CW) loosening at the abscission zone can occur through a combination chain breakage of apoplastic polysaccharides and tension release of cellulose microfibrils. Two distinctly regulated abscission cleavage events are amenable to study in small water ferns of the genus Azolla; one is a rapid abscission induced by environmental stimuli such as heat or chemicals, and the other is an ethylene-induced process occurring more slowly through the action of hydrolytic enzymes. Although free radicals are suggested to be involved in the induction of rapid root abscission, its mechanism is not fully understood. The apoplast contains peroxidases, metal-binding proteins and phenolic compounds that potentially generate free radicals from H2O2 to cleave polysaccharides in the CW and middle lamella. Effects of various thiol-reactive agents implicate the action of apoplastic peroxidases having accessible cysteine thiols in rapid abscission. The Ca(2+) dependency of rapid abscission may reflect the stabilization Ca(2+) confers to peroxidase structure and binding to pectin. To spur further investigation, we present a hypothetical model for small signaling molecules H2O2 and NO and their derivatives in regulating, via modification of putative protein thiols, free radical attack of apoplastic polysaccharides. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Protective effect and mechanism of glutaredoxin 1 on coronary arteries endothelial cells damage induced by high glucose.

PubMed

Li, Shuyan; Sun, Yan; Qi, Xiaodan; Shi, Yan; Gao, Han; Wu, Qi; Liu, Xiucai; Yu, Haitao; Zhang, Chunjing

2014-01-01

In recent years, diabetes and its associated complications have become a major public health concern. The cardiovascular risk increases significantly in diabetes patients. It is a complex disease characterized by multiple metabolic derangements and is known to impair cardiac function by disrupting the balance between pro-oxidants and antioxidants at the cellular level. The subsequent generation of reactive oxygen species (ROS) and accompanying oxidative stress are hallmarks of the molecular mechanisms responsible for cardiovascular disease. Protein thiols act as redox-sensitive switches and are believed to be a key element in maintaining the cellular redox balance. The redox state of protein thiols is regulated by oxidative stress and redox signaling and is important to cellular functions. The potential of the thiol-disulfide oxidoreductase enzymes (thioredoxin and glutaredoxin systems) in defense against oxidative stress has been noted previously. Increasing evidence demonstrates that glutaredoxin 1 (Grx1), a cytosolic enzyme responsible for the catalysis of protein deglutathionylation, plays distinct roles in inflammation and apoptosis by inducing changes in the cellular redox system. This study investigates whether and how Grx1 protects coronary artery vascular endothelial cells against high glucose (HG) induced damage. Results indicate that the activation of eNOS/NO system is regulated by Grx 1 and coupled with inhibition of JNK and NF-κB signaling pathway which could alleviate the oxidative stress and apoptosis damage in coronary arteries endothelial cells induced by HG.

Immunodetection of S-Nitrosoglutathione Reductase Protein in Plant Samples.

PubMed

Tichá, Tereza; Luhová, Lenka; Petřivalský, Marek

2018-01-01

S-nitrosation, the attachment of a nitroso group to cysteine thiols, has been recognized as an important posttranslational modification of proteins by nitric oxide and related reactive nitrogen species. Mechanisms and significance of S-nitrosation in the regulation of the structure and activity of proteins have been extensively studied in animal and plant systems. In plants, protein S-nitrosation is involved in signaling pathways of plant hormones and regulators during plant growth and development and in responses to abiotic and biotic stress stimuli. S-nitrosoglutathione reductase (GSNOR) has been identified as a key enzyme controlling the intracellular level of S-nitrosothiols. GSNOR irreversibly degrades S-nitrosoglutathione (GSNO), the major low molecular weight S-nitrosothiol involved in the formation of protein S-nitrosothiols through transnitrosylation. GSNOR level and activity in plant cells are modulated during plant development and in response to external stimuli such as pathogen infection. In this chapter, we give a detailed description of the immunochemical detection of the GSNOR protein in plant samples.

The Application of Clove Extract Protects Chinese-style Sausages against Oxidation and Quality Deterioration

PubMed Central

Peng, Xinyan

2017-01-01

This study was conducted to evaluate the effects of clove extract (CE) (0.25%, 0.5%, 1%, and 2%) on the oxidative stability and quality deterioration of Chinese-style sausage stored for 21 d at 4°C. The addition of clove extract to sausages significantly retarded increases in Thiobarbituric Reactive Substances (TBARS) values (p<0.05), while also controlling the production of protein carbonyls (p<0.05). However, the addition of clove extract promoted reduced thiol group content in sausages (p<0.05). Sausages amended with clove extract also had decreased L* values (p<0.05) and increased a* values (p<0.05) when compared with the control. Similarly, texture deterioration was retarded in sausage containing added clove extract when compared with the control during refrigerated storage. Moreover, the addition of clove extract had no negative effects on the sensory properties of sausages. These results suggested that clove extract was effective at protecting sausages from oxidation and quality deterioration during refrigerated storage for 21 d. PMID:28316478

The Application of Clove Extract Protects Chinese-style Sausages against Oxidation and Quality Deterioration.

PubMed

Zhang, Huiyun; Peng, Xinyan; Li, Xinling; Wu, Jingjuan; Guo, Xinyu

2017-01-01

This study was conducted to evaluate the effects of clove extract (CE) (0.25%, 0.5%, 1%, and 2%) on the oxidative stability and quality deterioration of Chinese-style sausage stored for 21 d at 4°C. The addition of clove extract to sausages significantly retarded increases in Thiobarbituric Reactive Substances (TBARS) values ( p <0.05), while also controlling the production of protein carbonyls ( p <0.05). However, the addition of clove extract promoted reduced thiol group content in sausages ( p <0.05). Sausages amended with clove extract also had decreased L* values ( p <0.05) and increased a* values ( p <0.05) when compared with the control. Similarly, texture deterioration was retarded in sausage containing added clove extract when compared with the control during refrigerated storage. Moreover, the addition of clove extract had no negative effects on the sensory properties of sausages. These results suggested that clove extract was effective at protecting sausages from oxidation and quality deterioration during refrigerated storage for 21 d.

Resin-Bound Crypto-Thioester for Native Chemical Ligation.

PubMed

Naruse, Naoto; Ohkawachi, Kento; Inokuma, Tsubasa; Shigenaga, Akira; Otaka, Akira

2018-04-20

The resin-bound N-sulfanylethylanilide (SEAlide) peptide was found to function as a crypto-thioester peptide. Exposure of the peptide resin to an aqueous solution under neutral conditions in the presence of thiols affords thioesters without accompanying racemization of C-terminal amino acids. Furthermore, the resin-bound SEAlide peptides react with N-terminal cysteinyl peptides in the absence of phosphate salts to afford ligated products, whereas soluble SEAlide peptides do not. This unexpected difference in reactivity of the SEAlide peptides allows for a one-pot/three-fragment ligation using resin-bound and unbound peptides.

Hmb(off/on) as a switchable thiol protecting group for native chemical ligation.

PubMed

Qi, Yun-Kun; Tang, Shan; Huang, Yi-Chao; Pan, Man; Zheng, Ji-Shen; Liu, Lei

2016-05-04

A new thiol protecting group Hmb(off/on) is described, which has a switchable activity that may be useful in the chemical synthesis of proteins. When placed on the side chain of Cys, Cys(Hmb(off)) is stable to trifluoroacetic acid (TFA) in the process of solid-phase peptide synthesis. When Cys(Hmb(off)) is treated with neutral aqueous buffers, it is cleanly converted to acid-labile Cys(Hmb(on)), which can later be fully deprotected by TFA to generate free Cys. The utility of Cys(Hmb(off/on)) is demonstrated by the chemical synthesis of an erythropoietin segment, EPO[Cys(98)-Arg(166)]-OH through native chemical ligation.

Preactivated thiolated glycogen as mucoadhesive polymer for drug delivery.

PubMed

Perrone, Mara; Lopalco, Antonio; Lopedota, Angela; Cutrignelli, Annalisa; Laquintana, Valentino; Douglas, Justin; Franco, Massimo; Liberati, Elisa; Russo, Vincenzo; Tongiani, Serena; Denora, Nunzio; Bernkop-Schnürch, Andreas

2017-10-01

The purpose of this study was to synthesize and characterize a novel thiolated glycogen, so-named S-preactivated thiolated glycogen, as a mucosal drug delivery systems and the assessment of its mucoadhesive properties. In this regard, glycogen-cysteine and glycogen-cysteine-2-mercaptonicotinic acid conjugates were synthesized. Glycogen was activated by an oxidative ring opening with sodium periodate resulting in reactive aldehyde groups to which cysteine was bound via reductive amination. The obtained thiolated polymer displayed 2203.09±200μmol thiol groups per gram polymer. In a second step, the thiol moieties of thiolated glycogen were protected by disulfide bond formation with the thiolated aromatic residue 2-mercaptonicotinic acid (2MNA). In vitro screening of mucoadhesive properties was performed on porcine intestinal mucosa using different methods. In particular, in terms of rheology investigations of mucus/polymer mixtures, the S-preactivated thiolated glycogen showed a 4.7-fold increase in dynamic viscosity over a time period of 5h, in comparison to mucus/Simulated Intestinal Fluid control. The S-preactivated polymer remained attached on freshly excised porcine mucosa for 45h. Analogous results were obtained with tensile studies demonstrating a 2.7-fold increase in maximum detachment force and 3.1- fold increase in total work of adhesion for the S-preactivated polymer compared to unmodified glycogen. Moreover, water-uptake studies showed an over 4h continuing weight gain for the S-preactivated polymer, whereas disintegration took place for the unmodified polymer within the first hour. Furthermore, even in the highest tested concentration of 2mg/ml the new conjugates did not show any cytotoxicity on Caco-2 cell monolayer using an MTT assay. According to these results, S-preactivated glycogen represents a promising type of mucoadhesive polymers useful for the development of various mucosal drug delivery systems. Copyright © 2017 Elsevier B.V. All rights reserved.

A more accurate profile of Achyrocline satureioides hypocholesterolemic activity.

PubMed

Espiña, Débora Corrêa; Carvalho, Fabiano Barbosa; Zanini, Daniela; Schlemmer, Josiane Bizzi; Coracini, Juliane Dors; Rubin, Maribel Antonello; Morsch, Vera Maria; Schetinger, Maria Rosa Chitolina; Leal, Daniela Bitencourt Rosa; Baiotto, Cléia Rosani; Jaques, Jeandre Augusto dos Santos

2012-06-01

The aim of this study was to investigate the effect of the aqueous extract (AE) of Achyrocline satureioides on serum lipid profile, liver oxidative profile and Na(+),K(+)-ATPase activity of rats submitted to a hyperlipidic diet. The animals were divided into four groups: control (C), AE 10% (A(10)), hyperlipidic (H) and hyperlipidic/AE 10% (HA(10)). In serum, we measured the levels of total cholesterol (TC), high-density lipoprotein, very-low-density lipoprotein, low-density lipoprotein (LDL) and triglyceride (TG). In liver homogenates, we measured the thiobarbituric acid reactive substances, the carbonyl proteins, the non-protein thiols (NPSHs) and the activity of superoxide dismutase, catalase (CAT) and Na(+),K(+)-ATPase. We observed a significant increase in the TC and LDL levels in the H group. A. satureioides prevented these effects, decreased the TG levels in the HA(10) group and increased the NPSH levels in the A(10) and HA(10) groups. The H group showed an increase in the carbonyl protein level and a decrease in CAT and Na(+),K(+)-ATPase activities. With the use of this model, results show that increased levels of lipids are related to a redox imbalance in the liver, which is also related to the inhibition of Na(+),K(+)-ATPase activity, and that chronic administration of the AE of A. satureioides is capable of changing this profile. Copyright © 2012 John Wiley & Sons, Ltd.

Octadecyl Chains Immobilized onto Hyaluronic Acid Coatings by Thiol-ene "Click Chemistry" Increase the Surface Antimicrobial Properties and Prevent Platelet Adhesion and Activation to Polyurethane.

PubMed

Felgueiras, Helena P; Wang, L M; Ren, K F; Querido, M M; Jin, Q; Barbosa, M A; Ji, J; Martins, M C L

2017-03-08

Infection and thrombus formation are still the biggest challenges for the success of blood contact medical devices. This work aims the development of an antimicrobial and hemocompatible biomaterial coating through which selective binding of albumin (passivant protein) from the bloodstream is promoted and, thus, adsorption of other proteins responsible for bacterial adhesion and thrombus formation can be prevented. Polyurethane (PU) films were coated with hyaluronic acid, an antifouling agent, that was previously modified with thiol groups (HA-SH), using polydopamine as the binding agent. Octadecyl acrylate (C18) was used to attract albumin since it resembles the circulating free fatty acids and albumin is a fatty acid transporter. Thiol-ene "click chemistry" was explored for C18 immobilization on HA-SH through a covalent bond between the thiol groups from the HA and the alkene groups from the C18 chains. Surfaces were prepared with different C18 concentrations (0, 5, 10, and 20%) and successful immobilization was demonstrated by scanning electron microscopy (SEM), water contact angle determinations, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). The ability of surfaces to bind albumin selectively was determined by quartz crystal microbalance with dissipation (QCM-D). Albumin adsorption increased in response to the hydrophobic nature of the surfaces, which augmented with C18 saturation. HA-SH coating reduced albumin adsorption to PU. C18 immobilized onto HA-SH at 5% promoted selective binding of albumin, decreased Staphylococcus aureus adhesion and prevented platelet adhesion and activation to PU in the presence of human plasma. C18/HA-SH coating was established as an innovative and promising strategy to improve the antimicrobial properties and hemocompatibility of any blood contact medical device.

Polythiol-containing, recombinant mannosylated-albumin is a superior CD68+/CD206+ Kupffer cell-targeted nanoantioxidant for treatment of two acute hepatitis models.

PubMed

Maeda, Hitoshi; Hirata, Kenshiro; Watanabe, Hiroshi; Ishima, Yu; Chuang, Victor Tuan Giam; Taguchi, Kazuaki; Inatsu, Akihito; Kinoshita, Manabu; Tanaka, Motohiko; Sasaki, Yutaka; Otagiri, Masaki; Maruyama, Toru

2015-02-01

Since reactive oxygen species (ROS) derived from Kupffer cells (KC), especially CD68(+) KC, play a key role in the induction of hepatic oxidative stress and injuries, we developed a polythiolated- and mannosylated human serum albumin (SH-Man-HSA), which functions as a novel nanoantioxidant for delivering thiol to CD68(+) KC. In vitro electron paramagnetic resonance coupled with pharmacokinetics and immunohistochemical studies showed that SH-Man-HSA possessed powerful radical-scavenging activity and rapidly and selectively delivered thiols to the liver via mannose receptor (CD206) on CD68(+) cells. SH-Man-HSA significantly improved the survival rate of concanavalin-A (Con-A)-treated mice. Moreover, SH-Man-HSA exhibited excellent hepatoprotective functions, not by decreasing tumor necrosis factor or interferon-γ production that is closely associated with Con-A-induced hepatitis, but by suppressing ROS production. Interestingly, the protective effect of SH-Man-HSA was superior to N-acetyl cysteine (NAC). This could be attributed to the difference in the inhibition of hepatic oxidative stress between the two antioxidants depending on their potential for thiol delivery to the liver. Similar results were also observed for acetaminophen (APAP)-induced hepatopathy models. Flow cytometric data further confirmed that an increase in F4/80(+)/ROS(+) cells was dramatically decreased by SH-Man-HSA. The administration of SH-Man-HSA at 4 hours following a Con-A or APAP injection also exhibited a profound hepatoprotective action against these hepatitis models, whereas this was not observed for NAC. It can be concluded therefore that SH-Man-HSA has great potential for use in a rescue therapy for hepatopathy as a nanoantioxidant because of its ability to efficiently and rapidly deliver thiols to CD68(+)/CD206(+) KC. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Cellular Metabolic and Autophagic Pathways: Traffic Control by Redox Signaling

PubMed Central

Dodson, Matthew; Darley-Usmar, Victor; Zhang, Jianhua

2013-01-01

It has been established that the key metabolic pathways of glycolysis and oxidative phosphorylation are intimately related to redox biology through control of cell signaling. Under physiological conditions glucose metabolism is linked to control of the NADH/NAD redox couple, as well as providing the major reductant, NADPH, for thiol-dependent antioxidant defenses. Retrograde signaling from the mitochondrion to the nucleus or cytosol controls cell growth and differentiation. Under pathological conditions mitochondria are targets for reactive oxygen and nitrogen species and are critical in controlling apoptotic cell death. At the interface of these metabolic pathways, the autophagy-lysosomal pathway functions to maintain mitochondrial quality, and generally serves an important cytoprotective function. In this review we will discuss the autophagic response to reactive oxygen and nitrogen species that are generated from perturbations of cellular glucose metabolism and bioenergetic function. PMID:23702245

A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Avonto, Cristina; Chittiboyina, Amar G.; Rua, Diego

2015-12-01

Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles aftermore » incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, ‘HTS-DCYA assay’, is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time. - Highlights: • A novel fluorescence-based method to detect electrophilic sensitizers is proposed. • A model fluorescent thiol was used to directly quantify the reaction products. • A discussion of the reaction workflow and critical parameters is presented. • The method could provide a useful tool to complement existing chemical assays.« less

Cytochrome P{sub 450}-dependent toxic effects of primaquine on human erythrocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganesan, Shobana; Department of Pharmacology, School of Pharmacy, University of Mississippi, University MS 38677; Tekwani, Babu L., E-mail: btekwani@olemiss.ed

Primaquine, an 8-aminoquinoline, is the drug of choice for radical cure of relapsing malaria. Use of primaquine is limited due to its hemotoxicity, particularly in populations with glucose-6-phosphate dehydrogenase deficiency [G6PD(-)]. Biotransformation appears to be central to the anti-infective and hematological toxicities of primaquine, but the mechanisms are still not well understood. Metabolic studies with primaquine have been hampered due to the reactive nature of potential hemotoxic metabolites. An in vitro metabolism-linked hemotoxicity assay has been developed. Co-incubation of the drug with normal or G6PD(-) erythrocytes, microsomes or recombinant cytochrome P{sub 450} (CYP) isoforms has allowed in situ generation ofmore » potential hemotoxic metabolite(s), which interact with the erythrocytes to generate hemotoxicity. Methemoglobin formation, real-time generation of reactive oxygen intermediates (ROIs) and depletion of reactive thiols were monitored as multiple biochemical end points for hemotoxicity. Primaquine alone did not produce any hemotoxicity, while a robust increase was observed in methemoglobin formation and generation of ROIs by primaquine in the presence of human or mouse liver microsomes. Multiple CYP isoforms (CYP2E1, CYP2B6, CYP1A2, CYP2D6 and CYP3A4) variably contributed to the hemotoxicity of primaquine. This was further confirmed by significant inhibition of primaquine hemotoxicity by the selective CYP inhibitors, namely thiotepa (CYP2B6), fluoxetine (CYP2D6) and troleandomycin (CYP3A4). Primaquine caused similar methemoglobin formation in G6PD(-) and normal human erythrocytes. However, G6PD(-) erythrocytes suffered higher oxidative stress and depletion of thiols than normal erythrocytes due to primaquine toxicity. The results provide significant insights regarding CYP isoforms contributing to hemotoxicity and may be useful in controlling toxicity of primaquine to increase its therapeutic utility.« less

High Yield Production and Radiochemical Isolation of Isotopically Pure Arsenic-72 and Novel Radioarsenic Labeling Strategies for the Development of Theranostic Radiopharmaceuticals.

PubMed

Ellison, Paul A; Barnhart, Todd E; Chen, Feng; Hong, Hao; Zhang, Yin; Theuer, Charles P; Cai, Weibo; Nickles, Robert J; DeJesus, Onofre T

2016-01-20

Radioisotopes of arsenic are of considerable interest to the field of nuclear medicine with unique nuclear and chemical properties making them well-suited for use in novel theranostic radiopharmaceuticals. However, progress must still be made in the production of isotopically pure radioarsenic and in its stable conjugation to biological targeting vectors. This work presents the production and irradiation of isotopically enriched (72)Ge(m) discs in an irrigation-cooled target system allowing for the production of isotopically pure (72)As with capability on the order of 10 GBq. A radiochemical separation procedure isolated the reactive trivalent radioarsenic in a small volume buffered aqueous solution, while reclaiming (72)Ge target material. The direct thiol-labeling of a monoclonal antibody resulted in a conjugate exhibiting exceptionally poor in vivo stability in a mouse model. This prompted further investigations to alternative radioarsenic labeling strategies, including the labeling of the dithiol-containing chelator dihydrolipoic acid, and thiol-modified mesoporous silica nanoparticles (MSN-SH). Radioarsenic-labeled MSN-SH showed exceptional in vivo stability toward dearsenylation.

High Yield Production and Radiochemical Isolation of Isotopically Pure Arsenic-72 and Novel Radioarsenic Labeling Strategies for the Development of Theranostic Radiopharmaceuticals

PubMed Central

Ellison, Paul A.; Barnhart, Todd E.; Chen, Feng; Hong, Hao; Zhang, Yin; Theuer, Charles P.; Cai, Weibo; Nickles, Robert J.; DeJesus, Onofre T.

2016-01-01

Radioisotopes of arsenic are of considerable interest to the field of nuclear medicine with unique nuclear and chemical properties making them well-suited for use in novel theranostic radiopharmaceuticals. However, progress must still be made in the production of isotopically pure radioarsenic and in its stable conjugation to biological targeting vectors. This work presents the production and irradiation of isotopically enriched 72Ge(m) discs in an irrigation-cooled target system allowing for the production of isotopically pure 72As with capability on the order of 10 GBq. A radiochemical separation procedure isolated the reactive trivalent radioarsenic in a small volume buffered aqueous solution, while reclaiming 72Ge target material. The direct thiol-labeling of a monoclonal antibody resulted in a conjugate exhibiting exceptionally poor in vivo stability in a mouse model. This prompted further investigations to alternative radioarsenic labeling strategies, including the labeling of the dithiol-containing chelator dihydrolipoic acid, and thiol-modified mesoporous silica nanoparticles (MSN-SH). Radioarsenic-labeled MSN-SH showed exceptional in vivo stability toward dearsenylation. PMID:26646989

Cage-like bifunctional chelators, copper-64 radiopharmaceuticals and PET imaging using the same

DOEpatents

Conti, Peter S.; Cai, Hancheng; Li, Zibo; Liu, Shuanglong

2016-08-02

Disclosed is a class of versatile Sarcophagine based bifunctional chelators (BFCs) containing a hexa-aza cage for labeling with metals having either imaging, therapeutic or contrast applications radiolabeling and one or more linkers (A) and (B). The compounds have the general formula ##STR00001## where A is a functional group selected from group consisting of an amine, a carboxylic acid, an ester, a carbonyl, a thiol, an azide and an alkene, and B is a functional group selected from the group consisting of hydrogen, an amine, a carboxylic acid, and ester, a carbonyl, a thiol, an azide and an alkene. Also disclosed are conjugate of the BFC and a targeting moiety, which may be a peptide or antibody. Also disclosed are metal complexes of the BFC/targeting moiety conjugates that are useful as radiopharmaceuticals, imaging agents or contrast agents.

In Vitro Inhibitory Activity of Acca sellowiana Fruit Extract on End Products of Advanced Glycation.

PubMed

Muñiz, Alethia; Garcia, Abraham H; Pérez, Rosa M; García, Efren V; González, Daphne E

2018-02-01

Hyperglycemia plays an important role in the pathogenesis of diabetic complications, as it increases protein glycation, as well as the progressive accumulation of advanced glycation end products (AGEs), which are complex structures that produce fluorescence. The glycation reaction raises the levels of protein carbonyl, N ε -(carboxymethyl)lysine (CML), and fructosamine and decreases the level of thiol groups. In the present study, the antiglycation activity was determined by fluorescence intensity using the bovine serum albumin (BSA)/glucose, CML method, and the level of fructosamine. The oxidation of proteins was determined by the carbonyl protein content and thiol groups. The results show that the hexane extract of Acca sellowiana (FOH) at different concentrations (0.30-5 mg/ml) significantly inhibited the formation of AGEs in the BSA/glucose model during the 4 weeks of the study. FOH reduced the levels of fructosamine and CML. Our results showed a significant effect of FOH in the prevention of oxidative damage of proteins, as well as an effect on the oxidation of thiol groups and carbonyl proteins. The present study indicates that FOH is effective in inhibiting the glycation of proteins in vitro, so it can prevent or ameliorate the chronic conditions of diabetes associated with the formation of AGEs.

Cysteine analogues potentiate glucose-induced insulin release in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ammon, H.P.; Hehl, K.H.; Enz, G.

1986-12-01

In rat pancreatic islets, cysteine analogues, including glutathione, acetylcysteine, cysteamine, D-penicillamine, L-cysteine ethyl ester, and cysteine-potentiated glucose (11.1 mM) induced insulin secretion in a concentration-dependent manner. Their maximal effects were similar and occurred at approximately 0.05, 0.05, 0.1, 0.5, 1.0, 1.0 mM, respectively. At substimulatory glucose levels (2.8 mM), insulin release was not affected by these compounds. In contrast, thiol compounds, structurally different from cysteine and its analogues, such as mesna, tiopronin, meso-2,3-dimercaptosuccinic acid (DMSA), dimercaprol (BAL), beta-thio-D-glucose, as well as those cysteine analogues that lack a free-thiol group, including L-cystine, cystamine, D-penicillamine disulfide, S-carbocysteine, and S-carbamoyl-L-cysteine, did not enhancemore » insulin release at stimulatory glucose levels (11.1 mM); cystine (5 mM) was inhibitory. These in vitro data indicate that among the thiols tested here, only cysteine and its analogues potentiate glucose-induced insulin secretion, whereas thiols that are structurally not related to cysteine do not. This suggests that a cysteine moiety in the molecule is necessary for the insulinotropic effect. For their synergistic action to glucose, the availability of a sulfhydryl group is also a prerequisite. The maximal synergistic action is similar for all cysteine analogues tested, whereas the potency of action is different, suggesting similarity in the mechanism of action but differences in the affinity to the secretory system.« less

Surface-functionalized polymethacrylic acid based hydrogel microparticles for oral drug delivery.

PubMed

Sajeesh, S; Bouchemal, K; Sharma, C P; Vauthier, C

2010-02-01

Aim of the present work was to develop novel thiol-functionalized hydrogel microparticles based on poly(methacrylic acid)-chitosan-poly(ethylene glycol) (PCP) for oral drug delivery applications. PCP microparticles were prepared by a modified ionic gelation process in aqueous medium. Thiol modification of surface carboxylic acid groups of PCP micro particles was carried out by coupling l-cysteine with a water-soluble carbodiimide. Ellman's method was adopted to quantify the sulfhydryl groups, and dynamic light-scattering technique was used to measure the average particle size. Cytotoxicity of the modified particles was evaluated on Caco 2 cells by MTT assay. Effect of thiol modification on permeability of paracellular marker fluorescence dextran (FD4) was evaluated on Caco 2 cell monolayers and freshly excised rat intestinal tissue with an Ussing chamber set-up. Mucoadhesion experiments were carried out by an ex vivo bioadhesion method with excised rat intestinal tissue. The average size of the PCP microparticles was increased after thiol modification. Thiolated microparticles significantly improved the paracellular permeability of FD4 across Caco 2 cell monolayers, with no sign of toxicity. However, the efficacy of thiolated system remained low when permeation experiments were carried out across excised intestinal membrane. This was attributed to the high adhesion of the thiolated particles on the gut mucosa. Nevertheless, it can be concluded that surface thiolation is an interesting strategy to improve paracellular permeability of hydrophilic macromolecules. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Size control and catalytic activity of bio-supported palladium nanoparticles.

PubMed

Søbjerg, Lina Sveidal; Lindhardt, Anders T; Skrydstrup, Troels; Finster, Kai; Meyer, Rikke Louise

2011-07-01

The development of nanoparticles has greatly improved the catalytic properties of metals due to the higher surface to volume ratio of smaller particles. The production of nanoparticles is most commonly based on abiotic processes, but in the search for alternative protocols, bacterial cells have been identified as excellent scaffolds of nanoparticle nucleation, and bacteria have been successfully employed to recover and regenerate platinum group metals from industrial waste. We report on the formation of bio-supported palladium (Pd) nanoparticles on the surface of two bacterial species with distinctly different surfaces: the gram positive Staphylococcus sciuri and the gram negative Cupriavidus necator. We investigated how the type of bacterium and the amount of biomass affected the size and catalytic properties of the nanoparticles formed. By increasing the biomass:Pd ratio, we could produce bio-supported Pd nanoparticles smaller than 10nm in diameter, whereas lower biomass:Pd ratios resulted in particles ranging from few to hundreds of nm. The bio-supported Pd nanoparticle catalytic properties were investigated towards the Suzuki-Miyaura cross coupling reaction and hydrogenation reactions. Surprisingly, the smallest nanoparticles obtained at the highest biomass:Pd ratio showed no reactivity towards the test reactions. The lack of reactivity appears to be caused by thiol groups, which poison the catalyst by binding strongly to Pd. Different treatments intended to liberate particles from the biomass, such as burning or rinsing in acetone, did not re-establish their catalytic activity. Sulphur-free biomaterials should therefore be explored as more suitable scaffolds for Pd(0) nanoparticle formation. Copyright © 2011 Elsevier B.V. All rights reserved.

Evaluation of an in situ chemically crosslinked hydrogel as a long-term vitreous substitute material.

PubMed

Tao, Yong; Tong, Xinming; Zhang, Yan; Lai, Jingjing; Huang, Yanbin; Jiang, Yan-Rong; Guo, Bao-Hua

2013-02-01

Currently there is no material that can be used as a long-term vitreous substitute, and this remains an unmet clinical need in ophthalmology. In this study, we developed an injectable, in situ chemically crosslinked hydrogel system and evaluated it in a rabbit model. The system consisted of two components, both based on multi-functional poly(ethylene glycol) (PEG) but with complementarily reactive end groups of thiol and active vinyl groups, respectively. The two components are mixed and injected as a solution mixture, react in vivo via the Michael addition route and form a chemically crosslinked hydrogel in situ. The linkages between the end groups and the backbone PEG chains are specially designed to ensure that the final network structure is hydrolysis-resistant. In the rabbit study and with an optimized operation protocol, we demonstrated that the hydrogel indeed formed in situ after injection, and remained transparent and stable during the study period of 9 months without significant adverse reactions. In addition, the hydrogel formed in situ showed rheological properties very similar to the natural vitreous. Therefore, our study demonstrated that this in situ chemically crosslinked PEG gel system is suitable as a potential long-term vitreous substitute. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

The 2-Cys Peroxiredoxin Alkyl Hydroperoxide Reductase C Binds Heme and Participates in Its Intracellular Availability in Streptococcus agalactiae*

PubMed Central

Lechardeur, Delphine; Fernandez, Annabelle; Robert, Bruno; Gaudu, Philippe; Trieu-Cuot, Patrick; Lamberet, Gilles; Gruss, Alexandra

2010-01-01

Heme is a redox-reactive molecule with vital and complex roles in bacterial metabolism, survival, and virulence. However, few intracellular heme partners were identified to date and are not well conserved in bacteria. The opportunistic pathogen Streptococcus agalactiae (group B Streptococcus) is a heme auxotroph, which acquires exogenous heme to activate an aerobic respiratory chain. We identified the alkyl hydroperoxide reductase AhpC, a member of the highly conserved thiol-dependent 2-Cys peroxiredoxins, as a heme-binding protein. AhpC binds hemin with a Kd of 0.5 μm and a 1:1 stoichiometry. Mutagenesis of cysteines revealed that hemin binding is dissociable from catalytic activity and multimerization. AhpC reductase activity was unchanged upon interaction with heme in vitro and in vivo. A group B Streptococcus ahpC mutant displayed attenuation of two heme-dependent functions, respiration and activity of a heterologous catalase, suggesting a role for AhpC in heme intracellular fate. In support of this hypothesis, AhpC-bound hemin was protected from chemical degradation in vitro. Our results reveal for the first time a role for AhpC as a heme-binding protein. PMID:20332091

Soft Cysteine Signaling Network: The Functional Significance of Cysteine in Protein Function and the Soft Acids/Bases Thiol Chemistry That Facilitates Cysteine Modification.

PubMed

Wible, Ryan S; Sutter, Thomas R

2017-03-20

The unique biophysical and electronic properties of cysteine make this molecule one of the most biologically critical amino acids in the proteome. The defining sulfur atom in cysteine is much larger than the oxygen and nitrogen atoms more commonly found in the other amino acids. As a result of its size, the valence electrons of sulfur are highly polarizable. Unique protein microenvironments favor the polarization of sulfur, thus increasing the overt reactivity of cysteine. Here, we provide a brief overview of the endogenous generation of reactive oxygen and electrophilic species and specific examples of enzymes and transcription factors in which the oxidation or covalent modification of cysteine in those proteins modulates their function. The perspective concludes with a discussion of cysteine chemistry and biophysics, the hard and soft acids and bases model, and the proposal of the Soft Cysteine Signaling Network: a hypothesis proposing the existence of a complex signaling network governed by layered chemical reactivity and cross-talk in which the chemical modification of reactive cysteine in biological networks triggers the reorganization of intracellular biochemistry to mitigate spikes in endogenous or exogenous oxidative or electrophilic stress.

Andrographolide sodium bisulphite-induced inactivation of urease: inhibitory potency, kinetics and mechanism.

PubMed

Mo, Zhi-Zhun; Wang, Xiu-Fen; Zhang, Xie; Su, Ji-Yan; Chen, Hai-Ming; Liu, Yu-Hong; Zhang, Zhen-Biao; Xie, Jian-Hui; Su, Zi-Ren

2015-07-16

The inhibitory effect of andrographolide sodium bisulphite (ASB) on jack bean urease (JBU) and Helicobacter pylori urease (HPU) was performed to elucidate the inhibitory potency, kinetics and mechanism of inhibition in 20 mM phosphate buffer, pH 7.0, 2 mM EDTA, 25 °C. The ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. The inhibitory effect of ASB was characterized with IC50 values. Lineweaver-Burk and Dixon plots for JBU inhibition of ASB was constructed from the kinetic data. SH-blocking reagents and competitive active site Ni2+ binding inhibitors were employed for mechanism study. Molecular docking technique was used to provide some information on binding conformations as well as confirm the inhibition mode. The IC50 of ASB against JBU and HPU was 3.28±0.13 mM and 3.17±0.34 mM, respectively. The inhibition proved to be competitive and concentration- dependent in a slow-binding progress. The rapid formation of initial ASB-JBU complex with an inhibition constant of Ki=2.86×10(-3) mM was followed by a slow isomerization into the final complex with an overall inhibition constant of Ki*=1.33×10(-4) mM. The protective experiment proved that the urease active site is involved in the binding of ASB. Thiol reagents (L-cysteine and dithiothreithol) strongly protect the enzyme from the loss of enzymatic activity, while boric acid and fluoride show weaker protection, indicating that the active-site sulfhydryl group of JBU was potentially involved in the blocking process. Moreover, inhibition of ASB proved to be reversible since ASB-inactivated JBU could be reactivated by dithiothreitol application. Molecular docking assay suggested that ASB made contacts with the important sulfhydryl group Cys-592 residue and restricted the mobility of the active-site flap. ASB was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for the treatment of urease-related diseases.

Drug interactions with potential rubber closure extractables. Identification of thiol-disulfide exchange reaction products of captopril and thiurams.

PubMed

Corredor, Claudia; Tomasella, Frank P; Young, Joel

2009-01-02

Mixtures of thiuram disulfides are frequently used as accelerators in rubber stoppers for injectables and sterilized powders for injection. Rapid reactions of thiuram disulfides between themselves and with thiols yield mixed disulfides due to thiol-disulfide exchange. The possibility of exchange reactions of thiuram disulfides extracted from rubber stoppers and drug products containing pendant thiol groups have not been reported in the analysis of potential stopper extractables. In this paper we report the formation and identification of mixed thiuram disulfides of N,N,N',N'-dimethylthiuram disulfide (TMTD), N,N,N',N'-dibutylthiuram disulfide (TBTD), and captopril (a thiol-containing drug). A reversed-phase HPLC method was developed for the determination of TMTD, TBTD, captopril and their disulfides in aqueous vehicles, using a YMC ODS AQ column at 35 degrees C and mobile phases A and B consisting of acetonitrile:water:trifluoroacetic acid (TFA) (20:80:0.1) and acetonitrile:TFA (100:0.1), respectively. The captopril-TBTD and captopril-TMTD disulfides were identified by MS, with molecular ions at m/z 420.9 and m/z of 337.1, respectively. Possible structures for the fragment ions in the spectra are provided. Mixed captopril-thiuram formation was studied as a function of pH. Captopril-TMTD formation was enhanced at pH 6.0, reaching a maximum of 31.3% in 4.1h. At pH 4.0 and 2.2, the mixed captopril adduct product was still detected in solution after 20h. The impact of the formation of mixed disulfide products of thiol-containing drugs with thiurams in the HPLC profile of extractables and leachables studies is discussed.

Surface charge fine tuning of reversed-phase/weak anion-exchange type mixed-mode stationary phases for milder elution conditions.

PubMed

Zimmermann, Aleksandra; Horak, Jeannie; Sánchez-Muñoz, Orlando L; Lämmerhofer, Michael

2015-08-28

A series of new mixed-mode reversed-phase/weak anion-exchange (RP/WAX) phases have been synthesized by immobilization of N-undecenyl-3-α-aminotropane onto thiol-modified silica gel by thiol-ene click chemistry and subsequent introduction of acidic thiol-endcapping functionalities of different type and surface densities. Click chemistry allowed to adjust a controlled surface concentration of the RP/WAX ligand in such a way that a sufficient quantity of residual thiols remained unmodified which have been capped by thiol click with either 3-butenoic acid or allylsulfonic acid as co-ligands. In another embodiment, performic acid oxidation of N-undecenyl-3-α-aminotropane-derivatized thiol-modified silica gave a RP/WAX phase with high density of sulfonic acid end-capping groups. ζ-Potential determinations confirmed the fine-tuned pI of these mixed-mode stationary phases which was shifted from 9.5 to 8.2, 7.8, and 6.5 with 3-butenoic acid and allylsulfonic acid end-capping as well as performic acid oxidation. For acidic solutes, the co-ionic endcapping leads to strongly reduced retention times and clearly allowed elution of these analytes under lower ionic strength thus milder elution conditions. In spite of the acidic endcapping, the new mixed-mode phases maintained their hydrophobic and anion-exchange selectivity as well as their multimodal nature featuring RP and HILIC elution domains at acetonitrile percentages below and above 50%, respectively. Column classification by principal component analysis of an extended retention map in comparison to a set of polar commercial and in-house synthesized stationary phases confirmed complementarity of the new mixed-mode phases with respect to HILIC, polar RP, amino and commercial mixed-mode phases. Copyright © 2015 Elsevier B.V. All rights reserved.

Trivalent methylated arsenical-induced phosphatidylserine exposure and apoptosis in platelets may lead to increased thrombus formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bae, Ok-Nam; Lim, Kyung-Min; AMOREPACIFIC CO/R and D Center, Gyeonggi-do 446-729

2009-09-01

Trivalent methylated metabolites of arsenic, monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}), have been found highly reactive and toxic in various cells and in vivo animal models, suggesting their roles in the arsenic-associated toxicity. However, their effects on cardiovascular system including blood cells, one of the most important targets for arsenic toxicity, remain poorly understood. Here we found that MMA{sup III} and DMA{sup III} could induce procoagulant activity and apoptosis in platelets, which play key roles in the development of various cardiovascular diseases (CVDs) through excessive thrombus formation. In freshly isolated human platelets, treatment of MMA{sup III} resultedmore » in phosphatidylserine (PS) exposure, a hallmark of procoagulant activation, accompanied by distinctive apoptotic features including mitochondrial membrane potential disruption, cytochrome c release, and caspase-3 activation. These procoagulant activation and apoptotic features were found to be mediated by the depletion of protein thiol and intracellular ATP, and flippase inhibition by MMA{sup III}, while the intracellular calcium increase or reactive oxygen species generation was not involved. Importantly, increased platelet procoagulant activity by MMA{sup III} resulted in enhanced blood coagulation and excessive thrombus formation in a rat in vivo venous thrombosis model. DMA{sup III} also induced PS-exposure with apoptotic features mediated by protein thiol depletion, which resulted in enhanced thrombin generation. In summary, we believe that this study provides an important evidence for the role of trivalent methylated arsenic metabolites in arsenic-associated CVDs, giving a novel insight into the role of platelet apoptosis in toxicant-induced cardiovascular toxicity.« less

Oxidative Stress Assessment in Response to Ultraendurance Exercise: Thiols Redox Status and ROS Production according to Duration of a Competitive Race

PubMed Central

Vezzoli, Alessandra; Dellanoce, Cinzia; Montorsi, Michela; Tonini, Annamaria; Accinni, Roberto

2016-01-01

Purpose. Response to an ultraendurance competitive race on thiols redox status, reactive oxygen species (ROS) production, and oxidative stress (OxS) was investigated according to duration. Methods. Twenty-four elite runners were examined: six completed 50 km and eighteen 100 km. Blood and urine samples were collected before and immediately after the race. Erythrocytes and plasma aminothiols by high-performance liquid chromatography, total antioxidant capacity (TAC), and OxS biomarkers (protein carbonyl (PC), thiobarbituric acid-reactive substances (TBARS), 8-isoprostane (8-iso-PGF2α), and 8-OH-2-deoxyguanosine (8-OH-dG)) by immunoenzymatic assays and ROS production by Electron Paramagnetic Resonance were assessed. Results. Significant increases (P between <0.05 and <0.0001) were recorded in plasma total and oxidized aminothiols concentration and TAC (P < 0.0001) only after 100 km: plasmatic (ROS production (+12 versus +29%), PC (+54 versus +115%), and TBARS (+28 versus +55%)) and urinary (8-OH-dG.creatinine−1 (+71 versus +158%) and 8-iso-PGF2α.creatinine−1 (+43 versus +135%)) concentrations for 50 and 100 km (duration 4 h 3′ versus 8 h 42′), respectively. Conclusion. Very prolonged ultraendurance exercise causes an increase in ROS production and OxS depending on specific biomarker examined but always linearly and directly related to exercise duration. Redox status of erythrocytes was preserved. A relationship between running performance and both prerace ROS production and antioxidant-redox status was found in 100 km race. PMID:27504148

Dietary consumption of monosodium L-glutamate induces adaptive response and reduction in the life span of Drosophila melanogaster.

PubMed

Abolaji, Amos O; Olaiya, Charles O; Oluwadahunsi, Oluwagbenga J; Farombi, Ebenezer O

2017-04-01

Adaptive response is the ability of an organism to better counterattack stress-induced damage in response to a number of different cytotoxic agents. Monosodium L-glutamate (MSG), the sodium salt of amino acid glutamate, is commonly used as a food additive. We investigated the effects of MSG on the life span and antioxidant response in Drosophila melanogaster (D. melanogaster). Both genders (1 to 3 days old) of flies were fed with diet containing MSG (0.1, 0.5, and 2.5-g/kg diet) for 5 days to assess selected antioxidant and oxidative stress markers, while flies for longevity were fed for lifetime. Thereafter, the longevity assay, hydrogen peroxide (H 2 O 2 ), and reactive oxygen and nitrogen species levels were determined. Also, catalase, glutathione S-transferase and acetylcholinesterase activities, and total thiol content were evaluated in the flies. We found that MSG reduced the life span of the flies by up to 23% after continuous exposure. Also, MSG increased reactive oxygen and nitrogen species and H 2 O 2 generations and total thiol content as well as the activities of catalase and glutathione S-transferase in D. melanogaster (P < .05). In conclusion, consumption of MSG for 5 days by D. melanogaster induced adaptive response, but long-term exposure reduced life span of flies. This study may therefore have public health significance in humans, and thus, moderate consumption of MSG is advocated by the authors. Copyright © 2017 John Wiley & Sons, Ltd.

Disulfide S-monoxides convert xanthine dehydrogenase into oxidase in rat liver cytosol more potently than their respective disulfides.

PubMed

Sakuma, Satoru; Fujita, Junko; Nakanishi, Masahiko; Wada, Shun-ich; Fujimoto, Yohko

2008-05-01

Xanthine oxidase (XO)/xanthine dehydrogenase (XD) oxidizes oxypurines to uric acid, with only the XO form producing reactive oxygen species. In the present study, the effects of cystamine S-monoxide and cystine S-monoxide (disulfide S-monoxides) on the conversion of XD to XO in rat liver were examined. A partially purified enzyme fraction from the rat liver was incubated with xanthine in the presence or absence of NAD+, and the uric acid formed was measured by HPLC. Under basal conditions, XO activity represented about 15% of the total XO plus XD activity. Cystamine S-monoxide and cystine S-monoxide converted XD into XO in a dose-dependent manner, and the concentrations required to increase XO activity by 50% were approximately 1 and 2 microM, respectively. Their respective thiols (cysteamine and cysteine) and disulfides (cystamine and cystine) up to 10 microM showed weak or no effects on the activities of XO and XD and their conversion. Experiments utilizing a sulfhydryl reducing reagent (dithiothreitol) and sulfhydryl modifiers (4,4'-dithiodipyridine and 1-fluoro-2,4-dinitrobenzene) indicated that disulfide S-monoxides-induced conversion of XD to XO occurs via disulfide bridge formation in XD, but not the modification of sulfhydryl groups. These results suggest that disulfide S-monoxides have the potential to increase the generation of reactive oxygen species through the conversion of XD to XO in liver.

Silver ion (Ag+)-induced increases in cell membrane K+ and Na+ permeability in the renal proximal tubule: reversal by thiol reagents.

PubMed

Kone, B C; Kaleta, M; Gullans, S R

1988-04-01

The initial mechanisms of injury to the proximal tubule following exposure to nephrotoxic heavy metals are not well established. We studied the immediate effects of silver (Ag+) on K+ transport and respiration with extracellular K+ and O2 electrodes in suspensions of renal cortical tubules. Addition of silver nitrate (AgNO3) to tubules suspended in bicarbonate Ringer's solution caused a rapid, dose-dependent net K+ efflux (Km = 10(-4) M, Vmax = 379 nmol K+/min/mg protein) which was not inhibited by furosemide, barium chloride, quinine, tetraethylammonium, or tolbutamide. An increase in the ouabain-sensitive oxygen consumption rate (QO2) (13.9 +/- 1.1 to 25.7 +/- 4.4 nmol O2/min/mg, P less than 0.001), was observed 19 sec after the K+ efflux induced by AgNO3 (10(-4) M), suggesting a delayed increase in Na+ entry into the cell. Ouabain-insensitive QO2, nystatin-stimulated QO2, and CCCP-uncoupled QO2 were not significantly affected, indicating preserved function of the Na+,K+-ATPase and mitochondria. External addition of the thiol reagents dithiothreitol (1 mM) and reduced glutathione (1 mM) prevented and/or immediately reversed the effects on K+ transport and QO2. We conclude that Ag+ causes early changes in the permeability of the cell membrane to K+ and then to Na+ at concentrations that do not limit Na+,K+-ATPase activity or mitochondrial function. These alterations are likely the result of a reversible interaction of Ag+ with sulfhydryl groups of cell membrane proteins and may represent initial cytotoxic effects common to other sulfhydryl-reactive heavy metals on the proximal tubule.

Melatonin reverses H-89 induced spatial memory deficit: Involvement of oxidative stress and mitochondrial function.

PubMed

Sharif, Rojin; Aghsami, Mehdi; Gharghabi, Mehdi; Sanati, Mehdi; Khorshidahmad, Tina; Vakilzadeh, Gelareh; Mehdizadeh, Hajar; Gholizadeh, Shervin; Taghizadeh, Ghorban; Sharifzadeh, Mohammad

2017-01-01

Oxidative stress and mitochondrial dysfunction play indispensable role in memory and learning impairment. Growing evidences have shed light on anti-oxidative role for melatonin in memory deficit. We have previously reported that inhibition of protein kinase A by H-89 can induce memory impairment. Here, we investigated the effect of melatonin on H-89 induced spatial memory deficit and pursued their interactive consequences on oxidative stress and mitochondrial function in Morris Water Maze model. Rats received melatonin (50 and 100μg/kg/side) and H-89(10μM) intra-hippocampally 30min before each day of training. Animals were trained for 4 consecutive days, each containing one block from four trials. Oxidative stress indices, including thiobarbituric acid (TBARS), reactive oxygen species (ROS), thiol groups, and ferric reducing antioxidant power (FRAP) were assessed using spectrophotometer. Mitochondrial function was evaluated through measuring ROS production, mitochondrial membrane potential (MMP), swelling, outer membrane damage, and cytochrome c release. As expected from our previous report, H-89 remarkably impaired memory by increasing the escape latency and traveled distance. Intriguingly, H-89 significantly augmented TBARS and ROS levels, caused mitochondrial ROS production, swelling, outer membrane damage, and cytochrome c release. Moreover, H-89 lowered thiol, FRAP, and MMP values. Intriguingly, melatonin pre-treatment not only effectively hampered H-89-mediated spatial memory deficit at both doses, but also reversed the H-89 effects on mitochondrial and biochemical indices upon higher dose. Collectively, these findings highlight a protective role for melatonin against H-89-induced memory impairment and indicate that melatonin may play a therapeutic role in the treatment of oxidative- related neurodegenerative disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

Nanoparticle-Based Receptors Mimic Protein-Ligand Recognition.

PubMed

Riccardi, Laura; Gabrielli, Luca; Sun, Xiaohuan; De Biasi, Federico; Rastrelli, Federico; Mancin, Fabrizio; De Vivo, Marco

2017-07-13

The self-assembly of a monolayer of ligands on the surface of noble-metal nanoparticles dictates the fundamental nanoparticle's behavior and its functionality. In this combined computational-experimental study, we analyze the structure, organization, and dynamics of functionalized coating thiols in monolayer-protected gold nanoparticles (AuNPs). We explain how functionalized coating thiols self-organize through a delicate and somehow counterintuitive balance of interactions within the monolayer itself and with the solvent. We further describe how the nature and plasticity of these interactions modulate nanoparticle-based chemosensing. Importantly, we found that self-organization of coating thiols can induce the formation of binding pockets in AuNPs. These transient cavities can accommodate small molecules, mimicking protein-ligand recognition, which could explain the selectivity and sensitivity observed for different organic analytes in NMR chemosensing experiments. Thus, our findings advocate for the rational design of tailored coating groups to form specific recognition binding sites on monolayer-protected AuNPs.

Thiol Modification of Psyllium Husk Mucilage and Evaluation of Its Mucoadhesive Applications

PubMed Central

Bhatia, Meenakshi

2013-01-01

Thiol functionalization of psyllium was carried out to enhance its mucoadhesive potential. Thiolation of psyllium was achieved by esterification with thioglycolic acid. Thiolation was observed to change the surface morphology of psyllium from fibrous to granular and result in a slight increase in the crystallinity and swelling. Thiolated psyllium was found to contain 3.282 m moles of thiol groups/g of the polymer. Mucoadhesive applications of thiolated psylium were explored by formulating gels using metronidazole as the model drug. On comparative evaluation thiolated psyllium gels showed 3-fold higher mucoadhesive strength than the psyllium gels as determined by modified physical balance using chicken buccal pouch. The results of in vitro release study revealed that thiolated psyllium gels provided a prolonged release of metronidazole. Further, the psyllium and thiolated psyllium gels were found to release the drug following first-order kinetics by combination of polymer relaxation and diffusion through the matrix. PMID:24348147

Selenium-containing organic nanoparticles as silent precursors for ultra-sensitive thiol-responsive transmembrane anion transport.

PubMed

Lang, Chao; Zhang, Xin; Dong, Zeyuan; Luo, Quan; Qiao, Shanpeng; Huang, Zupeng; Fan, Xiaotong; Xu, Jiayun; Liu, Junqiu

2016-02-07

An anion transporter with a selenoxide group was able to form nanoparticles in water, whose activity was fully turned off due to the aggregation effect. The formed nanoparticles have a uniform size and can be readily dispersed in water at high concentrations. Turn-on of the nanoparticles by reducing molecules is proposed to be a combined process, including the reduction of selenoxide to selenide, disassembly of the nanoparticles and location of the transporter to the lipid membrane. Accordingly, a special acceleration phase can be observed in the turn-on kinetic curves. Since turn-on of the nanoparticles is quantitatively related to the amount of reductant, the nanoparticles can be activated in a step-by-step manner. Due to the sensibility of this system to thiols, cysteine can be detected at low nanomolar concentrations. This ultra-sensitive thiol-responsive transmembrane anion transport system is quite promising in biological applications.

ortho- and meta-substituted aromatic thiols are efficient redox buffers that increase the folding rate of a disulfide-containing protein.

PubMed

Gough, Jonathan D; Barrett, Elvis J; Silva, Yenia; Lees, Watson J

2006-08-20

Thiol based redox buffers are used to enhance the folding rates of disulfide-containing proteins in vitro. Traditionally, small molecule aliphatic thiols such as glutathione are employed. Recently, we have demonstrated that aromatic thiols can further enhance protein-folding rates. In the presence of para-substituted aromatic thiols the folding rate of a disulfide-containing protein was increased by 4-23 times over that measured for glutathione. However, several important practical issues remain to be addressed. Aromatic thiols have never been tested in the presence of denaturants such as guanidine hydrochloride. Only two of the para-substituted aromatic thiols previously examined are commercially available. To expand the number of aromatic thiols for protein folding, several commercially available meta- and ortho-substituted aromatic thiols were studied. Furthermore, an ortho-substituted aromatic thiol, easily obtained from inexpensive starting materials, was investigated. Folding rates of scrambled ribonuclease A at pH 6.0, 7.0 and 7.7, with ortho- and meta-substituted aromatic thiols, were up to 10 times greater than those with glutathione. In the presence of the common denaturant guanidine hydrochloride (0.5M) aromatic thiols provided 100% yield of active protein while maintaining equivalent folding rates.

Attenuation of glycation-induced multiple protein modifications by Indian antidiabetic plant extracts.

PubMed

Tupe, Rashmi S; Kemse, Nisha G; Khaire, Amrita A; Shaikh, Shamim A

2017-12-01

Protein glycation is the major contributing factor in the development of diabetic complications. The antiglycation potential of medicinal plants provides a promising opportunity as complementary interventions for complications. To investigate the antiglycation potential of 19 medicinal plants extracts using albumin by estimating different indicators: (1) glycation (early and late), (2) albumin oxidation, and (3) amyloid aggregation. The effect of aqueous plant extracts (1% w/v) on protein glycation was assessed by incubating albumin (10 mg/mL) with fructose (250 mM) for 4 days. Degree of protein glycation in the absence and presence of plant extracts was assessed by estimating fructosamine, advanced glycation end products (AGEs), carbonyls, free thiol group and β-amyloid aggregation. Petroselinum crispum, Boerhavia diffusa, Terminalia chebula, Swertia chirayita and Glycyrrhiza glabra showed significant antiglycating activity. P. crispum and A. barbadensis inhibited the carbonyl stress and protected the thiol group from oxidative damage. There was significant correlation between protein thiols and amyloid inhibition (R = -.69, p < .001). P. crispum, B. diffusa and T. chebula had the most potent antiglycation activity. These plant exerted noticeable antiglycation activity at different glycation modifications of albumin. These findings are important for identifying plants with potential to combat diabetic complications.

Hydrogen Abstraction from Individual Thiophenol Molecules Adsorbed on Cu(111)

NASA Astrophysics Data System (ADS)

Rao, Bommisetty; Kwon, Ki-Young; Liu, Anwei; Zhang, Jin-Tao; Bartels, Ludwig

2004-03-01

Thiol compounds on metal surfaces have been studied intensively because of their ability to form self-assembled monolayers (SAMs). However, the transition from the thiol to the surface thiolate is difficult to investigate in detail in the solution phase. Here we report on STM measurements that address the adsorption of a variety of substituted thiophenols on Cu(111) at 15K in vacuum. At this temperature, adsorption does not cause immediate scission of the S-H bond. We confirmed this by STM-based vibrational spectroscopy. Consequently, the sulfur atom of the thiol group adsorbs on-top of a substrate atom, which results in a sufficient separation of the aryl group from the substrate to allow its free rotation even at 15K. Asymmetrically substituted thiophenols result in STM images of pronounced helicity, which indicates that the molecules cannot tilt upright to exchange their adsorption side. Attachment of electrons from the tunneling current can cause hydrogen abstraction from the thiophenols, which locks them into the substrate. We investigated the dependence of the yield of the hydrogen abstraction on the thiophenol substituent identity and position. We find pronounced variations which may follow the Hammett Equation known from Standard Organic Chemistry.

Protoapigenone, a natural derivative of apigenin, induces mitogen-activated protein kinase-dependent apoptosis in human breast cancer cells associated with induction of oxidative stress and inhibition of glutathione S-transferase π.

PubMed

Chen, Wen-Ying; Hsieh, Yu-An; Tsai, Ching-I; Kang, Ya-Fei; Chang, Fang-Rong; Wu, Yang-Chang; Wu, Chin-Chung

2011-12-01

Protoapigenone, a natural derivative of the flavonoid apigenin, has been shown to exhibit potent antitumor activity in vitro and in vivo; the precise mechanism of action, however, is not fully elucidated. In this study, we investigated and compared the mechanisms by which protoapigenone and apigenin caused cell death in the human breast cancer MDA-MB-231 cells. Flow cytometry analysis revealed that protoapigenone induced apoptosis with 10-fold greater potency than apigenin. Cancer cells treated with protoapigenone resulted in persistent activation of mitogen-activated protein kinase (MAPK) ERK, JNK, and p38, hyperphosphorylation of Bcl-2 and Bcl-xL, and loss of mitochondrial membrane potential (MMP). The MAPK inhibitors effectively prevented the loss of MMP and apoptosis induced by protoapigenone. Treatment of cells with protoapigenone led to increased levels of reactive oxygen species (ROS) and decreased levels of intracellular glutathione. The thiol-antioxidant N-acetylcysteine abolished protoapigenone-induced MAPK activation, mitochondrial dysfunction, and apoptosis. These results suggest that the induction of oxidative stress preceding the activation of MAPK is required to initiate the mitochondria-mediated apoptosis induced by protoapigenone. Additionally, protoapigenone-induced JNK activation was linked to thiol modification of glutathione S-transferase π (GSTpi), which impeded GSTpi inhibition of JNK. In contrast to protoapigenone, apigenin-induced apoptosis was neither dependent on ROS nor on MAPK. Structure-activity relationship studies suggested that the thiol reacting effect of protoapigenone might be associated with an α, β-unsaturated ketone moiety in the structure of ring B.

The effects of chromium(VI) on the thioredoxin system: Implications for redox regulation

PubMed Central

Myers, Charles R.

2014-01-01

Hexavalent chromium [Cr(VI)] compounds are highly redox active and have long been recognized as potent cytotoxins and carcinogens. The intracellular reduction of Cr(VI) generates reactive Cr intermediates, which are themselves strong oxidants, as well as superoxide, hydrogen peroxide, and hydroxyl radical. These probably contribute to the oxidative damage and effects on redox-sensitive transcription factors that have been reported. However, the identification of events that initiate these signaling changes has been elusive. More recent studies show that Cr(VI) causes irreversible inhibition of thioredoxin reductase (TrxR) and oxidation of thioredoxin (Trx) and peroxiredoxin (Prx). Mitochondrial Trx2/Prx3 are more sensitive to Cr(VI) treatment than cytosolic Trx1/Prx1, although both compartments show thiol oxidation with higher doses or longer treatments. Thiol redox proteomics demonstrate that Trx2, Prx3, and Trx1 are among the most sensitive proteins in cells to Cr(VI) treatment. Their oxidation could therefore represent initiating events that have widespread implications for protein thiol redox control and for multiple aspects of redox signaling. This review summarizes the effects of Cr(VI) on the TrxR/Trx system and how these events could influence a number of downstream redox signaling systems that are influenced by Cr(VI) exposure. Some of the signaling events discussed include the activation of apoptosis signal regulating kinase and MAP kinases (p38 and JNK) and the modulation of a number of redox-sensitive transcription factors including AP-1, NF-κB, p53, and Nrf2. PMID:22542445

Involvement of Redox State in the Aging of Drosophila melanogaster

PubMed Central

Radyuk, Svetlana N.; Sohal, Rajindar S.

2013-01-01

Abstract Significance: The main objective of this review was to provide an exposition of investigations, conducted in Drosophila melanogaster, on the role of reactive oxygen species and redox state in the aging process. While early transgenic studies did not clearly support the validity of the oxidative stress hypothesis of aging, predicated on the accumulation of structural damage, they spawned a broader search for redox-related effects that might impact the aging process. Recent Advances: Initial evidence implicating the thiol redox state as a possible causative factor in aging has been obtained in Drosophila. Overexpression of genes, such as GCL, G6PD, Prx2, and Prx5, which are involved in the maintenance of thiol redox homeostasis, has strong positive effects on longevity. Further, the depletion of peroxiredoxin activity in the mitochondria through the double knockdown of Prx5 and Prx3 not only results in a redox crisis but also elicits a rapid aging phenotype. Critical Issues: Herein, we summarize the present status of knowledge about the main components of the machinery controlling thiol redox homeostasis and describe how age-related redox fluctuations might impact aging more acutely through disruption of the redox-sensitive signaling mechanisms rather than via the simple accumulation of structural damage. Future Directions: Based on these initial insights into the plausible impact of redox fluctuations on redox signaling, future studies should focus on the pathways that have been explicitly implicated in aging, such as insulin signaling, TOR, and JNK/FOXO, with particular attention to elements that are redox sensitive. Antioxid. Redox Signal. 19, 788–803. PMID:23458359

New reactive polymer for protein immobilisation on sensor surfaces.

PubMed

Kyprianou, Dimitris; Guerreiro, Antonio R; Chianella, Iva; Piletska, Elena V; Fowler, Steven A; Karim, Kal; Whitcombe, Michael J; Turner, Anthony P F; Piletsky, Sergey A

2009-01-01

Immobilisation of biorecognition elements on transducer surfaces is a key step in the development of biosensors. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. A novel protocol for the covalent immobilisation of biomolecules containing primary amines using an inexpensive and simple polymer is presented. This tri-dimensional (3D) network leads to a random immobilisation of antibodies on the polymer and ensures the availability of a high percentage of antibody binding sites. The reactivity of the polymer is based on the reaction between primary amines and thioacetal groups included in the polymer network. These functional groups (thioacetal) do not need any further activation in order to react with proteins, making it attractive for sensor fabrication. The novel polymer also contains thiol derivative groups (disulphide groups or thioethers) that promote self-assembling on a metal transducer surface. For demonstration purposes the polymer was immobilised on Au Biacore chips. The resulting polymer layer was characterised using contact angle meter, atomic force microscopy (AFM) and ellipsometry. A general protocol suitable for the immobilisation of bovine serum albumin (BSA), enzymes and antibodies such as polyclonal anti-microcystin-LR antibody and monoclonal anti-prostate specific antigen (anti-PSA) antibody was then optimised. The affinity characteristics of developed immunosensors were investigated in reaction with microcystin-LR, and PSA. The calculated detection limit for analytes depended on the properties of antibodies. The detection limit for microcystin-LR was 10 ngmL(-1) and for PSA 0.01 ngmL(-1). The non-specific binding of analytes to synthesised polymers was very low. The polymer-coated chips were stored for up to 2 months without any noticeable deterioration in their ability to react with proteins. These findings make this new polymer very promising for the development of low-cost, easy to prepare and sensitive biosensors.

Tailoring protein nanomechanics with chemical reactivity

PubMed Central

Beedle, Amy E. M.; Mora, Marc; Lynham, Steven; Stirnemann, Guillaume; Garcia-Manyes, Sergi

2017-01-01

The nanomechanical properties of elastomeric proteins determine the elasticity of a variety of tissues. A widespread natural tactic to regulate protein extensibility lies in the presence of covalent disulfide bonds, which significantly enhance protein stiffness. The prevalent in vivo strategy to form disulfide bonds requires the presence of dedicated enzymes. Here we propose an alternative chemical route to promote non-enzymatic oxidative protein folding via disulfide isomerization based on naturally occurring small molecules. Using single-molecule force-clamp spectroscopy, supported by DFT calculations and mass spectrometry measurements, we demonstrate that subtle changes in the chemical structure of a transient mixed-disulfide intermediate adduct between a protein cysteine and an attacking low molecular-weight thiol have a dramatic effect on the protein's mechanical stability. This approach provides a general tool to rationalize the dynamics of S-thiolation and its role in modulating protein nanomechanics, offering molecular insights on how chemical reactivity regulates protein elasticity. PMID:28585528

S-protected thiolated hydroxyethyl cellulose (HEC): Novel mucoadhesive excipient with improved stability.

PubMed

Leonaviciute, Gintare; Bonengel, Sonja; Mahmood, Arshad; Ahmad Idrees, Muneeb; Bernkop-Schnürch, Andreas

2016-06-25

The aim of this study was the design of novel S-protected thiolated hydroxyethyl cellulose (HEC) and the assessment of its mucoadhesive properties and biodegradability compared to the corresponding unmodified polymer. Thiolated HEC was S-protected via disulfide bond formation between 6-mercaptonicotinamide (6-MNA) and the thiol substructures of the polymer. In vitro screening of mucoadhesive properties was accomplished using two different methods: rotating cylinder studies and viscosity measurements. Moreover, biodegradability of these polymers by cellulase, xylanase and lysozyme was evaluated. MTT and LDH assays were performed on Caco-2 cells to determine the cytotoxicity of S-protected thiolated HEC. Thiolated HEC displayed 280.09±1.70μmol of free thiol groups per gram polymer. S-protected thiolated HEC exhibiting 270.8±21.11μmol immobilized 6-MNA ligands per gram of polymer was shown being 2.4-fold more mucoadhesive compared to thiolated HEC. No mucoadhesion was observed in case of unmodified HEC. Results were in a good agreement with rheological studies. The presence of free thiol moieties likely caused lower degree of hydrolysis by xylanase, whereas the degradation by both enzymes cellulase and xylanase was more hampered when 6-MNA was introduced as ligand for thiol group's protection. Findings in cell viability revealed that all three conjugates were non-toxic. S-protection of thiolated hydroxyethyl cellulose improved mucoadhesive properties and provided pronounced stability towards enzymatic attack, that makes this excipient superior for non-invasive drug administration over thiolated and unmodified forms. Copyright © 2016 Elsevier Ltd. All rights reserved.

Probing Conformational Changes in Human DNA Topoisomerase IIα by Pulsed Alkylation Mass Spectrometry*

PubMed Central

Chen, Yu-tsung; Collins, Tammy R. L.; Guan, Ziqiang; Chen, Vincent B.; Hsieh, Tao-Shih

2012-01-01

Type II topoisomerases are essential enzymes for solving DNA topological problems by passing one segment of DNA duplex through a transient double-strand break in a second segment. The reaction requires the enzyme to precisely control DNA cleavage and gate opening coupled with ATP hydrolysis. Using pulsed alkylation mass spectrometry, we were able to monitor the solvent accessibilities around 13 cysteines distributed throughout human topoisomerase IIα by measuring the thiol reactivities with monobromobimane. Most of the measured reactivities are in accordance with the predicted ones based on a homology structural model generated from available crystal structures. However, these results reveal new information for both the residues not covered in the structural model and potential differences between the modeled and solution holoenzyme structures. Furthermore, on the basis of the reactivity changes of several cysteines located at the N-gate and DNA gate, we could monitor the movement of topoisomerase II in the presence of cofactors and detect differences in the DNA gate between two closed clamp enzyme conformations locked by either 5′-adenylyl β,γ-imidodiphosphate or the anticancer drug ICRF-193. PMID:22679013

Chiral mercaptoacetamides display enantioselective inhibition of histone deacetylase 6 and exhibit neuroprotection in cortical neuron models of oxidative stress.

PubMed

Kalin, Jay H; Zhang, Hankun; Gaudrel-Grosay, Sophie; Vistoli, Giulio; Kozikowski, Alan P

2012-03-05

Mercaptoacetamide-based ligands have been designed as a new class of histone deacetylase (HDAC) inhibitors for possible use in the treatment of neurodegenerative diseases. The thiol group of these compounds provides a key binding element for interaction with the catalytic zinc ion, and thus differs from the more typically employed hydroxamic acid based zinc binding groups. Herein we disclose the chemistry and biology of some substituted mercaptoacetamides with the intention of increasing HDAC6 isoform selectivity while maintaining potency similar to their hydroxamic acid analogues. The introduction of a stereocenter α to the thiol group was found to have a considerable impact on HDAC inhibitor potency. These new compounds were also profiled for their therapeutic potential in an in vitro model of stress-induced neuronal injury and were found to act as nontoxic neuroprotective agents. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of pro-oxidant-antioxidant balance (PAB) and its association with inflammatory cytokines in polycystic ovary syndrome (PCOS).

PubMed

Artimani, T; Karimi, J; Mehdizadeh, M; Yavangi, M; Khanlarzadeh, E; Ghorbani, M; Asadi, S; Kheiripour, N

2018-02-01

Chronic low-grade inflammation has been suggested as a key contributor of the pathogenesis and development of polycystic ovary syndrome (PCOS). To investigate the association between oxidative stress status and inflammatory cytokines in follicular fluid of 21 PCOS women compared to 21 women with normal ovarian function who underwent intra-cytoplasmic sperm injection. Concentration of IL-6, IL-8, IL-10, and TNF-α was measured using sandwich ELISA. Oxidative stress was examined by measuring total oxidant status (TOS), malondialdehyde (MDA), total antioxidant capacity (TAC), and thiol groups. PCOS women had an elevated concentration of MDA and TOS compared to controls. Levels of TAC and thiol groups were lower in PCOS compared to controls. PCOS patients had a higher concentration of IL-6, IL-8, and TNF-α compared to controls. Concentration of IL-10 was lower in PCOS compared to controls. Significant correlations were found between MDA and TOS concentration with TNF-α and between IL-6 and MDA, IL-8 and TAC, IL-10 and TOS levels and also between IL-10 and TAC levels. TAC and thiol groups were negatively correlated with TNF-α. Increased oxidative stress in PCOS is associated with inflammation which is closely linked. Inflammation can induce production of inflammatory cytokines in this syndrome and directly stimulates excess ovarian androgen production.

Influence of reagents reacting with metal, thiol and amino sites of catalytic activity and l-phenylalanine inhibition of rat intestinal alkaline phosphatase

PubMed Central

Fishman, William H.; Ghosh, Nimai K.

1967-01-01

1. Studies on the inactivation of rat intestinal alkaline phosphatase by several metal-binding agents, namely EDTA, 8-hydroxyquinoline, pyridine-2,6-dicarboxylic acid, αα′-bipyridyl, o-phenanthroline and sodium cyanide, indicated the functional role of a metal, probably zinc, in the catalysis. The metal ligands lowered stereospecific uncompetitive inhibition of the enzyme by l-phenylalanine by an extent that paralleled the decline in enzyme activity. 2. The thiol reagents p-hydroxymercuribenzoate, iodoacetamide and iodine inactivated rat intestinal phosphatase. The enzyme could be protected from inactivation by either cysteine or substrate. The l-phenylalanine inhibition remained unchanged only in the presence of moderately inactivating concentrations of the thiol reagents. 3. Inactivation of the enzyme by the amino-group-blocking reagent, O-methylisourea, provided ample evidence for the participation in the catalysis of the ∈-amino group of lysine. At the same time, l-phenylalanine inhibition remained unaltered even when the enzyme was strongly inactivated. This ∈-amino-group-blocked enzyme exhibited no change in migration in starch gel, in contrast with enzyme treated with acetic anhydride, formaldehyde or succinic anhydride. The Michaelis constant of the enzyme was enhanced by such modifications, but the optimum pH remained the same. 4. d-Phenylalanine acted as a competitive or `co-operative' activator for intestinal alkaline phosphatase after it had been modified by acetylation. PMID:16742542

IR spectroscopy study of SBA-15 silicas functionalized with the ethylthiocarbamidepropyl groups and their interactions with Ag(I) and Hg(II) ions

NASA Astrophysics Data System (ADS)

Melnyk, Inna V.; Nazarchuk, Galyna I.; Václavíková, Miroslava; Zub, Yuriy L.

2018-04-01

Mesoporous structure of silica is determined by the type of template, but the introduction of functional groups during the synthesis has additional influence. The structure of SBA-15 may be violated by the introduction of long functions, such as ≡Si(CH2)3NHC(=S)NHC2H5. These ethylthiocarbamidepropyl groups can form complexes with metal ions in thiol or thione tautomeric forms. We determined that the 2D hexagonal p6 mm structure is preserved for SBA-15 with thiourea groups at maximal TEOS:trifunctional silane ratio (mol) = 10:2, which was confirmed by TEM and by the presence of an intense reflex in the small-angle region of diffractograms of the final product. It was shown that the obtained sorbents possess high kinetic characteristics. The experimental data fit pseudo-second-order kinetic equation, but the rate constants depend on the content of functional groups in the surface layer. Template Pluronic P-123 defines the porosity of functional mesoporous silica materials even at increasing content of trifunctional silane in the initial solution. Infrared spectroscopy analysis showed that thione form of thiourea ligand is prevalent on the surface of pores of mesoporous samples. However, during the sorption of silver(I) ions, there are both thione and thiol forms on the surface. Thione form is transformed into thiol with increasing concentration of mercury(II) ions in the sorption solution. Adsorption experiments showed that the SBA-15 silicas functionalized with ethylthiocarbamidepropyl groups had high selectivity for silver(I) ions and could concentrate Ag(I) ions from metal ions mixture at pH 2.

Endocrine Imbalance Associated With Proteome Changes in Diabetes.

PubMed

Khairallah, Ahmed; Farag, Abo-Alela; Johar, Dina; Bernstein, Larry

2017-11-01

The dynamics of cellular metabolism involves rapid interactions between proteins and nucleic acids, proteins and proteins, and signaling. These involve the interactions with respect to the sulfur bond, noncovalent electrostatic interactions, protein structure stabilization and protein-ligand binding, weak electrostatic interactions in proteins, oxygen radicals that initiate a change in conformation and a chain of events. We review a development in molecular medicine that is a very promising work in progress. We also review the current and future research methods involving mitochondria. Long-term effects of diabetes include glycation of proteins, for example, glycohemoglobin (HbA1c), increased risk of cardiovascular diseases, atherosclerosis, retinopathy, nephropathy, and neurological dysfunctions. Tissues are exposed to significant quantities of highly reactive chemical species including nitric oxide • NO and reactive oxygen species ROS over months to years, to an extent generated by mitochondrial activities. The reactions of • NO can be broadly discussed with reference to three main processes which control their fate in biological systems: (1) diffusion and intra-cellular consumption; (2) autooxidation to form nitrous anhydride N 2 O 3 ; and (3) reaction with superoxide O2 • - to form peroxynitrite ONOO-. Reactive nitrogen species produced by macrophages and neutrophils in the interstitial space, with emphasis on • NO, N 2 O 3 , ONOO-, and nitrogen dioxide radicals • NO 2 generate protein and DNA damage. Serum thiol (-SH) groups act as an important extracellular scavenger of peroxides and are therefore helpful in protecting the surrounding tissues. The events described here are a homeostatic endocrine imbalance that is associated with proteostasis. The advances we have seen in untangling this web of interactions are sure to continue at a breathtaking pace. J. Cell. Biochem. 118: 3569-3576, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The Effect of Allium cepa Extract on Lung Oxidant, Antioxidant, and Immunological Biomarkers in Ovalbumin-Sensitized Rats

PubMed Central

Marefati, N.; Eftekhar, N.; Kaveh, M.; Boskabadi, J.; Beheshti, F.; Boskabady, M.H.

2018-01-01

Objectives To evaluate the effects of Allium cepa (A. cepa) on levels of oxidants, antioxidants, and immunological markers in bronchoalveolar lavage fluids (BALF) of sensitized rats. Materials and Methods Oxidant/antioxidant markers and cytokines in BALF of control rats treated with saline (group C), ovalbumin-sensitized rats (group S), rats treated with 1.25 μg/mL dexamethasone and 3 doses of A. cepa extract (35, 70, and 140 mg/kg body weight [BW]/day) (S + AC) were investigated. Comparison of the results between groups was performed using analysis of variance with the Tukey-Kramer post hoc test. Results The oxidant markers nitrogen dioxide (NO2), nitrate (NO3–), and malondialdehyde (MDA), and immunological markers interleukin (IL)-4 and immunoglobulin E (IgE) were significantly higher, but the antioxidant markers superoxide dismutase (SOD), catalase (CAT), thiol, and interferon (IFN)-γ, and the IFN-γ/IL-4 ratio were lower in sensitized rats compared to control rats (p < 0.001 to p < 0.01). Compared to group S, the levels of the following markers were significantly lower: NO2, NO3–, and IgE in groups treated with the A. cepa extract, MDA and IL-4 levels in groups treated with 70 and 140 mg/kg BW/day of the A. cepa extract, and all these markers as well as IFN-γ in rats treated with dexamethasone (p < 0.001 to p < 0.05). However, there were significantly higher levels of SOD and CAT and an increased IFN-γ/IL-4 ratio (groups treated with 70 and 140 mg/kg BW/day of the A. cepa extract), and levels of thiol and IFN-γ (group treated with 140 mg/kg BW/day of the A. cepa extract) as well as SOD, CAT, and thiol (dexamethasone-treated group) versus group S (p < 0.00 to p < 0.05). Conclusion A. cepa showed antioxidant and immunomodulatory properties in sensitized rats. PMID:29471299

Fluorescence labelling of NADPH-cytochrome P-450 reductase with the monobromomethyl derivative of syn-9,10-dioxabimane.

PubMed Central

Vogel, F; Lumper, L

1983-01-01

The kinetics of thiol-group alkylation in NADPH-cytochrome P-450 reductase during its inactivation by monobromobimane has been studied using the fluorimetric determination of S-bimane-L-cysteine by high-performance liquid chromatography. Loss of activity during the reaction of NADPH-cytochrome P-450 reductase with monobromobimane is caused by the alkylation of one single critical cysteine residue, which can be protected against thiol-specific reagents by NADP(H). The chemical stability of the bimane group allows the digestion of bimane-labelled NADPH-cytochrome P-450 reductase by CNBr. The critical cysteine residue could be located in a CNBr-cleaved peptide purified to homogeneity with Mr 10 500 +/- 1 000 and valine as N-terminus. Images Fig. 2. PMID:6414464

Highly sensitive, colorimetric detection of mercury(II) in aqueous media by quaternary ammonium group-capped gold nanoparticles at room temperature.

PubMed

Liu, Dingbin; Qu, Weisi; Chen, Wenwen; Zhang, Wei; Wang, Zhuo; Jiang, Xingyu

2010-12-01

We provide a highly sensitive and selective assay to detect Hg(2+) in aqueous solutions using gold nanoparticles modified with quaternary ammonium group-terminated thiols at room temperature. The mechanism is the abstraction of thiols by Hg(2+) that led to the aggregation of nanoparticles. With the assistance of solar light irradiation, the detection limit can be as low as 30 nM, which satisfies the guideline concentration of Hg(2+) in drinking water set by the WHO. In addition, the dynamic range of detection is wide (3 × 10(-8)-1 × 10(-2) M). This range, to our best knowledge, is the widest one that has been reported so far in gold nanoparticle (AuNP)-based assays for Hg(2+).

Thiol reactivity and its impact on the ciliate toxicity of α,β-unsaturated aldehydes, ketones, and esters.

PubMed

Böhme, Alexander; Thaens, Diana; Schramm, Franziska; Paschke, Albrecht; Schüürmann, Gerrit

2010-12-20

A recently introduced chemoassay has been used to determine second-order rate constants of the electrophile-nucleophile reaction of 15 α,β-unsaturated aldehydes with glutathione. The respective kGSH values vary for more than 3 orders of magnitude, and are within the range determined previously for 31 α,β-unsaturated ketones and esters. Structure-reactivity analyses yield distinct relationships between kGSH and structural features of the compounds. Moreover, increasing kGSH increases the aldehyde toxicity toward ciliates in terms of 48 h-EC50 values (effective concentration yielding 50% growth inhibition of Tetrahymena pyriformis within 48 h). A respective log-log regression equation including both kGSH and the octanol/water partition coefficient, Kow, yields a squared correlation coefficient of 0.96. Comparative analysis with corresponding data for 15 ketones and 16 esters reveals systematic differences between the three compound classes with regard to the individual contributions of hydrophobicity and electrophilic reactivity to aquatic toxicity. The former is particularly pronounced for aldehydes, while the ester toxicity is largely governed by reactivity, with ketones showing an intermediate pattern that is more similar to the one of esters than of aldehydes. It follows that within the Michael acceptor domain of α,β-unsaturated carbonyls, a distinction between aldehydes and nonaldehydic derivatives appears necessary when employing electrophilic reactivity as a component for the quantitative prediction of their reactive toxicity toward aquatic organisms.

Molecular Characterization of Thiols in Fossil Fuels by Michael Addition Reaction Derivatization and Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

PubMed

Wang, Meng; Zhao, Suoqi; Liu, Xuxia; Shi, Quan

2016-10-04

Thiols widely occur in sediments and fossil fuels. However, the molecular composition of these compounds is unclear due to the lack of appropriate analytical methods. In this work, a characterization method for thiols in fossil fuels was developed on the basis of Michael addition reaction derivatization followed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS). Model thiol compound studies showed that thiols were selectively reacted with phenylvinylsulfone and transformed to sulfones with greater than 98% conversions. This method was applied to a coker naphtha, light and heavy gas oils, and crude oils from various geological sources. The results showed that long alkyl chain thiols are readily present in petroleum, which have up to 30 carbon atoms. Large DBE dispersity of thiols indicates that naphthenic and aromatic thiols are also present in the petroleum. This method is capable of detecting thiol compounds in the part per million range by weight. This method allows characterization of thiols in a complex hydrocarbon matrix, which is complementary to the comprehensive analysis of sulfur compounds in fossil fuels.

Hydrolysis-controlled protein adsorption and antifouling behaviors of mixed charged self-assembled monolayer: A molecular simulation study.

PubMed

Liu, Jie; Zhou, Jian

2016-08-01

Understanding the mechanism of the antimicrobial and antifouling properties of mixed charged materials is of great significance. The interactions between human gamma fibrinogen (γFg) and mixed carboxylic methyl ether-terminated (COOCH3-) and trimethylamino-terminated (N(CH3)3(+)-) SAMs and the influence of hydrolysis were studied by molecular simulations. After hydrolysis, the mixed SAMs exhibit behaviors from antimicrobial to antifouling, since the COOCH3-thiols were translated into carboxylic acid (COO(-)-) terminated thiols, which carried a net charge of -1 e. Simulation results showed that the main differences between COOCH3-/N(CH3)3(+)-SAM and COO(-)-/N(CH3)3(+)-SAM are the charged property and the hydration layer above the surface. γFg could stably adsorb on the positively-charged COOCH3-/N(CH3)3(+)-SAM. The adsorption behavior is mainly induced by the strong electrostatic attraction. There is a single hydration layer bound to the surface, which is related to the N(CH3)3(+) groups. The van der Waals repulsion between γFg and the single hydration layer are not strong enough to compensate the strong electrostatic attraction. After hydrolysis, the positively-charged SAM was transferred to a neutral mixed charged surface, the electrostatic attraction between γFg and the surface disappears. Meanwhile, the SAM surface is covered by double hydration layers, which is induced by the N(CH3)3(+) and COO(-) groups; water molecules around COO(-) groups are obviously denser than that around N(CH3)3(+) groups. With the combined contribution from double hydration layers and the vanishment of electrostatic attraction, γFg is forced to desorb from the surface. After hydrolysis, the internal structure of mixed SAM appears more ordered due to the electrostatic interactions between charged groups on the top of SAMs. The antimicrobial and antifouling materials are of great importance in many biological applications. The strong hydration property of surfaces and the interactions between proteins and surfaces play a key role in resisting protein adsorption. The mixed SAMs, constructed from a 1:1 combination of COOCH3- and N(CH3)3(+)-terminated thiols, can induce protein adsorption mainly through the electrostatic interaction. When the COOCH3-terminated thiols were hydrolyzed to negatively charged COO(-)-terminated thiols, the mixed-charged SAMs switched from antimicrobial to antifouling. Due to the strong hydration property of the mixed charged SAMs, the adsorbed γFg moved away from the surface. Understanding the interactions between protein and mixed-charged SAMs in the atomistic level is important for the practical design and development of new antimicrobial and antifouling materials. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Biochemical and oxidative stress markers in the liver and kidneys of rats submitted to different protocols of anabolic steroids.

PubMed

Dornelles, Guilherme Lopes; Bueno, Andressa; de Oliveira, Juliana Sorraila; da Silva, Aleksandro Schafer; França, Raqueli Teresinha; da Silva, Cássia Bagolin; Machado, Márcia Silveira Netto; Petry, Letícia do Santos; Abdalla, Fátima Husein; Lhamas, Cibele Lima; de Andrade, Cinthia Melazzo

2017-01-01

The objective of this study was to evaluate the effects of different protocols (P1, P2, and P3) of boldenone undecylenate (BU) and stanozolol (ST) on markers of liver and kidney function and variables of oxidative stress in these organs. For this, 54 male Wistar rats were divided into nine groups of six animals each. Each animal received intramuscularly 5.0 mg kg -1 of BU or ST once a week for 4 weeks (P1); 2.5 mg kg -1 of BU or ST once a week for 8 weeks (P2); and 1.25 mg kg -1 of BU or ST once a week for 12 weeks (P3). For each protocol, a control group was used, and they received 0.1 ml of olive oil intramuscularly. Blood and fragments of liver and kidney were collected for alanine aminotransferase activity (ALT), alkaline phosphatase, albumin, creatinine, cholesterol, total protein, triglycerides, urea, reactive oxygen species, thiobarbituric acid reactive substances, total thiols, and glutathione evaluation. The results show that the BU in doses of 5 (day 30) and 2.5 mg kg -1 (day 60) changes the ALT seric activity, possibly showing a hepatotoxic effect. High doses of BU may lead to increased levels of cholesterol (protocol P1) possibly due to inhibition of the normal steroid biosynthesis process. All protocols used caused changes in the redox balance of the organs studied (except in the liver, protocol P2), which indicates that these drugs might be harmful even at low doses.

Hydrogen Sulfide Sensing through Reactive Sulfur Species (RSS) and Nitroxyl (HNO) in Enterococcus faecalis.

PubMed

Shen, Jiangchuan; Walsh, Brenna J C; Flores-Mireles, Ana Lidia; Peng, Hui; Zhang, Yifan; Zhang, Yixiang; Trinidad, Jonathan C; Hultgren, Scott J; Giedroc, David P

2018-05-17

Recent studies of hydrogen sulfide (H 2 S) signaling implicate low molecular weight (LMW) thiol persulfides and other reactive sulfur species (RSS) as signaling effectors. Here, we show that a CstR protein from the human pathogen Enterococcus faecalis ( E. faecalis), previously identified in Staphylococcus aureus ( S. aureus), is an RSS-sensing repressor that transcriptionally regulates a cst-like operon in response to both exogenous sulfide stress and Angeli's salt, a precursor of nitroxyl (HNO). E. faecalis CstR reacts with coenzyme A persulfide (CoASSH) to form interprotomer disulfide and trisulfide bridges between C32 and C61', which negatively regulate DNA binding to a consensus CstR DNA operator. A Δ cstR strain exhibits deficiency in catheter colonization in a catheter-associated urinary tract infection (CAUTI) mouse model, suggesting sulfide regulation and homeostasis is critical for pathogenicity. Cellular polysulfide metabolite profiling of sodium sulfide-stressed E. faecalis confirms an increase in both inorganic polysulfides and LMW thiols and persulfides sensed by CstR. The cst-like operon encodes two authentic thiosulfate sulfurtransferases and an enzyme we characterize here as an NADH and FAD-dependent coenzyme A (CoA) persulfide reductase (CoAPR) that harbors an N-terminal CoA disulfide reductase (CDR) domain and a C-terminal rhodanese homology domain (RHD). Both cysteines in the CDR (C42) and RHD (C508) domains are required for CoAPR activity and complementation of a sulfide-induced growth phenotype of a S. aureus strain lacking cstB, encoding a nonheme Fe II persulfide dioxygenase. We propose that S. aureus CstB and E. faecalis CoAPR employ orthogonal chemistries to lower CoASSH that accumulates under conditions of cellular sulfide toxicity and signaling.

Metabolism and toxicity of arsenicals in mammals.

PubMed

Sattar, Adeel; Xie, Shuyu; Hafeez, Mian Abdul; Wang, Xu; Hussain, Hafiz Iftikhar; Iqbal, Zahid; Pan, Yuanhu; Iqbal, Mujahid; Shabbir, Muhammad Abubakr; Yuan, Zonghui

2016-12-01

Arsenic (As) is a metalloid usually found in organic and inorganic forms with different oxidation states, while inorganic form (arsenite As-III and arsenate As-v) is considered to be more hazardous as compared to organic form (methylarsonate and dimethylarsinate), with mild or no toxicity in mammals. Due to an increasing trend to using arsenicals as growth promoters or for treatment purposes, the understanding of metabolism and toxicity of As gets vital importance. Its toxicity is mainly depends on oxi-reduction states (As-III or As-v) and the level of methylation during the metabolism process. Currently, the exact metabolic pathways of As have yet to be confirmed in humans and food producing animals. Oxidative methylation and glutathione conjugation is believed to be major pathways of As metabolism. Oxidative methylation is based on conversion of Arsenite in to mono-methylarsonic acid and di-methylarsenic acid in mammals. It has been confirmed that As is only methylated in the presence of glutathione or thiol compounds, suggesting that As is being methylated in trivalent states. Subsequently, non-conjugated trivalent arsenicals are highly reactive with thiol which converts the trivalent arsenicals in to less toxic pentavalent forms. The glutathione conjugate stability of As is the most important factor for determining the toxicity. It can lead to DNA damage by alerting enzyme profile and production of reactive oxygen and nitrogen species which causes the oxidative stress. Moreover, As causes immune-dysfunction by hindering cellular and humeral immune response. The present review discussed different metabolic pathways and toxic outcomes of arsenicals in mammals which will be helpful in health risk assessment and its impact on biological world. Copyright © 2016. Published by Elsevier B.V.

Antioxidants, endothelial dysfunction, and DCS: in vitro and in vivo study.

PubMed

Wang, Qiong; Mazur, Aleksandra; Guerrero, François; Lambrechts, Kate; Buzzacott, Peter; Belhomme, Marc; Theron, Michaël

2015-12-15

Reactive oxygen species (ROS) production is a well-known effect in individuals after an undersea dive. This study aimed to delineate the links between ROS, endothelial dysfunction, and decompression sickness (DCS) through the use of antioxidants in vitro and in vivo. The effect of N-acetylcysteine (NAC) on superoxide and peroxynitrite, nitric oxide (NO) generation, and cell viability during in vitro diving simulation were analyzed. Also analyzed was the effect of vitamin C and NAC on plasma glutathione thiol and thiobarbituric acid reactive substances (TBARS), plasma angiotensin-converting enzyme (ACE) activity, and angiotensin-II and DCS morbidity during in vivo diving simulation. During an in vitro diving simulation, vascular endothelial cells showed overproduction of superoxide and peroxynitrite, obvious attenuation of NO generation, and promotion of cell death, all of which were reversed by NAC treatment. After in vivo diving simulation, plasma ACE activity and angiotensin-II level were not affected. The plasma level of glutathione thiol was downregulated after the dive, which was attenuated partially by NAC treatment. Plasma TBARS level was upregulated; however, either NAC or vitamin C treatment failed to prevent DCS morbidity. During in vitro simulation, endothelial superoxide and peroxynitrite-mediated oxidative stress were involved in the attenuation of NO availability and cell death. This study is the first attempt to link oxidative stress and DCS occurrence, and the link could not be confirmed in vivo. Even in the presence of antioxidants, ROS and bubbles generated during diving and/or decompression might lead to embolic or biochemical stress and DCS. Diving-induced oxidative stress might not be the only trigger of DCS morbidity. Copyright © 2015 the American Physiological Society.

Leinamycin E1 acting as an anticancer prodrug activated by reactive oxygen species.

PubMed

Huang, Sheng-Xiong; Yun, Bong-Sik; Ma, Ming; Basu, Hirak S; Church, Dawn R; Ingenhorst, Gudrun; Huang, Yong; Yang, Dong; Lohman, Jeremy R; Tang, Gong-Li; Ju, Jianhua; Liu, Tao; Wilding, George; Shen, Ben

2015-07-07

Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140, featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. Upon reductive activation in the presence of cellular thiols, LNM exerts its antitumor activity by an episulfonium ion-mediated DNA alkylation. Previously, we have cloned the lnm gene cluster from S. atroolivaceus S-140 and characterized the biosynthetic machinery responsible for the 18-membered lactam backbone and the alkyl branch at C3 of LNM. We now report the isolation and characterization of leinamycin E1 (LNM E1) from S. atroolivacues SB3033, a ΔlnmE mutant strain of S. atroolivaceus S-140. Complementary to the reductive activation of LNM by cellular thiols, LNM E1 can be oxidatively activated by cellular reactive oxygen species (ROS) to generate a similar episulfonium ion intermediate, thereby alkylating DNA and leading to eventual cell death. The feasibility of exploiting LNM E1 as an anticancer prodrug activated by ROS was demonstrated in two prostate cancer cell lines, LNCaP and DU-145. Because many cancer cells are under higher cellular oxidative stress with increased levels of ROS than normal cells, these findings support the idea of exploiting ROS as a means to target cancer cells and highlight LNM E1 as a novel lead for the development of anticancer prodrugs activated by ROS. The structure of LNM E1 also reveals critical new insights into LNM biosynthesis, setting the stage to investigate sulfur incorporation, as well as the tailoring steps that convert the nascent hybrid peptide-polyketide biosynthetic intermediate into LNM.

Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor-Ca2+ release channel by NADPH oxidase 4

PubMed Central

Sun, Qi-An; Hess, Douglas T.; Nogueira, Leonardo; Yong, Sandro; Bowles, Dawn E.; Eu, Jerry; Laurita, Kenneth R.; Meissner, Gerhard; Stamler, Jonathan S.

2011-01-01

Physiological sensing of O2 tension (partial O2 pressure, pO2) plays an important role in some mammalian cellular systems, but striated muscle generally is not considered to be among them. Here we describe a molecular mechanism in skeletal muscle that acutely couples changes in pO2 to altered calcium release through the ryanodine receptor–Ca2+-release channel (RyR1). Reactive oxygen species are generated in proportion to pO2 by NADPH oxidase 4 (Nox4) in the sarcoplasmic reticulum, and the consequent oxidation of a small set of RyR1 cysteine thiols results in increased RyR1 activity and Ca2+ release in isolated sarcoplasmic reticulum and in cultured myofibers and enhanced contractility of intact muscle. Thus, Nox4 is an O2 sensor in skeletal muscle, and O2-coupled hydrogen peroxide production by Nox4 governs the redox state of regulatory RyR1 thiols and thereby governs muscle performance. These findings reveal a molecular mechanism for O2-based signaling by an NADPH oxidase and demonstrate a physiological role for oxidative modification of RyR1. PMID:21896730

The early research and development of ebselen.

PubMed

Parnham, Michael J; Sies, Helmut

2013-11-01

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one; PZ-51, DR-3305), is an organoselenium compound with glutathione peroxidase (GPx)-like, thiol-dependent, hydroperoxide reducing activity. As an enzyme mimic for activity of the selenoenzyme GPx, this compound has proved to be highly useful in research on mechanisms in redox biology. Furthermore, the reactivity of ebselen with protein thiols has helped to identify novel, selective targets for inhibitory actions on several enzymes of importance in pharmacology and toxicology. Importantly, the selenium in ebselen is not released and thus is not bioavailable, ebselen metabolites being excreted in bile and urine. As a consequence, initial concerns about selenium toxicity, fortunately, were unfounded. Potential applications in medical settings have been explored, notably in brain ischemia and stroke. More recently, there has been a surge in interest as new medical applications have been taken into consideration. The first publication on the biochemical effects of ebselen appeared 30 years ago (Müller et al.), which prompted the authors to retrace the early development from their perspective. It is a fascinating example of fruitful interaction between research-oriented industry and academia. Copyright © 2013 Elsevier Inc. All rights reserved.

Investigation of molybdenum-crosslinker interfaces for affinity based electrochemical biosensing applications

NASA Astrophysics Data System (ADS)

Kamakoti, Vikramshankar; Shanmugam, Nandhinee Radha; Tanak, Ambalika Sanjeev; Jagannath, Badrinath; Prasad, Shalini

2018-04-01

Molybdenum (Mo) has been investigated for implementation as an electrode material for affinity based biosensing towards devloping flexibe electronic biosensors. Treatment of the native oxide of molybdenum was investigated through two surface treatment strategies namely thiol and carbodiimide crosslinking methods. The binding interaction between cross-linker molecules and Mo electrode surface has been characterized using Fourier Transform Infrared Spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and optical microscopy. The efficacy of treatment of Mo with its native oxide using carbodiimide cross linking methodology was established. The carbodiimide cross-linking chemistry was found to possess better surface coverage and binding affinity with Molybdenum electrode surface when compared to thiol cross-linking chemistry.Electrochemical characterization of Mo electrode using Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltametry (CV) techniques was performed to evaluate the effect of ionic properties of solution buffer on the Mo electrode's performance. Affinity based biosensing of C-Reactive Protein (CRP) has been demonstrated on a flexible nanoporous polymeric substrate with detection threshold of 100 pg/ml in synthetic urine buffer medium. The biosensor has been evaluated to be developed as a dipstick based point of care device for detection of biomarkers in urine.

Chemical protection against ionizing radiation. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Livesey, J.C.; Reed, D.J.; Adamson, L.F.

1984-08-01

The scientific literature on radiation-protective drugs is reviewed. Emphasis is placed on the mechanisms involved in determining the sensitivity of biological material to ionizing radiation and mechanisms of chemical radioprotection. In Section I, the types of radiation are described and the effects of ionizing radiation on biological systems are reviewed. The effects of ionizing radiation are briefly contrasted with the effects of non-ionizing radiation. Section II reviews the contributions of various natural factors which influence the inherent radiosensitivity of biological systems. Inlcuded in the list of these factors are water, oxygen, thiols, vitamins and antioxidants. Brief attention is given tomore » the model describing competition between oxygen and natural radioprotective substances (principally, thiols) in determining the net cellular radiosensitivity. Several theories of the mechanism(s) of action of radioprotective drugs are described in Section III. These mechanisms include the production of hypoxia, detoxication of radiochemical reactive species, stabilization of the radiobiological target and the enhancement of damage repair processes. Section IV describes the current strategies for the treatment of radiation injury. Likely areas in which fruitful research might be performed are described in Section V. 495 references.« less

Blood modulates the kinetics of reactive oxygen release in pancreatic ischemia-reperfusion injury.

PubMed

Neeff, Hannes P; Sommer, Olaf; Meyer, Sebastian; Tinelli, Anja; Scholtes, Moritz; Hopt, Ulrich T; Drognitz, Oliver; von Dobschuetz, Ernst

2012-10-01

Reason for the unsuccessful use of antioxidants in transplantation might be the unknown kinetics of reactive oxygen species (ROS) release. In this study, we compared the kinetics of ROS release from rat pancreata in the presence and absence of blood. In vivo, ischemia-reperfusion injury (IRI) was induced in pancreata of male Wistar rats by occlusion of the arterial blood supply for 1 or 2 hours. In vitro, isolated pancreata were single-pass perfused with Krebs-Henseleit bicarbonate solution. Reactive oxygen species were quantified by electron spin resonance spectroscopy using CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) as spin label. Thiols (glutathione), nicotinamide adenine dinucleotide phosphate-oxidase activity, myeloperoxidase activity, and adenosine triphosphate content were measured. During reperfusion, an increase in IRI-induced ROS in arterial blood was noted after 2 hours of warm ischemia. In sharp contrast, ROS release was immediate and short lived in blood-free perfused organs. The degree of tissue damage correlated with nicotinamide adenine dinucleotide phosphate-oxidase activity and adenosine triphosphate content. Antioxidative capacity of tissues was reduced. Electron spin resonance spectroscopy in conjunction with spin labels allows for the detection of ROS kinetics in pancreatic IRI. Reactive oxygen species kinetics are dependent on the length of the ischemic period and the presence or absence of blood.

Formation of 4-ethoxy-4'-nitrosodiphenylamine in the reaction of the phenacetin metabolite 4-nitrosophenetol with glutathione.

PubMed

Klehr, H; Eyer, P; Schäfer, W

1987-08-01

Phenacetin, a constituent of several analgesic and antipyretic formulations has been made responsible for a variety of toxic and carcinogenic actions. 4-Nitrosophenetol, the N-oxydation product of intermediate 4-phenetidine, forms methemoglobin and binds covalently to sulfhydryl groups of proteins and glutathione. In the reaction of 4-nitrosophenetol with glutathione and other thiols an intermediate so-called "semimercaptal" is formed from which N-(thiol-S-yl)-4-phenetidine S-oxide, N-(thiol-S-yl)-4-phenetidine and 4-phenetidine derive. Besides thiol adducts, a yellow compound is formed which was isolated as a pure crystalline product (elemental analysis) and identified by FAB-MS, EI-MS, 13C-, 1H-NMR, and UV-VIS spectroscopy as 4-ethoxy-4'-nitrosodiphenylamine. This nitrosoarene is formed by an unknown mechanism from 4-nitrosophenetol and 4-phenetidine under liberation of ethanol. In human erythrocytes this compound is easily reduced to 4-amino-4'-ethoxydiphenylamine (FAB-MS, EI-MS, 13C-NMR). During the reaction of 4-nitrosophenetol with red cells only traces of 4-ethoxy-4'-nitrosodiphenylamine were formed, whereas up to 10% appeared as the reduction product 4-amino-4'-ethoxydiphenylamine. This latter compound is unstable in red cells and is metabolized further to unidentified products.

Syntheses, spectroscopic characterization, thermal study, molecular modeling, and biological evaluation of novel Schiff's base benzil bis(5-amino-1,3,4-thiadiazole-2-thiol) with Ni(II), and Cu(II) metal complexes

NASA Astrophysics Data System (ADS)

Chandra, Sulekh; Gautam, Seema; Rajor, Hament Kumar; Bhatia, Rohit

2015-02-01

Novel Schiff's base ligand, benzil bis(5-amino-1,3,4-thiadiazole-2-thiol) was synthesized by the condensation of benzil and 5-amino-1,3,4-thiadiazole-2-thiol in 1:2 ratio. The structure of ligand was determined on the basis of elemental analyses, IR, 1H NMR, mass, and molecular modeling studies. Synthesized ligand behaved as tetradentate and coordinated to metal ion through sulfur atoms of thiol ring and nitrogen atoms of imine group. Ni(II), and Cu(II) complexes were synthesized with this nitrogen-sulfur donor (N2S2) ligand. Metal complexes were characterized by elemental analyses, molar conductance, magnetic susceptibility measurements, IR, electronic spectra, EPR, thermal, and molecular modeling studies. All the complexes showed molar conductance corresponding to non-electrolytic nature, expect [Ni(L)](NO3)2 complex, which was 1:2 electrolyte in nature. [Cu(L)(SO4)] complex may possessed square pyramidal geometry, [Ni(L)](NO3)2 complex tetrahedral and rest of the complexes six coordinated octahedral/tetragonal geometry. Newly synthesized ligand and its metal complexes were examined against the opportunistic pathogens. Results suggested that metal complexes were more biological sensitive than free ligand.

Facile synthesis of thiol-polyethylene glycol functionalized magnetic titania nanomaterials for highly efficient enrichment of N-linked glycopeptides.

PubMed

Wang, Jiawen; Yao, Jizong; Sun, Nianrong; Deng, Chunhui

2017-08-25

As protein N-glycosylation involved in generation and development of various cancers and diseases, it is vital to capture glycopeptides from complex biological samples for biomarker discovery. In this work, by taking advantages of the interaction between titania and thiol groups, thiol-polyethylene glycol functionalized magnetic titania nanomaterials (denoted as Fe 3 O 4 @TiO 2 @PEG) were firstly fabricated as an excellent hydrophilic adsorbent of N-linked glycopeptides. On one hand, the special interaction of titanium-thiol makes the synthetic manipulation simple and provides a new idea for design and synthesis of novel nanomaterials; on the other hand, strong magnetic response could realize rapid separation and the outstanding hydrophilicity of polyethylene glycol makes Fe 3 O 4 @TiO 2 @PEG nanomaterials show superior performance for glycopeptides enrichment with ultralow limit of detection (0.1mol/μL) and high selectivity (1:100). As a result, 24 and 33 glycopeptides enriched from HRP and IgG digests were identified respectively by MALDI-TOF MS, and 300 glycopeptides corresponding to 106 glycoproteins were recognized from merely 2μL human serum, indicating a great potential of Fe 3 O 4 @TiO 2 @PEG nanomaterials for glycoproteomic research. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of thiolated polymers to textural and mucoadhesive properties of vaginal gel formulations prepared with polycarbophil and chitosan.

PubMed

Cevher, Erdal; Sensoy, Demet; Taha, Mohamed A M; Araman, Ahmet

2008-01-01

The aim of this study was to design and evaluate of mucoadhesive gel formulations for the vaginal application of clomiphene citrate (CLM) for local treatment of human papilloma virus (HPV) infections. Chitosan (CHI) and polycarbophil (PC) were covalently modified using the thioglycolic acid and L-cysteine, respectively. The formation of thiol conjugates of chitosan (CHI-TG) and polycarbophil (PC-CYS) were confirmed by FT-IR analysis and PC-CYS and CHI-TG were found to have 148.42 +/- 4.16 and 41.17 +/- 2.34 micromol of thiol groups per gram of polymer, respectively. One percent CLM gels were prepared by combination of various concentrations of PC and CHI with thiolated conjugates of these polymers. Hardness, compressibility, elasticity, adhesiveness and cohesiveness of the gels were measured by Texture profile analysis and the vaginal mucoadhesion was investigated by mucoadhesion test. The increasing in the amount of the thiol conjugates was found to enhance the elasticity, cohesiveness, adhesiveness and mucoadhesion of the gel formulations but not their hardness and compressibility when compared to gels prepared using their respective parent formulations. Slower release rate of CLM from gels was achieved when the polymer concentrations were increased in the gel formulations. PC and its thiol conjugate were found to prolong the release of CLM longer than 70 h unlike gel formulations prepared using CHI and its thiol conjugate which were able to release CLM up to 12 h. Stability of CLM was preserved during the 3 month stability analysis under controlled room temperature and accelerated conditions.

Albumin Antioxidant Response to Stress in Diabetic Nephropathy Progression

PubMed Central

Medina-Navarro, Rafael; Corona-Candelas, Itzia; Barajas-González, Saúl; Díaz-Flores, Margarita; Durán-Reyes, Genoveva

2014-01-01

Background A new component of the protein antioxidant capacity, designated Response Surplus (RS), was recently described. A major feature of this component is the close relationship between protein antioxidant capacity and molecular structure. Oxidative stress is associated with renal dysfunction in patients with renal failure, and plasma albumin is the target of massive oxidation in nephrotic syndrome and diabetic nephropathy. The aim of the present study was to explore the albumin redox state and the RS component of human albumin isolated from diabetic patients with progressive renal damage. Methods/Principal Findings Serum aliquots were collected and albumin isolated from 125 diabetic patients divided into 5 groups according to their estimated glomerular filtration rate (GFR). In addition to clinical and biochemical variables, the albumin redox state, including antioxidant capacity, thiol group content, and RS component, were evaluated. The albumin antioxidant capacity and thiol group content were reciprocally related to the RS component in association with GFR reduction. The GFR decline and RS component were significantly negatively correlated (R = –0.83, p<0.0001). Age, creatinine, thiol groups, and antioxidant capacity were also significantly related to the GFR decline (R = –0.47, p<0.001; R = –0.68, p<0.0001; R = 0.44, p<0.001; and R = 0.72, p<0.0001). Conclusion/Significance The response of human albumin to stress in relation to the progression of diabetic renal disease was evaluated. The findings confirm that the albumin molecular structure is closely related to its redox state, and is a key factor in the progression of diabetes nephropathy. PMID:25187963

Sulfur Hydrogen Bonding in Isolated Monohydrates: Furfuryl Mercaptan versus Furfuryl Alcohol.

PubMed

Juanes, Marcos; Lesarri, Alberto; Pinacho, Ruth; Charro, Elena; Rubio, José E; Enríquez, Lourdes; Jaraíz, Martín

2018-05-02

The hydrogen bonds involving sulfur in the furfuryl mercaptan monohydrate are compared with the interactions originating from the hydroxyl group in furfuryl alcohol. The dimers with water were created in a supersonic jet expansion and characterized using microwave spectroscopy and supporting molecular orbital calculations. In furfuryl alcohol-water, a single isomer is observed, in which the water molecule forms an insertion complex with two simultaneous hydrogen bonds to the alcohol (O-H⋅⋅⋅O w ) and the ring oxygen (O w -H⋅⋅⋅O r ). When the alcohol is replaced by a thiol group in furfuryl mercaptan-water, two isomers are observed, with the thiol group preferentially behaving as proton donor to water. The first isomer is topologically equivalent to the alcohol analog but the stronger hydrogen bond is now established by water and the ring oxygen, assisted by a thiol S-H⋅⋅⋅O w hydrogen bond. In the second isomer the sulfur group accepts a proton from water, forming a O w -H⋅⋅⋅S hydrogen bond. Binding energies for the mercaptan-water dimer are predicted around 12 kJ mol -1 weaker than in the alcohol hydrate (B3LYP-D3(BJ)). The non-covalent interactions in the furfuryl dimers are dominantly electrostatic according to a SAPT(0) energy decomposition, but with increasing dispersion components in the mercaptan dimers, which are larger for the isomer with the weaker O w -H⋅⋅⋅S interaction. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A rapid and fluorogenic TMP-AcBOPDIPY probe for covalent labeling of proteins in live cells.

PubMed

Liu, Wei; Li, Fu; Chen, Xi; Hou, Jian; Yi, Long; Wu, Yao-Wen

2014-03-26

Protein labeling is enormously useful for characterizing protein function in cells and organisms. Chemical tagging methods have emerged as a new generation protein labeling strategy in live cells. Here we have developed a novel and versatile TMP-AcBOPDIPY probe for selective and turn-on labeling of proteins in live cells. A small monomeric tag, E. coli dihydrofolate reductase (eDHFR), was rationally designed to introduce a cysteine in the vicinity of the ligand binding site. Trimethoprim (TMP) that specifically binds to eDHFR was linked to the BOPDIPY fluorophore containing a mildly thiol-reactive acrylamide group. TMP-AcBOPDIPY rapidly labeled engineered eDHFR tags via a reaction termed affinity conjugation (a half-life of ca. 2 min), which is one of the top fast chemical probes for protein labeling. The probe displays 2-fold fluorescence enhancement upon labeling of proteins. We showed that the probe specifically labeled intracellular proteins in live cells without and with washing out the dye. We demonstrated its utility in visualizing intracellular processes by fluorescence-lifetime imaging microscopy (FLIM) measurements.

Plasma Protein Oxidation and Its Correlation with Antioxidant Potential During Human Aging

PubMed Central

Pandey, Kanti Bhooshan; Mehdi, Mohd Murtaza; Maurya, Pawan Kumar; Rizvi, Syed Ibrahim

2010-01-01

Previous studies have indicated that the main molecular characteristic of aging is the progressive accumulation of oxidative damages in cellular macromolecules. Proteins are one of the main molecular targets of age-related oxidative stress, which have been observed during aging process in cellular systems. Reactive oxygen species (ROS) can lead to oxidation of amino acid side chains, formation of protein-protein cross-linkages, and oxidation of the peptide backbones. In the present study, we report the age-dependent oxidative alterations in biomarkers of plasma protein oxidation: protein carbonyls (PCO), advanced oxidation protein products (AOPPs) and plasma total thiol groups (T-SH) in the Indian population and also correlate these parameters with total plasma antioxidant potential. We show an age dependent decrease in T-SH levels and increase in PCO and AOPPs level. The alterations in the levels of these parameters correlated significantly with the total antioxidant capacity of the plasma. The levels of oxidized proteins in plasma provide an excellent biomarker of oxidative stress due to the relative long half-life of such oxidized proteins. PMID:20826915

Targeting cysteine residues of human immunodeficiency virus type 1 protease by reactive free radical species.

PubMed

Basu, A; Sehajpal, P K; Ogiste, J S; Lander, H M

1999-01-01

Nitric oxide (NO) is a naturally occurring free radical with many functions. The oxidized form of NO, the nitrosonium ion, reacts with the thiol group of cysteine residues resulting in their modification to S-nitrosothiols. The human immunodeficiency virus type 1 (HIV-1) protease (HIV-PR) has two cysteine residues that are conserved amongst different viral isolates found in patients with acquired immunodeficiency syndrome (AIDS). In an active dimer, these residues are located near the surface of the protease. We have found that treatment of HIV-PR with different NO congeners results in loss of its proteolytic activity and simultaneous formation of S-nitrosothiols. Sodium nitroprusside inhibited HIV-PR up to 70% and S-nitroso-N-acetylpenicillamine completely inhibited the protease within 5 min of treatment. The pattern of inhibition by NO donors is comparable to its inhibition by N-acetyl pepstatin. Using electrospray ionization-mass spectrometry, we identified the modification of HIV-PR by NO as that of S-nitrosation. Our findings point towards a possible role of NO in mediating resistance to HIV-1 infection.

Structural Basis for the Acyltransferase Activity of Lecithin:Retinol Acyltransferase-like Proteins*

PubMed Central

Golczak, Marcin; Kiser, Philip D.; Sears, Avery E.; Lodowski, David T.; Blaner, William S.; Palczewski, Krzysztof

2012-01-01

Lecithin:retinol acyltransferase-like proteins, also referred to as HRAS-like tumor suppressors, comprise a vertebrate subfamily of papain-like or NlpC/P60 thiol proteases that function as phospholipid-metabolizing enzymes. HRAS-like tumor suppressor 3, a representative member of this group, plays a key role in regulating triglyceride accumulation and energy expenditure in adipocytes and therefore constitutes a novel pharmacological target for treatment of metabolic disorders causing obesity. Here, we delineate a catalytic mechanism common to lecithin:retinol acyltransferase-like proteins and provide evidence for their alternative robust lipid-dependent acyltransferase enzymatic activity. We also determined high resolution crystal structures of HRAS-like tumor suppressor 2 and 3 to gain insight into their active site architecture. Based on this structural analysis, two conformational states of the catalytic Cys-113 were identified that differ in reactivity and thus could define the catalytic properties of these two proteins. Finally, these structures provide a model for the topology of these enzymes and allow identification of the protein-lipid bilayer interface. This study contributes to the enzymatic and structural understanding of HRAS-like tumor suppressor enzymes. PMID:22605381

2,4-Dichlorophenoxyacetic acid effects on nephrotoxicity in rats during late pregnancy and early postnatal periods.

PubMed

Troudi, Afef; Soudani, Nejla; Mahjoubi Samet, Amira; Ben Amara, Ibtissem; Zeghal, Najiba

2011-11-01

2,4-Dichlorophenoxyacetic acid (2,4-D) is largely used as a selective herbicide in Tunisia. The purpose of this study was to investigate the effects of 2,4-D on the kidneys of adult rats and their suckling pups. Female Wistar rats were divided into two groups: the controls and the treated rats that received 600 mg/L of 2,4-D in their drinking water from the 14th day of pregnancy until day 14 after delivery. Exposure to 2,4-D induced nephrotoxicity as evidenced by an increase in thiobarbituric acid reactive substances and protein carbonyl levels and a decrease in antioxidant enzyme activities such as catalase, superoxide dismutase, glutathione peroxidase in the kidneys of suckling pups and their mothers. In addition, a significant decline in kidney glutathione, non-protein thiol and vitamin C levels was also observed. Histological changes, seen in the kidney of mothers and their pups treated with 2,4-D are characterized by a narrowed Bowman's space, tubular epithelial cells degeneration, widened tubular lumen and vascular congestion. Copyright © 2011 Elsevier Inc. All rights reserved.

Organometallic Palladium Reagents for Cysteine Bioconjugation

PubMed Central

Vinogradova, Ekaterina V.; Zhang, Chi; Spokoyny, Alexander M.; Pentelute, Bradley L.; Buchwald, Stephen L.

2015-01-01

Transition-metal based reactions have found wide use in organic synthesis and are used frequently to functionalize small molecules.1,2 However, there are very few reports of using transition-metal based reactions to modify complex biomolecules3,4, which is due to the need for stringent reaction conditions (for example, aqueous media, low temperature, and mild pH) and the existence of multiple, reactive functional groups found in biopolymers. Here we report that palladium(II) complexes can be used for efficient and highly selective cysteine conjugation reactions. The bioconjugation reaction is rapid and robust under a range of biocompatible reaction conditions. The straightforward synthesis of the palladium reagents from diverse and easily accessible aryl halide and trifluoromethanesulfonate precursors makes the method highly practical, providing access to a large structural space for protein modification. The resulting aryl bioconjugates are stable towards acids, bases, oxidants, and external thiol nucleophiles. The broad utility of the new bioconjugation platform was further corroborated by the synthesis of new classes of stapled peptides and antibody-drug conjugates. These palladium complexes show potential as a new set of benchtop reagents for diverse bioconjugation applications. PMID:26511579

Enhanced Adsorption of Trivalent Arsenic from Water by Functionalized Diatom Silica Shells

PubMed Central

Zhang, Zhijian; Xu, Liping; Zhang, Chunlong

2015-01-01

The potential of porous diatom silica shells as a naturally abundant low-cost sorbent for the removal of arsenic in aqueous solutions was investigated in a batch study. The objective of this work was to chemically modify the silica shells of a diatom Melosira sp. with bifunctional (thiol and amino) groups to effectively remove arsenic in its toxic As(III) form (arsenite) predominant in the aquatic environment. Sorption experiments with this novel sorbent were conducted under varying conditions of pH, time, dosage, and As(III) concentration. A maximum adsorption capacity of 10.99 mg g-1 was achieved within 26 h for a solution containing 12 mg L-1 As(III) at pH 4 and sorbent dosage of 2 g L-1. The functionalized diatom silica shells had a surface morphological change which was accompanied by increased pore size at the expense of reduced specific surface area and total pore volume. As(III) adsorption was best fitted with the Langmuir-Freundlich model, and the adsorption kinetic data using pore surface diffusion model showed that both the external (film) and internal (intraparticle) diffusion can be rate-determining for As(III) adsorption. Fourier transform infrared spectroscopy (FTIR) indicated that the thiol and amino groups potentially responsible for As(III) adsorption were grafted on the surface of diatom silica shells. X-ray photoelectron spectroscopy (XPS) further verified that this unique sorbent proceeded via a chemisorption mechanism through the exchange between oxygen-containing groups of neutral As(III) and thiol groups, and through the surface complexation between As(III) and protonated nitrogen and hydroxyl groups. Results indicate that this functionalized bioadsorbent with a high As(III) adsorption capacity holds promise for the treatment of As(III) containing wastewater. PMID:25837498

Evidence for function of the ferredoxin/thioredoxin system in the reductive activation of target enzymes of isolated intact chloroplasts.

PubMed

Crawford, N A; Droux, M; Kosower, N S; Buchanan, B B

1989-05-15

Results obtained with isolated intact chloroplasts maintained aerobically under light and dark conditions confirm earlier findings with reconstituted enzyme assays and indicate that the ferredoxin/thioredoxin system functions as a light-mediated regulatory thiol chain. The results were obtained by application of a newly devised procedure in which a membrane-permeable thiol labeling reagent, monobromobimane (mBBr), reacts with sulfhydryl groups and renders the derivatized protein fluorescent. The mBBr-labeled protein in question is isolated individually from chloroplasts by immunoprecipitation and its thiol redox status is determined quantitatively by combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence measurements. The findings indicate that each member of the ferredoxin/thioredoxin system containing a catalytically active thiol group is reduced in isolated intact chloroplasts after a 2-min illumination. The extents of reduction were FTR, 38%; thioredoxin m, 75% (11-kDa form) and 87% (13-kDa form); thioredoxin f, 95%. Reduction of each of these components was negligible both in the dark and when chloroplasts were transferred from light to dark conditions. The target enzyme, NADP-malate dehydrogenase, also underwent net reduction in illuminated intact chloroplasts. Fructose-1,6-bisphosphatase showed increased mBBr labeling under these conditions, but due to interfering gamma globulin proteins it was not possible to determine whether this was a result of net reduction as is known to take place in reconstituted assays. Related experiments demonstrated that mBBr, as well as N-ethylmaleimide, stabilized photoactivated NADP-malate dehydrogenase and fructose-1,6-bisphosphatase so that they remained active in the dark. By contrast, phosphoribulokinase, another thioredoxin-linked enzyme, was immediately deactivated following mBBr addition. These latter results provide new information on the relation between the regulatory and active sites of these enzymes.

Real-time Kinetics of High-mobility Group Box 1 (HMGB1) Oxidation in Extracellular Fluids Studied by in Situ Protein NMR Spectroscopy*

PubMed Central

Zandarashvili, Levani; Sahu, Debashish; Lee, Kwanbok; Lee, Yong Sun; Singh, Pomila; Rajarathnam, Krishna; Iwahara, Junji

2013-01-01

Some extracellular proteins are initially secreted in reduced forms via a non-canonical pathway bypassing the endoplasmic reticulum and become oxidized in the extracellular space. One such protein is HMGB1 (high-mobility group box 1). Extracellular HMGB1 has different redox states that play distinct roles in inflammation. Using a unique NMR-based approach, we have investigated the kinetics of HMGB1 oxidation and the half-lives of all-thiol and disulfide HMGB1 species in serum, saliva, and cell culture medium. In this approach, salt-free lyophilized 15N-labeled all-thiol HMGB1 was dissolved in actual extracellular fluids, and the oxidation and clearance kinetics were monitored in situ by recording a series of heteronuclear 1H-15N correlation spectra. We found that the half-life depends significantly on the extracellular environment. For example, the half-life of all-thiol HMGB1 ranged from ∼17 min (in human serum and saliva) to 3 h (in prostate cancer cell culture medium). Furthermore, the binding of ligands (glycyrrhizin and heparin) to HMGB1 significantly modulated the oxidation kinetics. Thus, the balance between the roles of all-thiol and disulfide HMGB1 proteins depends significantly on the extracellular environment and can also be artificially modulated by ligands. This is important because extracellular HMGB1 has been suggested as a therapeutic target for inflammatory diseases and cancer. Our work demonstrates that the in situ protein NMR approach is powerful for investigating the behavior of proteins in actual extracellular fluids containing an enormous number of different molecules. PMID:23447529

Subchronic effects of methylmercury on plasma and organ biochemistries in great egret nestlings

USGS Publications Warehouse

Hoffman, D.J.; Spalding, M.G.; Frederick, P.C.

2005-01-01

In recent years, high concentrations of mercury have been found in wading birds in Florida, USA. Great egret (Ardea alba) chicks (2 weeks old) were dosed orally daily with the equivalent of 0, 0.5, or 5 ug/g Hg as methylmercury chloride in the diet for up to 12 weeks. Weakness of the legs or paralysis occurred in all high-dosed birds. Geometric mean blood Hg concentrations were 0.17, 10.3, and 78.5 ug/g (wet wt), respectively. Mercury concentrations for organs (ug/g wet wt), including brain (0.22, 3.4, and 35, respectively), liver (0.34, 15.1, 138, respectively), and kidney (0.28, 8.1, and 120, respectively), increased in a dose-dependent manner. Total glutathione (GSH) peroxidase activity was significantly lower in the plasma, brain, liver, and kidney of the high-dosed group. Plasma aspartate aminotransferase activity increased with mercury treatment, whereas lactate dehydrogenase activity decreased. Four other plasma chemistries were decreased significantly in the high-dosed group and included uric acid, total protein, albumin, and inorganic phosphorus. Lipid peroxidation increased in liver (low and high dose) and brain (high dose). Tissue changes in concentrations of reduced thiols included decreased total thiols and protein-bound thiols in liver, decreased protein-bound thiols in kidney, and increased GSH in kidney and brain. Activities of GSH S-transferase and oxidized glutathione reductase increased in liver. In kidney, GSH S-transferase and glucose-6-phosphate dehydrogenase activities increased with mercury dose. These findings, including apparent compensatory changes, are compared to other Hg studies where oxidative stress was reported in egrets, herons, and diving ducks in the field and mallards in the laboratory.

Chemical Modification of Cellulose Esters for Oral Drug Delivery

NASA Astrophysics Data System (ADS)

Meng, Xiangtao

Polymer functional groups have critical impacts upon physical, chemical and mechanical properties, and thus affect the specific applications of the polymer. Functionalization of cellulose esters and ethers has been under extensive investigation for applications including drug delivery, cosmetics, food ingredients, and automobile coating. In oral delivery of poorly water-soluble drugs, amorphous solid dispersion (ASD) formulations have been used, prepared by forming miscible blends of polymers and drugs to inhibit crystallization and enhance bioavailability of the drug. The Edgar and Taylor groups have revealed that some cellulose o-carboxyalkanoates were highly effective as ASD polymers, with the pendant carboxylic acid groups providing both specific polymer-drug interactions and pHtriggered release through swelling of the ionized polymer matrix. While a variety of functional groups such as hydroxyl and amide groups are also of interest, cellulose functionalization has relied heavily on classical methods such as esterification and etherification for appending functional groups. These methods, although they have been very useful, are limited in two respects. First, they typically employ harsh reaction conditions. Secondly, each synthetic pathway is only applicable for one or a narrow group of functionalities due to restrictions imposed by the required reaction conditions. To this end, there is a great impetus to identify novel reactions in cellulose modification that are mild, efficient and ideally modular. In the initial effort to design and synthesize cellulose esters for oral drug delivery, we developed several new methods in cellulose functionalization, which can overcome drawbacks of conventional synthetic pathways, provide novel cellulose derivatives that are otherwise inaccessible, and present a platform for structure-property relationship study. Cellulose o-hydroxyalkanoates were previously difficult to access as the hydroxyl groups, if not protected, react with carboxylic acid/carbonyl during a typical esterification reaction or ring opening of lactones, producing cellulose-g-polyester and homopolyester. We demonstrated the viability of chemoselective olefin hydroboration-oxidation in the synthesis of cellulose o-hydroxyesters in the presence of ester groups. Cellulose esters with terminally olefinic side chains were transformed to the target products by two-step, one-pot hydroborationoxidation reactions, using 9-borabicyclo[3.3.1]nonane (9-BBN) as hydroboration agent, followed by oxidizing the organoborane intermediate to a primary alcohol using mildly alkaline H2O2. The use of 9-BBN as hydroboration agent and sodium acetate as base catalyst in oxidation successfully avoided cleavage of ester linkages by borane reduction and base catalyzed hydrolysis. With the impetus of modular and efficient synthesis, we introduced olefin crossmetathesis (CM) in polysaccharide functionalization. Using Grubbs type catalyst, cellulose esters with terminally olefinic side chains were reacted with various CM partners including acrylic acid, acrylates and acrylamides to afford families of functionalized cellulose esters. Molar excesses of CM partners were used in order to suppress potential crosslinking caused by self-metathesis between terminally olefinic side chains. Amide CM partners can chelate with the ruthenium catalyst and cause low conversions in conventional solvents such as THF. While the inherent reactivity toward CM and tendency of acrylamides to chelate Ru is influenced by the acrylamide N-substituents, employing acetic acid as a solvent significantly improved the conversion of certain acrylamides. We observed that the CM products are prone to crosslinking during storage, and found that the crosslinking is likely caused by free radical abstraction of gamma-hydrogen of the alpha,beta-unsaturation and subsequent recombination. We further demonstrated successful hydrogenation of these alpha,beta-unsaturated acids, esters, and amides, thereby eliminating the potential for radical-induced crosslinking during storage. The alpha,beta-unsaturation on CM products can cause crosslinking due to gamma-H abstraction and recombination if not reduced immediately after reaction. Instead of eliminating the double bond by hydrogenation, we described a method to make use of these reactive conjugated olefins by post-CM thiol-Michael addition. Under amine catalysis, different CM products and thiols were combined and reacted. Using proper thiols and catalyst, complete conversion can be achieved under mild reaction conditions. The combination of the two modular reactions creates versatile access to multi-functionalized cellulose derivatives. Compared with conventional reactions, these reactions enable click or click-like conjugation of functional groups onto cellulose backbone. The modular profile of the reactions enables clean and informative structure-property relationship studies for ASD. These approaches also provide opportunities for the synthesis of chemically and architecturally diverse cellulosic polymers that are otherwise difficult to access, opening doors for many other applications such as antimicrobial, antifouling, in vivo drug delivery, and bioconjugation. We believe that the cellulose functionalization approaches we pioneered can be expanded to the modification of other polysaccharides and polymers, and that these reactions will become useful tools in the toolbox of polymer/polysaccharide chemists.

Alleviative effects of litchi (Litchi chinensis Sonn.) flower on lipid peroxidation and protein degradation in emulsified pork meatballs.

PubMed

Ding, Yi; Wang, Sheng-Yao; Yang, Deng-Jye; Chang, Ming-Hsu; Chen, Yi-Chen

2015-09-01

To avoid or retard the lipid peroxidation of meat products, antioxidants are commonly added. Considering the safety and health of additives in meat products, consumers prefer natural antioxidants rather than synthetic ones. Gentisic acid and epicatechin were identified as the major phenolic acid and flavonoid, respectively, of litchi flowers (LFs). The physicochemical properties of pork meatballs with or without dried LF powders (0.5%, 1.0%, and 1.5%, w/w) and tert-butylhydroquinone (TBHQ; 0.01%, w/w) were analyzed during a 4-week frozen storage period. LF and TBHQ decreased (p < 0.05) thiobarbituric acid reactive substance (TBARS) values but increased (p < 0.05) thiol group contents in meatballs. LF added to meatballs improved (p < 0.05) texture and water-holding capacity (centrifugation/purge losses) more than in the control group upon the storage. Although LF powders made meatballs redder and darker (p < 0.05) than the control and TBHQ groups, they did not affect the preference of panelists. The addition of 0.5% LF powders exhibited the best (p < 0.05) overall sensory panel acceptance. LFs may be an effective natural antioxidant to reduce lipid and protein oxidation for frozen cooked meat products. Copyright © 2015. Published by Elsevier B.V.

Ascorbyl stearate and ionizing radiation potentiate apoptosis through intracellular thiols and oxidative stress in murine T lymphoma cells.

PubMed

Mane, Shirish D; Kamatham, Akhilender Naidu

2018-02-01

Ascorbyl stearate (Asc-s) is a derivative of ascorbic acid with better anti-tumour efficacy compared to its parent compound ascorbic acid. In this study, we have examined radio-sensitizing effect of Asc-s in murine T cell lymphoma (EL4) cells at 4 Gy. Asc-s and radiation treatment reduced cell proliferation, induced apoptosis in a dose dependent manner by arresting the cells at S/G2-M phase of cell cycle. It also decreased the frequency of cancer stem cells per se, with significantly higher decrease in combination with radiation treatment./Further, Asc-s and radiation treatment increased the level of reactive oxygen species (ROS), drop in mitochondrial membrane potential (MMP) and increased caspase-3 activity resulting in apoptosis of EL4 cells. Further it also significantly decreased GSH/GSSG ratio due to binding of Asc-s with thiols. The increase in oxidative stress induced by Asc-s and radiation treatment was abrogated by thiol antioxidants in EL4 cells. Interestingly, this redox modulation triggered significant increase in protein glutathionylation in a time dependent manner. Asc-s treatment resulted in glutathionylation of IKK, p50-NF-kB and mutated p53, thereby inhibiting cancer progression during oxidative stress. Asc-s quenches GSH ensuing Asc-s + GSH adduct thereby further modulating GSH/GSSG ratio as evident from HPLC and docking studies. The anti-tumour effect of Asc-s along with radiation was studied by injecting EL4 cells in synegenicC57/BL6 male mice. Intraperitoneal injection of Asc-s followed by radiation exposure at 4 Gy to the tumour bearing mice resulted in radio-sensitization which is evident from significant regression of tumour as evident from tumour burden index. The survival study supports the data that Asc-s pre-treatment enhances radio-sensitization in murine lymphoma. Our data, suggest that Asc-s and ionizing radiation induced cell cycle arrest and apoptosis by perturbing redox balance through irreversible complexes of thiols with Asc-s, disturbed mitochondrial membrane permeability and activation of caspase-3 in EL4 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Synthesis of thiol-containing analogues of puromycin and a study of their interaction with N-acetylphenylalanyl-transfer ribonucleic acid on ribosomes to form thioesters

PubMed Central

Gooch, John; Hawtrey, Arthur O.

1975-01-01

1. The thiol-containing analogue of puromycin, 6-dimethylamino-9-{1′-[3′-(2″-mercapto-3″-phenylpropionamido)-3′-deoxy-β-d-ribofuranosyl]}purine (XVII) in which the primary amino group of the antibiotic is replaced with a thiol grouping, was synthesized chemically (compound XVII is abbreviated to thiopuromycin). 2. Thiopuromycin (XVII) was found to be active in releasing N-[3H]acetylphenylalanine from its tRNA carrier as the thioester, N-acetylphenylalanylthiopuromycin (XIX) in the Escherichia coli ribosomal system. The reaction product (XIX) was synthesized chemically from thiopuromycin and N-acetylphenylalanine and found to be stable to hydrolysis in the standard incubation medium at pH7.6. dl-Phenyl-lactylpuromycin (XXI), the hydroxy analogue of puromycin, was also synthesized chemically and shown to release N-acetylphenylalanine from its tRNA carrier in the E. coli ribosomal system, thus confirming the previous results of Fahnestock et al. [Biochemistry (1970) 9, 2477–2483]. 3. In marked contrast with the results obtained in the E. coli system, both thiopuromycin (XVII) and hydroxypuromycin (XXI) were found to be inactive in releasing N-acetylphenylalanine from its tRNA carrier in the rat liver ribosomal system. PMID:1103886

Incessant formation of chain-like mesoporous silica with a superior binding capacity for mercury.

PubMed

Ravi, S; Selvaraj, M

2014-04-14

A novel incessant formation of chain like mesoporous silica (ICMS) has been easily materialized using a mixed surfactant (Pluronic P123 and FC-4) as a structuring reagent in conjunction with a thiol precursor (3-MPS) through a one-pot synthetic method. A particular thiol concentration facilitated the interaction of the micelle head groups to form long-chain micelles, where FC-4 enhanced further growth. The rapid interactions of the micelles and the condensation of silicic acid and its oligomeric derivatives by coordinating 3-MPS through hydrogen bonding interactions leads to form ICMS. The characterization results for the ICMS illustrated that it has an ordered hexagonal pore geometry. The capability of the ICMS for Hg(2+) adsorption was extensively studied under different optimal parameters and the adsorption isothermal values clearly fit with the Langmuir and Freundlich isothermal plots. This novel material exhibited an unprecedentedly high binding affinity toward even microgram levels of mercury ions in wastewater, compared to other thiol-based mesoporous silica.

Synthesis, characterization and evaluation of thiolated tamarind seed polysaccharide as a mucoadhesive polymer.

PubMed

Kaur, Harmanmeet; Yadav, Shikha; Ahuja, Munish; Dilbaghi, Neeraj

2012-11-06

In the present study, thiol-functionalization of tamarind seed polysaccharide was carried out by esterification with thioglycolic acid. Thiol-functionalization was confirmed by SH stretch in Fourier-transformed infra-red spectra at 2586 cm(-1). It was found to possess 104.5 mM of thiol groups per gram. The results of differential scanning calorimetry and X-ray diffraction study indicate increase in crystallinity. Polymer compacts of thiolated tamarind seed polysaccharide required 6.85-fold greater force to detach from the mucin coated membrane than that of tamarind seed polysaccharide. Comparative evaluation of Carbopol-based metronidazole gels containing thiolated tamarind seed polysaccharide with gels containing tamarind seed polysaccharide for mucoadhesive strength using chicken ileum by modified balance method revealed higher mucoadhesion of gels containing thiolated tamarind seed polysaccharide. Further, the gels containing tamarind seed polysaccharide and thiolated tamarind seed polysaccharide released the drug by Fickian-diffusion following the first-order and Higuchi's-square root release kinetics, respectively. Copyright © 2012 Elsevier Ltd. All rights reserved.

Photodynamic therapy potential of thiol-stabilized CdTe quantum dot-group 3A phthalocyanine conjugates (QD-Pc).

PubMed

Tekdaş, Duygu Aydın; Durmuş, Mahmut; Yanık, Hülya; Ahsen, Vefa

2012-07-01

Thiol stabilized CdTe quantum dot (QD) nanoparticles were synthesized in aqueous phase and were used as energy donors to tetra-triethyleneoxythia substituted aluminum, gallium and indium phthalocyanines through fluorescence resonance energy transfer (FRET). Energy transfer occurred from the QDs to phthalocyanines upon photoexcitation of the QDs. An enhancement in efficiency of energy transfer with the nature of the carboxylic thiol stabilizer on the QDs was observed. As a result of the nanoparticle and the phthalocyanine mixing, the photoluminescence efficiency of the phthalocyanine moieties in the mixtures does not strictly follow the quantum yields of the bare phthalocyanines. The photochemistry study of phthalocyanines in the presence of the QDs revealed high singlet oxygen quantum yield, hence the possibility of using QDs in combination with phthalocyanines as photosensitizers in photodynamic therapy of cancer. The fluorescence of the CdTe quantum dots-phthalocyanine conjugates (QDs-Pc) were effectively quenched by addition of 1,4-benzoquinone. Copyright © 2012 Elsevier B.V. All rights reserved.

Full inhibition of enzymatic browning in the presence of thiol-functionalised silica nanomaterial.

PubMed

Muñoz-Pina, Sara; Ros-Lis, José V; Argüelles, Ángel; Coll, Carmen; Martínez-Máñez, Ramón; Andrés, Ana

2018-02-15

Darkening processed fruits and vegetables is caused mainly by enzymatic browning through polyphenol oxidase (PPO) action. Accordingly, we explored the potential of four silica-based materials (MCM-41 nanometric size, MCM-41 micrometric size, UVM-7 and aerosil), non-functionalised and functionalised with thiol groups, to inhibit PPO activity in the model system and apple juice. All materials showed relevant performance when immobilising and inhibiting PPO in model systems, and support topology is a main factor for enzyme immobilisation and inhibition. Thiol-containing silica UVM7-SH showed the greatest inactivation, and similar browning values to those obtained by acidification. The enzyme's kinetic parameters in the presence of UVM-7-SH suggested non-competitive inhibition, which indicated that the material interacted with the enzyme, but beyond the active centre. In real systems, UVM-7-SH completely inhibited enzymatic browning in apple juice (cv. Granny Smith and cv. Golden Delicious) up to 9days after 5min of contact. Copyright © 2017 Elsevier Ltd. All rights reserved.