Sample records for gamma-ifn-inducible-lysosomal thiol reductase

  1. Comparative study of the physiological roles of three peroxidases (NADH peroxidase, Alkyl hydroperoxide reductase and Thiol peroxidase) in oxidative stress response, survival inside macrophages and virulence of Enterococcus faecalis.

    La Carbona, Stephanie; Sauvageot, Nicolas; Giard, Jean-Christophe; Benachour, Abdellah; Posteraro, Brunella; Auffray, Yanick; Sanguinetti, Maurizio; Hartke, Axel


    The opportunistic pathogen Enterococcus faecalis is well equipped with peroxidatic activities. It harbours three loci encoding a NADH peroxidase, an alkyl hydroperoxide reductase and a protein (EF2932) belonging to the AhpC/TSA family. We present results demonstrating that ef2932 does encode a thiol peroxidase (Tpx) and show that it is part of the regulon of the hydrogen peroxide regulator HypR. Characterization of unmarked deletion mutants showed that all three peroxidases are important for the defence against externally provided H(2)O(2). Exposure to internal generated H(2)O(2) by aerobic growth on glycerol, lactose, galactose or ribose showed that Npr was absolutely required for aerobic growth on glycerol and optimal growth on the other substrates. Growth on glycerol was also dependent on Ahp. Addition of catalase restored growth of the mutants, and therefore, extracellular H(2)O(2) concentrations have been determined. This showed that the time point of growth arrest of the Deltanpr mutant correlated with the highest H(2)O(2) concentration measured. Analysis of the survival of the different strains inside peritoneal macrophages revealed that Tpx was the most important antioxidant activity for protecting the cells against the hostile phagocyte environment. Finally, the Deltatpx and the triple mutant showed attenuated virulence in a mouse peritonitis model.

  2. Distribution and abundance of organic thiols

    Fahey, R.


    The role of glutathione (GSH) in protecting against the toxicity of oxygen and oxygen by products is well established for all eukaryotes studied except Entamoeba histolytica which lacks mitochrondria, chloroplasts, and microtubules. The GSH is not universal among prokaryotes. Entamoeba histolytica does not produce GSH or key enzymes of GSH metabolism. A general method of thiol analysis based upon fluorescent labeling with monobromobimane and HPLC separation of the resulting thiol derivatives was developed to determine the occurrence of GSH and other low molecular weight thiols in bacteria. Glutathione is the major thiol in cyanobacteria and in most bacteria closely related to the purple photosynthetic bacteria, but GSH was not found in archaebacteria, green bacteria, or GRAM positive bacteria. It suggested that glutathione metabolism was incorporated into eukaryotes at the time that mitochondria and chloroplasts were acquired by endosymbiosis. In Gram positive aerobes, coenzyme A occurs at millimolar levels and CoA disulfide reductases are identified. The CoA, rather than glutathione, may function in the oxygen detoxification processes of these organisms.

  3. Thiol biochemistry of prokaryotes

    Fahey, Robert C.


    The present studies have shown that GSH metabolism arose in the purple bacteria and cyanobacteria where it functions to protect against oxygen toxicity. Evidence was obtained indicating that GSH metabolism was incorporated into eucaryotes via the endosymbiosis giving rise to mitochrondria and chloroplasts. Aerobic bacteria lacking GSH utilize other thiols for apparently similar functions, the thiol being coenzyme A in Gram positive bacteria and chi-glutamylcysteine in the halobacteria. The thiol biochemistry of prokaryotes is thus seen to be much more highly diversified than that of eucaryotes and much remains to be learned about this subject.

  4. Electroanalysis of Plant Thiols

    Rene Kizek


    Full Text Available Due to unique physico-chemical properties of –SH moiety thiols comprise widegroup of biologically important compounds. A review devoted to biological functions ofglutathione and phytochelatins with literature survey of methods used to analysis of thesecompounds and their interactions with cadmium(II ions and Murashige-Skoog medium ispresented. For these purposes electrochemical techniques are used. Moreover, we revealedthe effect of three different cadmium concentrations (0, 10 and 100 μM on cadmiumuptake and thiols content in maize plants during 192 hours long experiments usingdifferential pulse anodic stripping voltammetry to detect cadmium(II ions and highperformance liquid chromatography with electrochemical detection to determineglutathione. Cadmium concentration determined in tissues of the plants cultivated innutrient solution containing 10 μM Cd was very low up to 96 hours long exposition andthen the concentration of Cd markedly increased. On the contrary, the addition of 100 μMCd caused an immediate sharp increase in all maize plant parts to 96 hours Cd expositionbut subsequently the Cd concentration increased more slowly. A high performance liquidchromatography with electrochemical detection was used for glutathione determination intreated maize plants after 96 and 192 hours of treatment. The highest total content of glutathione per one plant was 6 μg (96 h, 10 μM Cd in comparison with non-treated plant (control where glutathione content was 1.5 μg. It can be concluded that electrochemical techniques have proved to be useful to analyse plant thiols.

  5. Histochemical Localization of Glutathione Dependent NBT-Reductase in Mouse Skin


    Objective Localization of the glutathione dependent Nitroblue tetrazolium (NBT) reductase in fresh frozen sections of mouse skin and possible dependence of NBT reductase on tissue thiol levels has been investigated. Methods The fresh frozen tissue sections (8m thickness) were prepared and incubated in medium containing NBT, reduced glutathione (GSH) and phosphate buffer. The staining for GSH was performed with mercury orange. Results  The activity of the NBT-reductase in mouse skin has been found to be localized in the areas rich in glutathione and actively proliferating area of the skin. Conclusion The activity of the NBT-reductase seems to be dependent on the glutathione contents.

  6. Naegleria fowleri: a free-living highly pathogenic amoeba contains trypanothione/trypanothione reductase and glutathione/glutathione reductase systems.

    Ondarza, Raúl N; Hurtado, Gerardo; Tamayo, Elsa; Iturbe, Angélica; Hernández, Eva


    This paper presents definitive data showing that the thiol-bimane compound isolated and purified by HPLC from Naegleria fowleri trophozoites unequivocally corresponds by matrix assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) [M(+)H(+)] of m/z 1104.57 and to the trypanothione-(bimane) of m/z 914.46. The trypanothione disulfide T(S)(2) was also found to have a molecular ion of m/z 723.37. Additionally HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were present in Naegleria. The ion patterns of the thiol-bimane compounds prepared from commercial trypanothione standard, Entamoeba histolytica and Crithidia luciliae are identical to the Naegleria thiol-bimane compound. Partially purified extracts from N. fowleri showed the coexistence of glutathione and trypanothione reductases activities. There is not doubt that the thiol compound trypanothione, which was previously thought to occur only in Kinetoplastida, is also present in the human pathogens E. histolytica and N. fowleri, as well as in the non-pathogenic euglenozoan E. gracilis. The presence of the trypanothione/trypanothione reductase system in N. fowleri creates the possibility of using this enzyme as a new "drug target" for rationally designed drugs to eliminate the parasite, without affecting the human host.

  7. Low-molecular-mass thiol compounds from a free-living highly pathogenic amoeba, Naegleria fowleri.

    Ondarza, Raúl N; Iturbe, Angélica; Hernández, Eva; Hurtado, Gerardo


    Acid extracts labelled with the fluorescent reagent monobromobimane and separated by HPLC have enabled the detection of low-molecular-mass thiol compounds in Naegleria fowleri for the first time. The amounts detected are expressed in nmol/1 x 10(6) trophozoites cultivated at various stages of growth in the appropriate culture medium. N. fowleri is a highly pathogenic free-living amoeba, in which we found important thiol compounds, some of them in their reduced and oxidized forms. Unlike cysteine and glutathione, a number of these are not represented in normal human lymphocytes. Some of these thiol compounds from Naegleria must have their respective disulphide reductases, although the presence of thiol-disulphide exchange reactions must be considered. Ovothiol A, with antioxidant properties, is an example of a compound that is kept reduced by trypanothione in trypanosomatids, although no disulphide reductase for ovothiol A has yet been discovered. In our case we were unable to detect this biothiol in Naegleria. The presence of thiol compounds that seem to be particular to this pathogen and which are not present in human lymphocytes opens the possibility of searching for disulphide-reducing enzymes that can serve as drug targets.


    Upadhyaya, Bijaya; Tian, Xiaoli; Wu, Hongli; Lou, Marjorie F.


    Glutaredoxin2 (Grx2) is a mitochondrial isozyme of the cytosolic glutaredoxin1 (thioltransferase or TTase). Both belong to the large oxidoreductase family and play an important role in maintaining thiol/disulfide redox homeostasis in the cells. Grx2 is recently found in the lens where its activities of disulfide reductase and peroxidase, similar to TTase, can protect the lens against oxidative stress. Since other eye tissues are also highly sensitive to oxidative stress, and TTase’s distribut...

  9. Quinone Reductase 2 Is a Catechol Quinone Reductase

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao (NYMEDCO)


    The functions of quinone reductase 2 have eluded researchers for decades even though a genetic polymorphism is associated with various neurological disorders. Employing enzymatic studies using adrenochrome as a substrate, we show that quinone reductase 2 is specific for the reduction of adrenochrome, whereas quinone reductase 1 shows no activity. We also solved the crystal structure of quinone reductase 2 in complexes with dopamine and adrenochrome, two compounds that are structurally related to catecholamine quinones. Detailed structural analyses delineate the mechanism of quinone reductase 2 specificity toward catechol quinones in comparison with quinone reductase 1; a side-chain rotational difference between quinone reductase 1 and quinone reductase 2 of a single residue, phenylalanine 106, determines the specificity of enzymatic activities. These results infer functional differences between two homologous enzymes and indicate that quinone reductase 2 could play important roles in the regulation of catecholamine oxidation processes that may be involved in the etiology of Parkinson disease.

  10. Histochemical Localization of Glutathione Dependent NBT—Reductase in Mouse Skin



    Objective:Localization of the glutathione dependent Nitroblue tetrazolium(NBT) reductase in fresh frozen sections of mouse skin and possible dependence of NBT reductase on tissue thiol levels has been investigated.Methods:The fresh frozen tissue sections(8m thickness)were prepared and incuated in medium containing NBT,reduced glutathione(GSH) and Phosphate uffer,The staining for GSH was performed with mercury orange.Results:The activity of the NBT-reductase in mouse skin has een found to be localized in the areas rich in glutatione and actively proliferating area of the skin.Conclusion:The activity of the NBT-reductase seems to be dependent on the glutatione contents.

  11. Thiol/disulfide homeostasis in asphalt workers.

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


    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.

  12. Mitochondrial Thioredoxin-Glutathione Reductase from Larval Taenia crassiceps (Cysticerci

    Alberto Guevara-Flores


    Full Text Available Mitochondrial thioredoxin-glutathione reductase was purified from larval Taenia crassiceps (cysticerci. The preparation showed NADPH-dependent reductase activity with either thioredoxin or GSSG, and was able to perform thiol/disulfide exchange reactions. At 25∘C specific activities were 437  ±  27 mU mg-1 and 840  ±  49 mU mg-1 with thioredoxin and GSSG, respectively. Apparent Km values were 0.87  ±  0.04  μM, 41  ±  6  μM and 19  ±  10  μM for thioredoxin, GSSG and NADPH, respectively. Thioredoxin from eukaryotic sources was accepted as substrate. The enzyme reduced H2O2 in a NADPH-dependent manner, although with low catalytic efficiency. In the presence of thioredoxin, mitochondrial TGR showed a thioredoxin peroxidase-like activity. All disulfide reductase activities were inhibited by auranofin, suggesting mTGR is dependent on selenocysteine. The reductase activity with GSSG showed a higher dependence on temperature as compared with the DTNB reductase activity. The variation of the GSSG- and DTNB reductase activities on pH was dependent on the disulfide substrate. Like the cytosolic isoform, mTGR showed a hysteretic kinetic behavior at moderate or high GSSG concentrations, but it was less sensitive to calcium. The enzyme was able to protect glutamine synthetase from oxidative inactivation, suggesting that mTGR is competent to contend with oxidative stress.

  13. 100% thiol-functionalized ethylene PMOs prepared by "thiol acid-ene" chemistry.

    Esquivel, Dolores; van den Berg, Otto; Romero-Salguero, Francisco J; Du Prez, Filip; Van der Voort, Pascal


    A novel thiol functionalized bis-silane PMO precursor was synthesized by highly efficient thiol acid-ene chemistry between the double bonds of 1,2-(E)-bis(triethoxysilyl)ethene and thioacetic acid. After aminolysis the self-assembly process of the formed SH-precursor with Pluronic P123 under acidic conditions yields the first 100% thiol-PMO material with good structural ordering.

  14. Evolution of thiol protective systems in prokaryotes

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


    Biological thiols are essential elements in most aspects of cell function but undergo rapid oxidation to disulfides in the presence of oxygen. The evolution of systems to protect against such oxygen toxicity was essential to the emergence of aerobic life. The protection system used by eukaryotes is based upon glutathione (GSH) and GSH-dependent enzymes but many bacteria lack GSH and apparently use other mechanisms. The objective of this research is to elaborate the thiol protective mechanisms employed by prokaryotes of widely divergent evolutionary origin and to understand why GSH became the central thiol employed in essentially all higher organisms. Thiol-selective fluorescent labeling and HPLC analysis has been used to determine key monothiol components.

  15. Hypochlorite-induced oxidation of thiols

    Davies, Michael Jonathan; Hawkins, C L


    -molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion...... to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented...... for the involvement of sulfenyl chlorides (RSCl) in the formation of these radicals, and studies with an authentic sulfenyl chloride have demonstrated that this compound readily decomposes in thermal-, metal-ion- or light-catalysed reactions to give thiyl radicals. The formation of thiyl radicals on oxidation...

  16. Protein-thiol substitution or protein dethiolation by thiol/disulfide exchange reactions: the albumin model.

    Summa, Domenico; Spiga, Ottavia; Bernini, Andrea; Venditti, Vincenzo; Priora, Raffaella; Frosali, Simona; Margaritis, Antonios; Di Giuseppe, Danila; Niccolai, Neri; Di Simplicio, Paolo


    Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb-SS-X+RSHAlb-SS-R+XSH) or dethiolation (Alb-SS-X+XSHAlb-SH+XSSX), depending on the different pK(a) values of thiols involved in protein-thiol mixed disulfides (Alb-SS-X). It appeared in these reactions that the compound with lower pK(a) in mixed disulfide was a good leaving group and that the pK(a) differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb-TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb-SS-X (peaks at 0.25-1 min). In turn, Alb-SS-X were dethiolated by the excess nonprotein SH groups because of the lower pK(a) value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb-SS-X was accompanied by formation of XSSX and Alb-SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma.

  17. Changes in plasma thiol levels induced by different phases of treatment in breast cancer; the role of commercial extract from black chokeberry.

    Kędzierska, Magdalena; Głowacki, Rafał; Czernek, Urszula; Szydłowska-Pazera, Katarzyna; Potemski, Piotr; Piekarski, Janusz; Jeziorski, Arkadiusz; Olas, Beata


    Different low-molecular-weight thiols, including glutathione, cysteine, and cysteinylglycine are physiological free radical scavengers. On the other hand, homocysteine may play a role as an oxidant. The aim of our present study was to establish in vitro the effects of the commercial extract of Aronia melanocarpa (Aronox(®)) on the amount of selected low-molecular-weight thiols and the activity of antioxidative enzymes (superoxide dismutase, glutathione peroxidase, and glutathione reductase) in plasma obtained from patients with invasive breast cancer during different phases of treatment [before or after the surgery and patients after different phases of chemotherapy (doxorubicin and cyclophosphamide)] and from healthy subjects. Patients were hospitalized in Department of Oncological Surgery and Department of Chemotherapy, Medical University of Lodz, Poland. The level of low-molecular-weight thiols was determined by high-performance liquid chromatography. We observed that in the presence of the Aronia extract changes in amount of thiols in plasma from breast cancer patients (at all tested groups) were significantly reduced. Our results showed that tested commercial extract reduced modifications of antioxidative enzymes activity in plasma from patients during different phases of treatment, but this effect was not statistical significant. Our results suggest that the Aronia extract supplementation in breast cancer patients has a beneficial effect on thiols concentration in plasma. Plasma, as reported in this work, could be used as an experimental model to evaluate the beneficial action of plant supplements, including phenolic extracts on thiols or other molecules during different phases of treatment.

  18. Genomics and X-ray microanalysis indicate that Ca2+ and thiols mediate the aggregation and adhesion of Xylella fastidiosa

    Leite B.


    Full Text Available The availability of the genome sequence of the bacterial plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, is accelerating important investigations concerning its pathogenicity. Plant vessel occlusion is critical for symptom development. The objective of the present study was to search for information that would help to explain the adhesion of X. fastidiosa cells to the xylem. Scanning electron microscopy revealed that adhesion may occur without the fastidium gum, an exopolysaccharide produced by X. fastidiosa, and X-ray microanalysis demonstrated the presence of elemental sulfur both in cells grown in vitro and in cells found inside plant vessels, indicating that the sulfur signal is generated by the pathogen surface. Calcium and magnesium peaks were detected in association with sulfur in occluded vessels. We propose an explanation for the adhesion and aggregation process. Thiol groups, maintained by the enzyme peptide methionine sulfoxide reductase, could be active on the surface of the bacteria and appear to promote cell-cell aggregation by forming disulfide bonds with thiol groups on the surface of adjacent cells. The enzyme methionine sulfoxide reductase has been shown to be an auxiliary component in the adhesiveness of some human pathogens. The negative charge conferred by the ionized thiol group could of itself constitute a mechanism of adhesion by allowing the formation of divalent cation bridges between the negatively charged bacteria and predominantly negatively charged xylem walls.

  19. Improvement of oxidized glutathione fermentation by thiol redox metabolism engineering in Saccharomyces cerevisiae.

    Hara, Kiyotaka Y; Aoki, Naoko; Kobayashi, Jyumpei; Kiriyama, Kentaro; Nishida, Keiji; Araki, Michihiro; Kondo, Akihiko


    Glutathione is a valuable tripeptide widely used in the pharmaceutical, food, and cosmetic industries. In industrial fermentation, glutathione is currently produced primarily using the yeast Saccharomyces cerevisiae. Intracellular glutathione exists in two forms; the majority is present as reduced glutathione (GSH) and a small amount is present as oxidized glutathione (GSSG). However, GSSG is more stable than GSH and is a more attractive form for the storage of glutathione extracted from yeast cells after fermentation. In this study, intracellular GSSG content was improved by engineering thiol oxidization metabolism in yeast. An engineered strain producing high amounts of glutathione from over-expression of glutathione synthases and lacking glutathione reductase was used as a platform strain. Additional over-expression of thiol oxidase ( genes ERV1 or ERO1 increased the GSSG content by 2.9-fold and 2.0-fold, respectively, compared with the platform strain, without decreasing cell growth. However, over-expression of thiol oxidase gene ERV2 showed almost no effect on the GSSG content. Interestingly, ERO1 over-expression did not decrease the GSH content, raising the total glutathione content of the cell, but ERV1 over-expression decreased the GSH content, balancing the increase in the GSSG content. Furthermore, the increase in the GSSG content due to ERO1 over-expression was enhanced by additional over-expression of the gene encoding Pdi1, whose reduced form activates Ero1 in the endoplasmic reticulum. These results indicate that engineering the thiol redox metabolism of S. cerevisiae improves GSSG and is critical to increasing the total productivity and stability of glutathione.

  20. Fabrication and bonding of thiol-ene-based microfluidic devices

    Sikanen, Tiina M; Lafleur, Josiane P.; Moilanen, Maria-Elisa


    In this work, the bonding strength of microchips fabricated by thiol-ene free-radical polymerization was characterized in detail by varying the monomeric thiol/allyl composition from the stoichiometric ratio (1:1) up to 100% excess of thiol (2:1) or allyl (1:2) functional groups. Four different...... properties for each application. Here, a capillary electrophoresis separation is performed to demonstrate the attractive properties of stoichiometric thiol-ene microchips....

  1. Total Thiols: Biomedical Importance And Their Alteration In Various Disorders

    Mungli Prakash


    Full Text Available Thiols are the organic compounds that contain a sulphydryl group. Among all the antioxidants that are available in the body, thiols constitute the major portion of the total body antioxidants and they play a significant role in defense against reactive oxygen species. Total thiols composed of both intracellular and extracellular thiols either in the free form as oxidized or reduced glutathione, or thiols bound to proteins. Among the thiols that are bound to proteins, albumin makes the major portion of the protein bound thiols, which binds to sufhydryl group at its cysteine-34 portion. Apart from their role in defense against free radicals, thiols share significant role in detoxification, signal transduction, apoptosis and various other functions at molecular level. The thiol status in the body can be assessed easily by determining the serum levels of thiols. Decreased levels of thiols has been noted in various medical disorders including chronic renal failure and other disorders related to kidney, cardiovascular disorders, stroke and other neurological disorders, diabetes mellitus, alcoholic cirrhosis and various other disorders. Therapy using thiols has been under investigation for certain disorders.

  2. A Search for Interstellar Monohydric Thiols

    Gorai, Prasanta; Das, Ankan; Das, Amaresh; Sivaraman, Bhalamurugan; Etim, Emmanuel E.; Chakrabarti, Sandip K.


    It has been pointed out by various astronomers that a very interesting relationship exists between interstellar alcohols and the corresponding thiols (sulfur analog of alcohols) as far as the spectroscopic properties and chemical abundances are concerned. Monohydric alcohols such as methanol and ethanol are widely observed and 1-propanol was recently claimed to have been seen in Orion KL. Among the monohydric thiols, methanethiol (chemical analog of methanol) has been firmly detected in Orion KL and Sgr B2(N2) and ethanethiol (chemical analog of ethanol) has been observed in Sgr B2(N2), though the confirmation of this detection is yet to come. It is very likely that higher order thiols could be observed in these regions. In this paper, we study the formation of monohydric alcohols and their thiol analogs. Based on our quantum chemical calculation and chemical modeling, we find that the Tg conformer of 1-propanethiol is a good candidate of astronomical interest. We present various spectroscopically relevant parameters of this molecule to assist in its future detection in the interstellar medium.

  3. Chiral thiols. Synthesis and enantiomeric excess determination

    Strijtveen, Bernardus Wilhelmus Maria


    Chiral thiols are a class of chira compounds, which is gaining mone and nore attention the last decenniun. Especially α-mercaptocarboxylic acids are frequently found as a structunal unit in (biologically) important products. As a consequence, many synthetic analoga or derivatives of these natural pr

  4. Evaluation of arene ruthenium(II) N-heterocyclic carbene complexes as organometallics interacting with thiol and selenol containing biomolecules.

    Oehninger, Luciano; Stefanopoulou, Maria; Alborzinia, Hamed; Schur, Julia; Ludewig, Stephanie; Namikawa, Kazuhiko; Muñoz-Castro, Alvaro; Köster, Reinhard W; Baumann, Knut; Wölfl, Stefan; Sheldrick, William S; Ott, Ingo


    Metal complexes with N-heterocyclic carbene (NHC) ligands have been widely used in catalytic chemistry and are now increasingly considered for the development of new chemical tools and metal based drugs. Ruthenium complexes of the type (p-cymene)(NHC)RuCl(2) interacted with biologically relevant thiols and selenols, which resulted in the inhibition of enzymes such as thioredoxin reductase or cathepsin B. Pronounced antiproliferative effects could be obtained provided that an appropriate cellular uptake was achieved. Inhibition of tumor cell growth was accompanied by a perturbation of metabolic parameters such as cellular respiration.

  5. Pathways of peroxynitrite oxidation of thiol groups.

    Quijano, C; Alvarez, B; Gatti, R M; Augusto, O; Radi, R


    Peroxynitrite mediates the oxidation of the thiol group of both cysteine and glutathione. This process is associated with oxygen consumption. At acidic pH and a cysteine/peroxynitrite molar ratio of < or = 1.2, there was a single fast phase of oxygen consumption, which increased with increasing concentrations of both cysteine and oxygen. At higher molar ratios the profile of oxygen consumption became biphasic, with a fast phase (phase I) that decreased with increasing cysteine concentration, followed by a slow phase (phase II) whose rate of oxygen consumption increased with increasing cysteine concentration. Oxygen consumption in phase I was inhibited by desferrioxamine and 5,5-dimethyl-1-pyrroline N-oxide, but not by mannitol; superoxide dismutase also inhibited oxygen consumption in phase I, while catalase added during phase II decreased the rate of oxygen consumption. For both cysteine and glutathione, oxygen consumption in phase I was maximal at neutral to acidic pH: in contrast, total thiol oxidation was maximal at alkaline pH. EPR spin-trapping studies using N-tert-butyl-alpha-phenylnitrone indicated that the yield of thiyl radical adducts had a pH profile comparable with that found for oxygen consumption. The apparent second-order rate constants for the reactions of peroxynitrite with cysteine and glutathione were 1290 +/- 30 M-1.S-1 and 281 +/- 6 M-1.S-1 respectively at pH 5.75 and 37 degrees C. These results are consistent with two different pathways participating in the reaction of peroxynitrite with low-molecular-mass thiols: (a) the reaction of the peroxynitrite anion with the protonated thiol group, in a second-order process likely to involve a two-electron oxidation, and (b) the reaction of peroxynitrous acid, or a secondary species derived from it, with the thiolate in a one-electron transfer process that yields thiyl radicals capable of initiating an oxygen-dependent radical chain reaction.

  6. Detection of thiol modifications by hydrogen sulfide.

    Williams, E; Pead, S; Whiteman, M; Wood, M E; Wilson, I D; Ladomery, M R; Teklic, T; Lisjak, M; Hancock, J T


    Hydrogen sulfide (H2S) is an important gasotransmitter in both animals and plants. Many physiological events, including responses to stress, have been suggested to involve H2S, at least in part. On the other hand, numerous responses have been reported following treatment with H2S, including changes in the levels of antioxidants and the activities of transcription factors. Therefore, it is important to understand and unravel the events that are taking place downstream of H2S in signaling pathways. H2S is known to interact with other reactive signaling molecules such as reactive oxygen species (ROS) and nitric oxide (NO). One of the mechanisms by which ROS and NO have effects in a cell is the modification of thiol groups on proteins, by oxidation or S-nitrosylation, respectively. Recently, it has been reported that H2S can also modify thiols. Here we report a method for the determination of thiol modifications on proteins following the treatment with biological samples with H2S donors. Here, the nematode Caenorhabditis elegans is used as a model system but this method can be used for samples from other animals or plants.

  7. Differential response of oxidative stress and thiol metabolism in contrasting rice genotypes for arsenic tolerance.

    Tripathi, Preeti; Mishra, Aradhana; Dwivedi, Sanjay; Chakrabarty, Debasis; Trivedi, Prabodh K; Singh, Rana Pratap; Tripathi, Rudra Deo


    The mechanism of arsenic (As) tolerance was investigated on two contrasting rice (Oryza sativa L.) genotypes, selected for As tolerance and accumulation. One tolerant (Triguna) and one sensitive (IET-4786) variety were exposed to various arsenate (0-50 μM) levels for 7 d for biochemical analyses. Arsenic induced oxidative stress was more pronounced in IET-4786 than Triguna especially in terms of reactive oxygen species, lipid peroxidation, EC and pro-oxidant enzymes (NADPH oxidase and ascorbate oxidase). However, Triguna tolerated As stress through the enhanced enzymes activities particularly pertaining to thiol metabolism such as serine acetyl transferase (SAT), cysteine synthase (CS), γ-glutamyl cysteine synthase (γ-ECS), γ-glutamyl transpeptidase (γ-GT), and glutathione-S-transferase (GST) as well as arsenate reductase (AR). Besides maintaining the ratio of redox couples GSH/GSSG and ASC/DHA, the level of phytochelatins (PCs) and phytochelatin synthase (PCS) activity were more pronounced in Triguna, in which harmonized responses of thiol metabolism was responsible for As tolerance in contrast to IET-4786 showing its susceptible nature towards As exposure.

  8. Functional and Structural Characterization of a Thiol Peroxidase from Mycobacterium tuberculosis

    Rho,B.; Hung, L.; Holton, J.; Vigil, D.; Kim, S.; Park, M.; Terwilliger, T.; Pedelacq, j.


    A thiol peroxidase (Tpx) from Mycobacterium tuberculosis was functionally analyzed. The enzyme shows NADPH-linked peroxidase activity using a thioredoxin-thioredoxin reductase system as electron donor, and anti-oxidant activity in a thiol-dependent metal-catalyzed oxidation system. It reduces H{sub 2}O{sub 2}, t-butyl hydroperoxide, and cumene hydroperoxide, and is inhibited by sulfhydryl reagents. Mutational studies revealed that the peroxidatic (Cys60) and resolving (Cys93) cysteine residues are critical amino acids for catalytic activity. The X-ray structure determined to a resolution of 1.75 Angstroms shows a thioredoxin fold similar to that of other peroxiredoxin family members. Superposition with structural homologues in oxidized and reduced forms indicates that the M. tuberculosis Tpx is a member of the atypical two-Cys peroxiredoxin family. In addition, the short distance that separates the Ca atoms of Cys60 and Cys93 and the location of these cysteine residues in unstructured regions may indicate that the M. tuberculosis enzyme is oxidized, though the side-chain of Cys60 is poorly visible. It is solely in the reduced Streptococcus pneumoniae Tpx structure that both residues are part of two distinct helical segments. The M. tuberculosis Tpx is dimeric both in solution and in the crystal structure. Amino acid residues from both monomers delineate the active site pocket.

  9. Isolated menthone reductase and nucleic acid molecules encoding same

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L


    The present invention provides isolated menthone reductase proteins, isolated nucleic acid molecules encoding menthone reductase proteins, methods for expressing and isolating menthone reductase proteins, and transgenic plants expressing elevated levels of menthone reductase protein.

  10. Quantifying the global cellular thiol-disulfide status

    Hansen, Rosa E; Roth, Doris; Winther, Jakob R


    It is widely accepted that the redox status of protein thiols is of central importance to protein structure and folding and that glutathione is an important low-molecular-mass redox regulator. However, the total cellular pools of thiols and disulfides and their relative abundance have never been...... redox pool than glutathione. Accordingly, protein thiols are likely to be directly involved in the cellular defense against oxidative stress....

  11. Trametes versicolor carboxylate reductase uncovered

    Winkler, Margit; Winkler, Christoph K.


    Abstract The first carboxylate reductase from Trametes versicolor was identified, cloned, and expressed in Escherichia coli. The enzyme reduces aromatic acids such as benzoic acid and derivatives, cinnamic acid, and 3-phenylpropanoic acid, but also aliphatic acids such as octanoic acid are reduced. Graphical abstract

  12. Antibodies with thiol-S-transferase activity

    Fan, E.; Oei, Yoko; Sweet, E.; Uno, Tetsuo; Schultz, P.G. [Univ. of California, Berkeley, CA (United States)


    A major detoxification pathway used by aerobic organisms involves the conjugation of the tripeptide glutathione (GSH) to the electrophilic center of toxic substances. This reaction is catalyzed by a class of enzymes referred to as the glutathione S-transferases (GST) (EC These enzymes activate the cysteine thiol group of GSH for nucleophilic addition to a variety of substrates, including aryl halides, {alpha}{beta}-unsaturated aldehydes and ketones, and epoxides. Despite the availability of X-ray crystal structures, the mechanism whereby glutathione transferases catalyze these addition reactions remains unclear. In order to gain a greater understanding of this important biological transformation, as well as to generate new detoxification catalysts, we have asked whether antibodies can be generated that catalyze similar nucleophilic addition reactions. Our initial efforts focused on the addition reaction of thiol nucleophiles to the nitro-substituted styrene derivative 1. The ratio of k{sub cat}/K{sub m} reported for the reaction of the isozyme 4-4` of rat liver GST with the good substance, 1-chloro-2,4-dinitrobenzene, is approximately 10{sup 4} M{sup -1} s{sup -1} compared to a calculated pseudo-first-order rate constant for the uncatalyzed reaction of approximately 3 x 10{sup -2} s{sup -1} (60 mM GSH, pH = 80). These comparisons suggest that with further improvements in hapten design, catalytic antibodies may prove a good source of detoxification catalysts. 19 refs., 1 fig.

  13. Colorimetric and fluorescent detection of biological thiols in aqueous solution

    Yin-Hui Li; Jin-Feng Yang; Chang-Hui Liu; Ji-Shan Li; Rong-Hua Yang


    A new colorimetric and fluorescent probe,2-(2,4-dinitrostyryl)-1,3,3-trimethyl-3H-indolium iodide (DTI),for selective and sensitive detection of biological thiols is reported.In aqueous solution at physiological pH 7.4,biological thiols react with DTI via Michael addition to give the brownish red adduct concomitant with fluorescence emission decrease.

  14. Effects of copper on induction of thiol-compounds and antioxidant enzymes by the fruiting body of Oudemansiella radicata.

    Jiang, Juan; Qin, Chuixin; Shu, Xueqin; Chen, Rong; Song, Haihai; Li, Qiao; Xu, Heng


    Oudemansiella radicata has been found to have ability to tolerate and accumulate heavy metals. In this study, to know about the metal tolerance and detoxification strategy of O. radicata, the tolerance responses in both cap and stipe of the fruiting body, including the copper content, the changes of thiol compounds production and antioxidant enzymes activities, caused by various copper stress (150-600 mg kg(-1)) during 2-6 days were investigated. Results showed that Cu content in the fruiting bodies increased with the increasing Cu concentrations and growing time, which was higher in cap than that in stipe. For thiols contents, the maximum level was in the sample at 300 mg kg(-1) Cu after 2 d both in cap and stipe, in accordance with superoxide dismutase (SOD) and glutathione reductase (GR) activities. Guaicol peroxidase (POD) activities reached maximum at 150 mg kg(-1) Cu after 4 d and 6 d, respectively in cap and stipe, while the maximum of catalase (CAT) activities was recorded at 300 and 600 mg kg(-1) Cu after 4 d in the cap and stipe, respectively. As a whole, low concentration of Cu stimulated the production of thiols and activated the antioxidant enzymes activities in the fruiting body of O. radicata after 2/4 d, while high-level Cu decreased the thiols production and enzymes activities after 4/6 d. Furthermore, the cap was more sensitive than the stipe to Cu exposure. Different indicators showed different responses to copper accumulation and the different fruiting part (cap and stipe) of O. radicata had ability to response the oxidative stress caused by Cu. Considering the metal accumulation and its own detoxification with short growing time, mushroom might have the potential to be used as bio-accumulator to deal with Cu exposure in the Cu-contaminated farmland soil.

  15. Serum paraoxonase activity and protein thiols in patients with hyperlipidemia

    Mungli Prakash; Jeevan K Shetty; Sudeshna Tripathy; Pannuri Vikram; Manish Verma


    Objective: In the present study we evaluated the paraoxonase activity and protein thiols level in south Indian population with newly diagnosed hyperlipidemia. Methods: The study was conducted on 55 newly diagnosed hyperlipidemic pa-tients and 57 healthy controls. Serum paraoxonase activity and protein thiols were estimated by spectrophotometeric method and lipid profile by enzymatic kinetic assay method. Results: Serum paraoxonase activity, protein thiols and high density lipoprotein levels were low and total cholesterol, triglycerides and low density lipoprutein levels were high in patients with hyperlipidemia compared to healthy controls ( P < 0.01 ). Serum paranxonase activity correlated positively with protein thiols and high density lipoprotein (P<0.01). Conclusion: Decreased paraoxonase activity and protein thiols were found in patients with hyperlipi-demia. This may indicate the susceptibility of this population to accelerated atherogenesis and protein oxidation.

  16. Efficient Functionalization of Oxide-Free Silicon(111) Surfaces: Thiol-yne versus Thiol-ene Click Chemistry

    Bhairamadgi, N.S.; Gangarapu, S.; Caipa Campos, M.A.; Paulusse, J.M.J.; Rijn, van C.J.M.; Zuilhof, H.


    Thiol-yne click (TYC) chemistry was utilized as a copper-free click reaction for the modification of alkyne-terminated monolayers on oxide-free Si(111) surfaces, and the results were compared with the analogous thiol–ene click (TEC) chemistry. A wide range of thiols such as 9-fluorenylmethoxy-carbon

  17. Nitrate Reductase: Properties and Regulation


    Nitrate Reductase (NR) is a rating-limit and key enzyme of nitrate assimilation in plants ,so ,NR activity is important for growth,development and the dry matter accumulation of plants. The regulation of NR activity appears to be rather complex and many studies have been devoted to the description of regulation and properties,but in this paper we focus on the properties and regulation of NR in higher plants.

  18. Purification and kinetic analysis of cytosolic and mitochondrial thioredoxin glutathione reductase extracted from Taenia solium cysticerci.

    Plancarte, Agustin; Nava, Gabriela


    Thioredoxin glutathione reductases (TGRs) (EC were purified to homogeneity from the cytosolic (cTsTGR) and mitochondrial (mTsTGR) fractions of Taenia solium, the agent responsible for neurocysticercosis, one of the major central nervous system parasitic diseases in humans. TsTGRs had a relative molecular weight of 132,000, while the corresponding value per subunit obtained under denaturing conditions, was of 62,000. Specific activities for thioredoxin reductase and glutathione reductase substrates for both TGRs explored were in the range or lower than values obtained for other platyhelminths and mammalian TGRs. cTsTGR and mTsTGR also showed hydroperoxide reductase activity using hydroperoxide as substrate. Km(DTNB) and Kcat(DTNB) values for cTsTGR and mTsTGR (88 µM and 1.9 s(-1); 45 µM and 12.6 s(-1), respectively) and Km(GSSG) and Kcat(GSSG) values for cTsTGR and mTsTGR (6.3 µM and 0.96 s(-1); 4 µM and 1.62 s(-1), respectively) were similar to or lower than those reported for mammalian TGRs. Mass spectrometry analysis showed that 12 peptides from cTsTGR and seven from mTsTGR were a match for gi|29825896 thioredoxin glutathione reductase [Echinococcus granulosus], confirming that both enzymes are TGRs. Both T. solium TGRs were inhibited by the gold compound auranofin, a selective inhibitor of thiol-dependent flavoreductases (I₅₀ = 3.25, 2.29 nM for DTNB and GSSG substrates, respectively for cTsTGR; I₅₀ = 5.6, 25.4 nM for mTsTGR toward the same substrates in the described order). Glutathione reductase activity of cTsTGR and mTsTGR exhibited hysteretic behavior with moderate to high concentrations of GSSG; this result was not observed either with thioredoxin, DTNB or NADPH. However, the observed hysteretic kinetics was suppressed with increasing amounts of both parasitic TGRs. These data suggest the existence of an effective substitute which may account for the lack of the detoxification enzymes glutathione reductase

  19. Fatty acyl-CoA reductase

    Reiser, Steven E.; Somerville, Chris R.


    The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

  20. Thiol-based redox signaling in the nitrogen-fixing symbiosis

    Pierre eFrendo


    Full Text Available In nitrogen poor soils legumes establish a symbiotic interaction with rhizobia that results in the formation of root nodules. These are unique plant organs where bacteria differentiate into bacteroids, which express the nitrogenase enzyme complex that reduces atmospheric N2 to ammonia. Nodule metabolism requires a tight control of the concentrations of reactive oxygen and nitrogen species (RONS so that they can perform useful signaling roles while avoiding nitro-oxidative damage. In nodules a thiol-dependent regulatory network that senses, transmits and responds to redox changes is starting to be elucidated. A combination of enzymatic, immunological, pharmacological and molecular analyses has allowed to conclude that glutathione and its legume-specific homolog, homoglutathione, are abundant in meristematic and infected cells, their spatio-temporally distribution is correlated with the corresponding (homoglutathione synthetase activities, and are crucial for nodule development and function. Glutathione is at high concentrations in the bacteroids and at moderate amounts in the mitochondria, cytosol and nuclei. Less information is available on other components of the network. The expression of multiple isoforms of glutathione peroxidases, peroxiredoxins, thioredoxins, glutaredoxins and NADPH-thioredoxin reductases has been detected in nodule cells using antibodies and proteomics. Peroxiredoxins and thioredoxins are essential to regulate and in some cases to detoxify RONS in nodules. Further research is necessary to clarify the regulation of the expression and activity of thiol redox-active proteins in response to abiotic, biotic and developmental cues, their interactions with downstream targets by disulfide-exchange reactions, and their participation in signaling cascades. The availability of mutants and transgenic lines will be crucial to facilitate systematic investigations into the function of the various proteins in the legume

  1. Facially amphiphilic thiol capped gold and silver nanoparticles

    Shreedhar Bhata; Uday Maitra


    A series of bile acid-derived facially amphiphilic thiols have been used to cap sliver and gold nanoparticles. The self-assembling properties of these steroid-capped nanoparticles have been investigated and reported in this article.

  2. A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S


    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

  3. Fast and Highly Efficient Solid State Oxidation of Thiols

    Nasrin Haghighat


    Full Text Available A fast and efficient solid state method for the chemoselective room temperature oxidative coupling of thiols to afford their corresponding disulfides using inexpensive and readily available moist sodiumperiodate as the reagent is described. The reaction was applicable to a variety of thiols giving high yields after short reaction times. Comparison of yield/time ratios of this method with some of those reported in the literature shows the superiority of this reagent over others under these conditions.

  4. Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection

    Stantchev Tzanko S


    Full Text Available Abstract Background The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/ reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI and/or thioredoxin (Trx have emerged as the two enzymes most often implicated in this process. Results We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets - primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid (DTNB, significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM, and also phytohemagglutinin (PHA-stimulated peripheral blood mononuclear cells (PBMC. Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb in HIV-1 envelope pseudotyped and wild type (wt virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells. Conclusions Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this

  5. Protein Thiols as an Indication of Oxidative Stress

    Yousef Rezaei Chianeh


    Full Text Available Thiol is an organic compound that contain sulphhydryl group that have a critical role in preventing any involvement of oxidative stress in the cell. These defensive functions are generally considered to be carried out by the low molecular weight thiol glutathione and by cysteine residues in the active sites of proteins such as thioredoxin and peroxiredoxin. In addition, there are thiols exposed on protein surfaces that are not directly involved with protein function, although they can interact with the intracellular environment.The process of protection of the cell against an oxidative damage occur by thiol and cystein residue that has a low molecular weight. These residue are present in the active sites of a protein like, peroxiredoxin and thioredoxin. Apart from intracellular antioxidant defense mechanism by protein thiol, there are presence of thiol in outer surface of protein that are not involved with the function of protein, even though they can interact with intracellular part of the cell. [Archives Medical Review Journal 2014; 23(3.000: 443-456

  6. Efficient functionalization of oxide-free silicon(111) surfaces: thiol-yne versus thiol-ene click chemistry.

    Bhairamadgi, Nagendra S; Gangarapu, Satesh; Caipa Campos, Mabel A; Paulusse, Jos M J; van Rijn, Cees J M; Zuilhof, Han


    Thiol-yne click (TYC) chemistry was utilized as a copper-free click reaction for the modification of alkyne-terminated monolayers on oxide-free Si(111) surfaces, and the results were compared with the analogous thiol-ene click (TEC) chemistry. A wide range of thiols such as 9-fluorenylmethoxy-carbonyl cysteine, thio-β-d-glucose tetraacetate, thioacetic acid, thioglycerol, thioglycolic acid, and 1H,1H,2H,2H-perfluorodecanethiol was immobilized using TYC under photochemical conditions, and all modified surfaces were characterized by static water contact angle measurements, X-ray photoelectron spectroscopy (including a simulation thereof by density functional calculations), and infrared absorption reflection spectroscopy. Surface-bound TYC proceeds with an efficiency of up to 1.5 thiols per alkyne group. This high surface coverage proceeds without oxidizing the Si surface. TYC yielded consistently higher surface coverages than TEC, due to double addition of thiols to alkyne-terminated monolayers. This also allows for the sequential and highly efficient attachment of two different thiols onto an alkyne-terminated monolayer.

  7. Copper-induced changes in intracellular thiols in two marine diatoms: Phaeodactylum tricornutum and Ceratoneis closterium.

    Smith, Cassandra L; Steele, Jessica E; Stauber, Jennifer L; Jolley, Dianne F


    Phytochelatins and glutathione (reduced (GSH) and oxidised (GSSG)) are important intracellular ligands involved in metal sequestration and detoxification in algae. Intracellular ratios of GSH:GSSG are sensitive indicators of metal stress in algae, and like phytochelatin production are influenced by metal speciation, concentration, exposure time and the biological species. This study investigated the effect of copper exposure on phytochelatin and glutathione content in two marine diatoms Phaeodactylum tricornutum and Ceratoneis closterium at various time intervals between 0.5 and 72h. Liberation of cellular glutathione and phytochelatins was optimised using freeze/thaw cycles and chemical extraction, respectively. Extracted phytochelatins were derivatised (by fluorescent tagging of thiol compounds), separated and quantified using HPLC with fluorescence detection. Glutathione ratios were determined using a commercially available kit, which uses the enzyme glutathione reductase to measure total and oxidised glutathione. Despite similarities in size and shape between the two diatoms, differences in internalised copper, phytochelatin production (both chain length and quantity) and reduced glutathione concentrations were observed. P. tricornutum maintained reduced glutathione at between 58 and 80% of total glutathione levels at all time points, which would indicate low cellular stress. In C. closterium reduced glutathione constituted phytochelatins and phytochelatins of longer chain length than C. closterium despite the latter species internalising significantly more copper.

  8. The impact of thiol peroxidases on redox regulation.

    Flohé, Leopold


    The biology of glutathione peroxidases and peroxiredoxins is reviewed with emphasis on their role in metabolic regulation. Apart from their obvious function in balancing oxidative challenge, these thiol peroxidases are not only implicated in orchestrating the adaptive response to oxidative stress, but also in regulating signaling triggered by hormones, growth factors and cytokines. The mechanisms presently discussed comprise dampening of redox-sensitive regulatory processes by elimination of hydroperoxides, suppression of lipoxygenase activity, committing suicide to save H2O2 for signaling, direct binding to receptors or regulatory proteins in a peroxidase activity-independent manner, or acting as sensors for hydroperoxides and as transducers of oxidant signals. The various mechanistic proposals are discussed in the light of kinetic data, which unfortunately are scarce. Taking into account pivotal criteria of a meaningful regulatory circuit, kinetic plausibility and specificity, the mechanistic concepts implying a direct sensor/transducer function of the thiol peroxidases appear most appealing. With rate constants for the reaction with hydroperoxide of 10(5)-10(8) M(-1) s(-1), thiol peroxidases are qualified as kinetically preferred hydroperoxide sensors, and the ability of the oxidized enzymes to react with defined protein thiols lends specificity to the transduction process. The versatility of thiol peroxidases, however, allows multiple ways of interaction with regulatory pathways.

  9. Surface functionalized thiol-ene waveguides for fluorescence biosensing in microfluidic devices

    Feidenhans'l, Nikolaj Agentoft; Lafleur, Josiane P.; Jensen, Thomas Glasdam;


    . The reactive functional groups present at the surface of the thiol-ene polymer are subsequently used for the rapid, one step, site-specific functionalization of the waveguide with biological recognition molecules. It was found that while the bulk properties and chemical surface properties of thiol......-ene waveguides were fabricated from 40% excess thiol thiol-ene to ensure the presence of thiol functional groups at the surface of the waveguide. Biotin alkyne was photografted at specific locations using a photomask, directly at the interface between the microfluidic channel and the thiol-ene waveguide prior...

  10. Suspended hybrid films assembled from thiol-capped gold nanoparticles.

    Zhang, Yu Xin; Huang, Ming; Hao, Xiao Dong; Dong, Meng; Li, Xin Lu; Huang, Jia Mu


    In this work, we explored the formation processes of suspended hybrid thin films of thiol-capped Au nanoparticles (AuNPs) inside metal oxide tubular structures. We found that a balance between in-film interactions of the AuNPs and boundary interactions with metal oxides is a key in making these special organic-inorganic thin films. The hybrid films process many processing advantages and flexibilities, such as controllable film thickness, interfacial shape and inter-AuNPs distance, tuning of particle sizes, thiol population, chain lengths, and other new properties by introducing functional groups to thiol chains. Among their many unique features, the assembly-disassembly property may be useful for future on-off or store-release applications.

  11. Highly tailorable thiol-ene based emulsion-templated monoliths

    Lafleur, J. P.; Kutter, J. P.


    The attractive surface properties of thiol-ene polymers combined with their ease of processing make them ideal substrates in many bioanalytical applications. We report the synthesis of highly tailorable emulsion-templated porous polymers and beads in microfluidic devices based on off-stoichiometr......The attractive surface properties of thiol-ene polymers combined with their ease of processing make them ideal substrates in many bioanalytical applications. We report the synthesis of highly tailorable emulsion-templated porous polymers and beads in microfluidic devices based on off......-stoichiometry thiolene chemistry. The method allows monolith synthesis and anchoring inside thiol-ene microchannels in a single step. Variations in the monomer stoichiometric ratios and/or amount of porogen used allow for the creation of extremely varied polymer morphologies, from foam-like materials to dense networks...

  12. General and practical formation of thiocyanates from thiols.

    Frei, Reto; Courant, Thibaut; Wodrich, Matthew D; Waser, Jerome


    A new method for the cyanation of thiols and disulfides using cyanobenziodoxol(on)e hypervalent iodine reagents is described. Both aliphatic and aromatic thiocyanates can be accessed in good yields in a few minutes at room temperature starting from a broad range of thiols with high chemioselectivity. The complete conversion of disulfides to thiocyanates was also possible. Preliminary computational studies indicated a low energy concerted transition state for the cyanation of the thiolate anion or radical. The developed thiocyanate synthesis has broad potential for various applications in synthetic chemistry, chemical biology and materials science.

  13. 5-Furan-2yl[1,3,4]oxadiazole-2-thiol, 5-Furan-2yl-4H [1,2,4] triazole-3-thiol and Their Thiol-Thione Tautomerism

    A. Cansız


    Full Text Available 5-Furan-2-yl[1,3,4]oxadiazole-2-thiol (Ia and 5-furan-2-yl-4H-[1,2,4]-triazole-3-thiol (Ib were synthesized from furan-2-carboxylic acid hydrazide. Mannich basesand methyl derivatives were then prepared. The structures of the synthesized compoundswere confirmed by elemental analyses, IR and 1H-NMR spectra. Their thiol-thione tautomericequilibrium is described.

  14. Quantification of protein-derived thiols during atmosphere-controlled brewing in laboratory scale

    Murmann, Anne Nordmark; Andersen, Preben; Mauch, Alexander;


    An atmosphere-controlled brewing system was built to study thiol oxidation during brewing in laboratory scale under conditions with limited oxygen exposure. Quantification of free and total thiols and protein showed that thiols were lost during wort boiling possibly owing to protein precipitation...

  15. Thiol reactive nitroimidazoles: radiosensitization studies in vitro and in vivo

    Stratford, I.J.; Adams, G.E.; Hardy, C.; Hoe, S.; O' Neill, P.; Sheldon, P.W. (Medical Research Council, Harwell (UK). Radiobiological Research Unit)


    Using Chinese hamster V79 cells in vitro a study was made of the radiosensitizing properties of 4- or 5-nitroimidazoles substituted in the 2,5 or 4 position with various halo, sulphur ether, sulphonamide, sulphonate, ether or nitro groups. Values of E/sub 7//sup 1/ (the one-electron reduction potential measured versus the normal hydrogen electrode at pH7) vary in the range -178 to -565 mV. All the compounds, with one exception, are more efficient radiosensitizers than would be predicted from redox potentials, and the factor Csub(1.6)/Csub(1.6), by which a compound is more efficient has been calculated. The second-order rate constants, k/sub 2/, for reaction of these nitroimidazoles with glutathione and/or dithiothreitol tended to increase with increasing redox potential. However, there is no clear trend between k/sub 2/ and Csub(1.6)/Csub(1.6). The concentration required to cause a 50% depletion of intracellular glutathione was determined for selected compounds, as was the ability of glutathione-S-transferase to catalyse reaction with thiols. These observations suggested the relative thiol reactivity measured under chemically controlled conditions does not necessarily indicate thiol reactivity intracellularly. Studies using MT tumour in mice showed that high levels of radiosensitization seen in vitro could not be duplicated in vivo (attributed to thiol reactivity).

  16. Preparation of Novel Hydrolyzing Urethane Modified Thiol-Ene Networks

    Bridget S. Confait


    Full Text Available Novel tetra-functional hydrolyzing monomers were prepared from the reaction of TEOS and select alkene-containing alcohols, ethylene glycol vinyl ether or 2-allyloxy ethanol, and combined with trimethylolpropane tris(3-mercaptopropionate (tri-thiol in a thiol-ene “click” polymerization reaction to produce clear, colorless thiol-ene networks using both radiation and thermal-cure techniques. These networks were characterized for various mechanical characteristics, and found to posses Tg’s (DSC, hardness, tack, and thermal stability (TGA consistent with their molecular structures. A new ene-modified urethane oligomer was prepared based on the aliphatic polyisocyanate Desmodur® N 3600 and added to the thiol-ene hydrolyzable network series in increasing amounts, creating a phase-segregated material having two Tg’s. An increase in water absorption in the ene-modified urethane formulations leading to a simultaneous increase in the rate of hydrolysis was supported by TGA data, film hardness measurements, and an NMR study of closely related networks. This phenomenon was attributed to the additional hydrogen bonding elements and polar functionality brought to the film with the addition of the urethane segment. SEM was utilized for visual analysis of topographical changes in the film’s surface upon hydrolysis and provides support for surface-driven erosion. Coatings prepared in this study are intended for use as hydrolyzing networks for marine coatings to protect against ship fouling.

  17. Are free radicals involved in thiol-based redox signaling?

    Winterbourn, Christine C


    Cells respond to many stimuli by transmitting signals through redox-regulated pathways. It is generally accepted that in many instances signal transduction is via reversible oxidation of thiol proteins, although there is uncertainty about the specific redox transformations involved. The prevailing view is that thiol oxidation occurs by a two electron mechanism, most commonly involving hydrogen peroxide. Free radicals, on the other hand, are considered as damaging species and not generally regarded as important in cell signaling. This paper examines whether it is justified to dismiss radicals or whether they could have a signaling role. Although there is no direct evidence that radicals are involved in transmitting thiol-based redox signals, evidence is presented that they are generated in cells when these signaling pathways are activated. Radicals produce the same thiol oxidation products as two electron oxidants, although by a different mechanism, and at this point radical-mediated pathways should not be dismissed. There are unresolved issues about how radical mechanisms could achieve sufficient selectivity, but this could be possible through colocalization of radical-generating and signal-transducing proteins. Colocalization is also likely to be important for nonradical signaling mechanisms and identification of such associations should be a priority for advancing the field.

  18. Corynebacterium diphtheriae methionine sulfoxide reductase a exploits a unique mycothiol redox relay mechanism.

    Tossounian, Maria-Armineh; Pedre, Brandán; Wahni, Khadija; Erdogan, Huriye; Vertommen, Didier; Van Molle, Inge; Messens, Joris


    Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen.

  19. New drug target in protozoan parasites: the role of thioredoxin reductase.

    Andrade, Rosa M; Reed, Sharon L


    Amebiasis causes approximately 70,000 deaths annually and is the third cause of death due to parasites worldwide. It is treated primarily with metronidazole, which has adverse side effects, is mutagenic and carcinogenic, and emergence of resistance is an increasing concern. Unfortunately, better therapeutic alternatives are lacking. Re-purposing of older FDA approved drugs is advantageous to drug discovery since safety and pharmacokinetic effects in humans are already known. In high throughput screening studies, we recently demonstrated that auranofin, a gold containing compound originally approved to treat rheumatoid arthritis, has activity against trophozoites of E. histolytica, the causative agent of amebiasis. Auranofin's anti-parasitic activity is attributed to its monovalent gold molecule that readily inhibits E. histolytica thioredoxin reductase. This anti-oxidant enzyme is the only thiol-dependent flavo-reductase present in E. histolytica. Auranofin has also shown promising activity against other protozoans of significant public health importance. Altogether, this evidence suggests that auranofin has the potential to become a broad spectrum alternative therapeutic agent for diseases with a large global burden.

  20. New drug target in protozoan parasites: the role of thioredoxin reductase

    Rosa M. Andrade


    Full Text Available Amebiasis causes approximately 70,000 deaths annually and is the third cause of death due to parasites worldwide. It is treated primarily with metronidazole, which has adverse side effects, is mutagenic and carcinogenic, and emergence of resistance is an increasing concern. Unfortunately, better therapeutic alternatives are lacking. Re-purposing of older FDA approved drugs is advantageous to drug discovery since safety and pharmacokinetic effects in humans are already known. In high throughput screening studies, we recently demonstrated that auranofin, a gold containing compound originally approved to treat rheumatoid arthritis, has activity against trophozoites of E. histolytica, the causative agent of amebiasis. Auranofin’s anti-parasitic activity is attributed to its monovalent gold molecule that readily inhibits E.histolytica thioredoxin reductase. This anti-oxidant enzyme is the only thiol-dependent flavo-reductase present in E.histolytica. Auranofin has also shown promising activity against other protozoans of significant public health importance. Altogether, this evidence suggests that auranofin has the potential to become a broad spectrum alternative therapeutic agent for diseases with a large global burden.

  1. The aldo-keto reductase superfamily homepage.

    Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M


    The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

  2. Influence of the microenvironment of thiol groups in low molecular mass thiols and serum albumin on the reaction with methylglyoxal.

    Aćimović, Jelena M; Stanimirović, Bojana D; Todorović, Nina; Jovanović, Vesna B; Mandić, Ljuba M


    Methylglyoxal (MG), a reactive alpha-oxoaldehyde that is produced in higher quantities in diabetes, uremia, oxidative stress, aging and inflammation, reacts with the thiol groups (in addition to the amino and guanidino groups) of proteins. This causes protein modification, formation of advanced glycated end products (AGEs) and cross-linking. Low molecular mass thiols can be used as competitive targets for MG, preventing the reactions mentioned above. Therefore, this paper investigated how the microenvironment of the thiol group in low molecular mass thiols (cysteine, N-acetylcysteine (NAcCys), carboxymethylcysteine (CMC) and glutathione (GSH)) and human serum albumin (HSA) affected the thiol reaction with MG. The SH group reaction course was monitored by (1)H-NMR spectroscopy and spectrophotometric quantification. Changes in the HSA molecules were monitored by SDS-PAGE. The microenvironment of the SH group had a major effect on its reactivity and on the product yield. The reactivity of SH groups decreased in the order Cys>GSH>NAcCys. CMC did not react. The percentages of the reacted SH groups in the equilibrium state were almost equal, regardless of the ratio of thiol compound/MG (1:1, 1:2, 1:5): 38.1 + or - 0.9%; 38.2 + or - 0.7% and 39.0 + or - 0.8% for Cys; 26.5 + or - 0.6%; 26.6 + or - 2.6% and 27.4 + or - 2.5% for GSH; 10.8 + or - 0.9%; and 11.2 + or - 0.7% and 12.2 + or - 0.9% for NAcCys, respectively. Our results explain why substances containing alpha-amino-beta-mercapto-ethane as a pharmacophore are successful scavengers of MG. In equilibrium, HSA SH reacted in high percentages both with an insufficient amount and with an excess of MG (55% and 65%, respectively). An analysis of the hydrophobicity of the microenvironment of the SH group on the HSA surface showed that it could contribute to high levels of SH modification, leading to an increase in the scavenging activity of the albumin thiol.

  3. Respiratory arsenate reductase as a bidirectional enzyme

    Richey, C.; Chovanec, P.; Hoeft, S.E.; Oremland, R.S.; Basu, P.; Stolz, J.F.


    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe–S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

  4. Evolution of plant defense mechanisms. Relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol and isoflavone reductases.

    Gang, D R; Kasahara, H; Xia, Z Q; Vander Mijnsbrugge, K; Bauw, G; Boerjan, W; Van Montagu, M; Davin, L B; Lewis, N G


    Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent Km and Vmax values and observed kinetic 3H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.

  5. Thiol metabolism and antioxidant systems complement each other during arsenate detoxification in Ceratophyllum demersum L.

    Mishra, Seema; Srivastava, Sudhakar; Tripathi, Rudra D; Trivedi, Prabodh K


    Ceratophyllum demersum L. is known to be a potential accumulator of arsenic (As), but mechanisms of As detoxification have not been investigated so far. In the present study, we analyzed the biochemical responses of Ceratophyllum plants to arsenate (As(V); 0-250 microM) exposure to explore the underlying mechanisms of As detoxification. Plants efficiently tolerated As toxicity up to concentrations of 50 microM As(V) and durations of 4 d with no significant effect on growth by modulating various pathways in a coordinated and complementary manner and accumulated about 76 microg As g(-1)dw. Significant increases were observed in the levels of various thiols including phytochelatins (PCs), the activities of enzymes of thiolic metabolism as well as arsenate reductase (AR). These primary responses probably enabled plants to detoxify at least some part of As(V) through its reduction and subsequent complexation. The maximum proportion of As chelated by PCs was found to be about 30% (at 50 microM As(V) after 2 d). Simultaneously, a significant increase in the activities of antioxidant enzymes was observed and hence plants did not experience oxidative stress when exposed to 50 microM As(V) for 4 d. Exposure of plants to higher concentrations (250 microM As(V)) and/or for longer durations (7 d) resulted in a significant increase in the level of As (maximum 525 microgg(-1)dw at 250 microM after 7 d) and an inverse relationship between As accumulation and various detoxification strategies was observed that lead to enhanced oxidative stress and hampered growth.

  6. Radical Scavenging Efficacy of Thiol Capped Silver Nanoparticles

    Kumudini Chandraker; Sandeep Kumar Vaishanav; Rekha Nagwanshi; Manmohan L Satnami


    Radical scavenging efficacy of L-cysteine (L-Cys), glutathione (GSH) and thioctic acid (TA) in the presence of silver nanoparticles (AgNPs) were determined by 1,1-diphenyl 2-picryl hydrazil (DPPH), nitric oxide (NO) and hydroxyl (OH) radicals as spectrophotometric assay. The hydrogen peroxide (H2O2) scavenging efficacy has been determined by titration method. Ascorbic acid has been used as standard for all radical scavenging efficacies. In general, antioxidant activity decreases in the presence of AgNPs. The covalent interactions of thiols (-SH) were found to be a key factor for the decreases in scavenging activity. The effect of thiol concentrations has been discussed. The size and shape of the nanoparticles and AgNP-SR interactions have been characterized through Transmission Electron Microscopy (TEM) and Fourier Transform Infrared (FTIR) spectroscopy, respectively.

  7. Dynamic Cyclic Thiodepsipeptide Libraries from Thiol-Thioester Exchange


    the reaction dynamics are discussed. Cyclic peptides have been described as “privileged structures” for drug design because so many natural and...inhibitors, which hold promise for treatment of cancer.4 As drug scaffolds, cyclic peptides are advantageous because they mimic native protein structure...but instead an influence of chirality on the accessibility of the thioester or thiol. We also investigated the effect of positively charged amino

  8. Investigation of thiol derivatized gold nanoparticle sensors for gas analysis

    Stephens, Jared S.

    Analysis of volatile organic compounds (VOCs) in air and exhaled breath by sensor array is a very useful testing technique. It can provide non-invasive, fast, inexpensive testing for many diseases. Breath analysis has been very successful in identifying cancer and other diseases by using a chemiresistor sensor or array with gold nanoparticles to detect biomarkers. Acetone is a biomarker for diabetes and having a portable testing device could help to monitor diabetic and therapeutic progress. An advantage to this testing method is it is conducted at room temperature instead of 200 degrees Celsius. 3. The objective of this research is to determine the effect of thiol derivatized gold nanoparticles based on sensor(s) detection of VOCs. The VOCs to be tested are acetone, ethanol, and a mixture of acetone and ethanol. Each chip is tested under all three VOCs and three concentration levels (0.1, 1, and 5.0 ppm). VOC samples are used to test the sensors' ability to detect and differentiate VOCs. Sensors (also referred to as a chip) are prepared using several types of thiol derivatized gold nanoparticles. The factors are: thiol compound and molar volume loading of the thiol in synthesis. The average resistance results are used to determine the VOC selectivity of the sensors tested. The results show a trend of increasing resistance as VOC concentration is increased relative to dry air; which is used as baseline for VOCs. Several sensors show a high selectivity to one or more VOCs. Overall the 57 micromoles of 4-methoxy-toluenethiol sensor shows the strongest selectivity for VOCs tested. 3. Gerfen, Kurt. 2012. Detection of Acetone in Air Using Silver Ion Exchanged ZSM-5 and Zinc Oxide Sensing Films. Master of Science thesis, University of Louisville.

  9. Organized thiol functional groups in mesoporous core shell colloids

    Marchena, Martin H. [Gerencia Quimica, Centro Atomico Constituyentes, Comision Nacional de Energia Atomica (CNEA), Avda. Gral. Paz 1499, B1650KNA Buenos Aires (Argentina); Granada, Mara [Centro Atomico Bariloche-CNEA, 8400 San Carlos de Bariloche (Argentina); Instituto Balseiro-Centro Atomico Bariloche-CNEA, San Carlos de Bariloche 8400 (Argentina); Bordoni, Andrea V. [Gerencia Quimica, Centro Atomico Constituyentes, Comision Nacional de Energia Atomica (CNEA), Avda. Gral. Paz 1499, B1650KNA Buenos Aires (Argentina); Joselevich, Maria [Asociacion Civil Expedicion Ciencia, Cabrera 4948, C1414BGP Buenos Aires (Argentina); Troiani, Horacio [Centro Atomico Bariloche-CNEA, 8400 San Carlos de Bariloche (Argentina); Instituto Balseiro-Centro Atomico Bariloche-CNEA, San Carlos de Bariloche 8400 (Argentina); Williams, Federico J. [DQIAQyF-INQUIMAE FCEN, Universidad de Buenos Aires, Ciudad Universitaria, Pabellon II, C1428EHA Buenos Aires (Argentina); Wolosiuk, Alejandro, E-mail: [Gerencia Quimica, Centro Atomico Constituyentes, Comision Nacional de Energia Atomica (CNEA), Avda. Gral. Paz 1499, B1650KNA Buenos Aires (Argentina)


    The co-condensation in situ of tetraethoxysilane (TEOS) and mercaptopropyltrimethoxysilane (MPTMS) using cetyltrimethylammonium bromide (CTAB) as a template results in the synthesis of multilayered mesoporous structured SiO{sub 2} colloids with 'onion-like' chemical environments. Thiol groups were anchored to an inner selected SiO{sub 2} porous layer in a bilayered core shell particle producing different chemical regions inside the colloidal layered structure. X-Ray Photoelectron Spectroscopy (XPS) shows a preferential anchoring of the -SH groups in the double layer shell system, while porosimetry and simple chemical modifications confirm that pores are accessible. We can envision the synthesis of interesting colloidal objects with defined chemical environments with highly controlled properties. - Graphical abstract: Mesoporous core shell SiO{sub 2} colloids with organized thiol groups. Highlights: Black-Right-Pointing-Pointer Double shell mesoporous silica colloids templated with CTAB. Black-Right-Pointing-Pointer Sequential deposition of mesoporous SiO{sub 2} layers with different chemistries. Black-Right-Pointing-Pointer XPS shows the selective functionalization of mesoporous layers with thiol groups.

  10. Occurrence of polyfunctional thiols in fresh lager beers.

    Vermeulen, C; Lejeune, I; Tran, T T H; Collin, S


    Polyfunctional thiols are known to have a strong impact on the overall aroma of many fermented foods. Surprisingly, very little data is available on their occurrence in beer. A specific extraction with p-hydroxymercuribenzoic acid was performed on four different fresh light-protected lager beers. gas chromatography-olfactometry, gas chromatography-mass spectrometry, and gas chromatography-pulsed-flame photometer detector analyses of the extracts revealed the presence of more than 10 polyfunctional thiols. All of them were absent from wort, suggesting a key role of the H(2)S excreted by yeasts. 3-Methyl-2-buten-1-thiol, 2-mercapto-3-methylbutanol, 3-mercapto-3-methylbutanol seem to be created from hop allylic alcohols via four different mechanisms: nucleophilic substitution, addition-elimination, and radical anti-Markovnikov or electrophilic Markovnikov additions. 1,4 Addition of hydrogen sulfide to wort alpha,beta-unsaturated aldehydes or ketones may explain the synthesis of 1-mercapto-3-pentanol, 3-mercaptohexanol, and 4-mercapto-4-methyl-2-pentanone through fermentation. Finally, 2-mercaptoethanol, 3-mercaptopropanol, and their corresponding acetates may derive from Ehrlich degradation of sulfur amino acids, while 2-methyl-3-furanthiol should be logically issued from Maillard reactions.

  11. Isoquinoline-mediated S-vinylation and N-vinylation of benzo[d]oxazole-2-thiol and benzo[d]thiazole-2-thiol

    Issa Yavari; Samira Nasiri-Gheidari; Anvar Mirzaei


    An effective route to S-vinylated andN-vinylated benzo[d]oxazole-2(3H)-thiones and benzo[d]thiazole-2(3H)-thionesis described via reaction ofacetylenic esters and benzo[d] oxazole-2-thiol and benzo [d]thiazole-2-thiol in the presence of 15 mol% of isoquinoline.

  12. Thermal and Mechanical Properties of Sequential and Simultaneous Thiol-Ene-Isocyanate Networks

    McNair, Olivia; Brent, Davis; Savin, Daniel


    Ternary networks containing having stoichiometrically balanced thiol /(ene+isocyanate) ranging from 0 to 20 mol% isocyanate were synthesized via sequential or simultaneous thiol/ene and thiol/isocyanate click reactions. The effects of cross-link density were studied using three thiols, GDMP (difunctional), 3T (trifunctional) and 4T (tetrafunctional) respectively. TEA catalyzes the isocyanate-thiol coupling and chain extension, while the photoinitiator DMPA initiates a radical thiol-ene crosslinking process. Real-time FTIR was used to study kinetics of both light and dark reactions utilizing thiol, ene and isocyanate peaks which appear independently. It was found that difunctional thiols and isocyanates reacted initially, forming chain extended prepolymers end-capped with thiol functionalities. Upon UV irradiation, thiol functionalized prepolymers reacted with TTT, a trifunctional ene, forming networks containing incorporated thiourethane linkages. Initial DSC results indicated higher Tgs for higher cross-linked networks; however, isocyanate content has significant effects on each system. Films were also be thermally characterized via DMA and mechanical properties measured using MTS.

  13. Thiol switches in redox regulation of chloroplasts: balancing redox state, metabolism and oxidative stress.

    Dietz, Karl-Josef; Hell, Rüdiger


    In photosynthesizing chloroplasts, rapidly changing energy input, intermediate generation of strong reductants as well as oxidants and multiple participating physicochemical processes and pathways, call for efficient regulation. Coupling redox information to protein function via thiol modifications offers a powerful mechanism to activate, down-regulate and coordinate interdependent processes. Efficient thiol switching of target proteins involves the thiol-disulfide redox regulatory network, which is highly elaborated in chloroplasts. This review addresses the features of this network. Its conditional function depends on specificity of reduction and oxidation reactions and pathways, thiol redox buffering, but also formation of heterogeneous milieus by microdomains, metabolite gradients and macromolecular assemblies. One major player is glutathione. Its synthesis and function is under feedback redox control. The number of thiol-controlled processes and involved thiol switched proteins is steadily increasing, e.g., in tetrapyrrole biosynthesis, plastid transcription and plastid translation. Thus chloroplasts utilize an intricate and versatile redox regulatory network for intraorganellar and retrograde communication.

  14. Neutron-gamma irradiation and protein thiols: development of a protein thiol evaluation micro-method and application to irradiated baboons; Irradiation neutron-gamma et groupements thiols proteiques: developpement d`une micromethode d`evaluation des thiols proteiques et application au babouin irradie

    Chancerelle, Y.; Lafond, J.L.; Della-Maura, L.; Faure, P.; Mathieu, J.; Costa, P.; Mestries, J.C.; Kergonou, J.F.


    The essential non-protein sulfhydryl compound implicated in cellular radioprotection is glutathione. Protein thiols seem to be also involved in this protection and might be scavengers for free radical injury. We developed an analytical procedure for protein thiols measurement and we applied this method in neutron-gamma irradiated baboons. Our results demonstrated the reliability and sensitivity of the procedure. They also a drastic decrease of in vivo protein thiols after irradiation. (author). 5 refs.

  15. Multi-chamber and multi-layer thiol-ene microchip for cell culture

    Tan, H. Y.; Hemmingsen, Mette; Lafleur, Josiane P.


    We present a multi-layer and multi-chamber microfluidic chip fabricated using two different thiol-ene mixtures. Sandwiched between the thiol-ene chip layers is a commercially available membrane whose morphology has been altered with coatings of thiol-ene mixtures. Experiments have been conducted ...... with the microchip and shown that the fabricated microchip is suitable for long term cell culture....

  16. Methods for the determination and quantification of the reactive thiol proteome

    Hill, Bradford G.; Reily, Colin; Oh, Joo-Yeun; Johnson, Michelle S.; Landar, Aimee


    Protein thiol modifications occur under both physiological and pathological conditions and have been shown to contribute to changes in protein structure, function, and redox signaling. The majority of protein thiol modifications occur on cysteine residues that have a low pKa; these nucleophilic proteins comprise the “reactive thiol proteome.” The most reactive members of this proteome are typically low abundance proteins. Therefore, sensitive and quantitative methods are needed to detect and ...

  17. Integration between anticipatory blocking and redox signaling by the peroxiredoxin/thioredoxin/thioredoxin-reductase system.

    Selvaggio, Gianluca; Coelho, Pedro M B M; Salvador, Armindo


    Cells are occasionally exposed to high H2O2 concentrations, often preceding exposure to other electrophylic compounds. Both H2O2 and these compounds can irreversibly modify protein thiols, with deleterious consequences. Induction of enzymatic defenses against those agents is too slow to avoid significant damage. Cells may solve this conundrum by reversibly "blocking" the thiols once H2O2 concentrations begin to increase. We term this mechanism "anticipatory blocking" because it acts in anticipation of irreversible damage upon detection of early signs of stress. Here we examine the design requirements for the Peroxiredoxin/Thioredoxin/Thioredoxin-Reductase/Protein-Dithiol System (PTTRDS) to effectively integrate H2O2 signaling and anticipatory blocking of protein dithiols as disulfides, and we compared them to the designs found in cells. To that effect, we developed a minimal model of the PTTRDS, and we defined a set of quantitative performance criteria that embody the requirements for (a) efficient scavenging capacity, (b) low NADPH consumption, (c) effective signal propagation, and (d) effective anticipatory blocking. We then sought the design principles (relationships among rate constants and species concentrations) that warrant fulfillment of all these criteria. Experimental data indicates that the design of the PTTRDS in human erythrocytes fulfills these principles and thus accomplishes effective integration between anticipatory blocking, antioxidant protection and redox signaling. A more general analysis suggests that the same principles hold in a wide variety of cell types and organisms. We acknowledge grants PEst-C/SAU/LA0001/2013-2014, PEst-OE/QUI/UI0612/2013, FCOMP-01-0124-FEDER-020978 (PTDC/QUI-BIQ/119657/2010) financed by FEDER through the "Programa Operacional Factores de Competitividade, COMPETE" and by national funds through "FCT, Fundação para a Ciência e a Tecnologia".

  18. Distributions of dissolved and particulate biogenic thiols in the subartic Pacific Ocean

    Dupont, Christopher L.; Moffett, James. W.; Bidigare, Robert R.; Ahner, Beth A.


    Dissolved and particulate concentrations of the biogenic thiols cysteine (Cys), arginine-cysteine (Arg-Cys), glutamine-cysteine (Gln-Cys), γ-glutamate-cysteine ( γ-Glu-Cys) and glutathione (GSH) were measured in the subartic Pacific Ocean in the summer of 2003 using high performance liquid chromatography (HPLC) with precolumn derivatization as reported in previous work. In this study, a preconcentration protocol for the derivatized thiols was utilized to extend detection limits of dissolved thiols to picomolar levels. The measured concentrations of particulate and dissolved thiols were uncoupled, with distinctive depth profiles and large differences in the particulate to dissolved ratios between individual compounds. Glutathione was the most abundant particulate thiol whereas the most abundant dissolved thiol was γ-Glu-Cys, with concentrations as high as 15 nM. Given the relatively small pool of intracellular γ-Glu-Cys and the very low dissolved concentrations of GSH, we hypothesize that glutathione released from cells is rapidly converted to the potentially degradation resistant γ-Glu-Cys outside the cell. The relatively high concentrations of other dissolved thiols compared to particulate concentrations implies both biological exudation and slow degradation rates. Some thiols appear to vary with changes in nutrient availability but this effect is difficult to decouple from changes in community structure inferred from pigment analyses. Dissolved thiol concentrations also exceed typical metal concentrations in the subartic Pacific, supporting previous arguments that they may be important in metal speciation.

  19. Chemical interaction between polyphenols and a cysteinyl thiol under radical oxidation conditions.

    Fujimoto, Aya; Masuda, Toshiya


    Chemical interaction between polyphenols and thiols was investigated under radical oxidation conditions using a model cysteinyl thiol derivative, N-benzoylcysteine methyl ester. The radical oxidation was carried out with a stoichiometric amount of 2,2-diphenyl-1-picrylhydrazyl (DPPH), and the decreases in the amounts of polyphenols and the thiol were measured by HPLC analysis. Cross-coupling products between various polyphenols and the thiol were examined by LC-MS in reactions that showed decreases in both the polyphenols and the thiol. The LC-MS results indicated that three phenolic acid esters (methyl caffeate, methyl dihydrocaffeate, and methyl protocatechuate) and six flavonoids (kaempferol, myricetin, luteolin, morin, taxifolin, and catechin) gave corresponding thiol adducts, whereas three polyphenols (methyl ferulate, methyl sinapate, and quercetin) gave only dimers or simple oxidation products without thiol substituents. Thiol adducts of the structurally related compounds methyl caffeate and methyl dihydrocaffeate were isolated, and their chemical structures were determined by NMR analysis. The mechanism for the thiol addition was discussed on the basis of the structures of the products.

  20. Transsulfuration pathway thiols and methylated arginines: the Hunter Community Study.

    Arduino A Mangoni

    Full Text Available BACKGROUND: Serum homocysteine, when studied singly, has been reported to be positively associated both with the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine [ADMA, via inhibition of dimethylarginine dimethylaminohydrolase (DDAH activity] and with symmetric dimethylarginine (SDMA. We investigated combined associations between transsulfuration pathway thiols, including homocysteine, and serum ADMA and SDMA concentrations at population level. METHODS: Data on clinical and demographic characteristics, medication exposure, C-reactive protein, serum ADMA and SDMA (LC-MS/MS, and thiols (homocysteine, cysteine, taurine, glutamylcysteine, total glutathione, and cysteinylglycine; capillary electrophoresis were collected from a sample of the Hunter Community Study on human ageing [n = 498, median age (IQR = 64 (60-70 years]. RESULTS: REGRESSION ANALYSIS SHOWED THAT: a age (P = 0.001, gender (P = 0.03, lower estimated glomerular filtration rate (eGFR, P = 0.08, body mass index (P = 0.008, treatment with beta-blockers (P = 0.03, homocysteine (P = 0.02, and glutamylcysteine (P = 0.003 were independently associated with higher ADMA concentrations; and b age (P = 0.001, absence of diabetes (P = 0.001, lower body mass index (P = 0.01, lower eGFR (P<0.001, cysteine (P = 0.007, and glutamylcysteine (P < 0.001 were independently associated with higher SDMA concentrations. No significant associations were observed between methylated arginines and either glutathione or taurine concentrations. CONCLUSIONS: After adjusting for clinical, demographic, biochemical, and pharmacological confounders the combined assessment of transsulfuration pathway thiols shows that glutamylcysteine has the strongest and positive independent associations with ADMA and SDMA. Whether this reflects a direct effect of glutamylcysteine on DDAH activity (for ADMA and/or cationic amino acid transport requires further investigations.

  1. Regulative roles of glutathione reductase and four glutaredoxins in glutathione redox, antioxidant activity, and iron homeostasis of Beauveria bassiana.

    Zhang, Long-Bin; Tang, Li; Ying, Sheng-Hua; Feng, Ming-Guang


    Multiple glutaredoxins (Grx) and glutathione reductase (Glr) are vital for the thiol-disulfide redox system in budding yeast but generally unexplored in filamentous fungi. Here we characterized the Beauveria bassiana redox system comprising dithiol Grx1, monothiol Grx2-4, Grx-like Grx5, and Glr orthologue. Each grx or glr deletion was compensated by increased transcripts of some other grx genes in normal cultures. Particularly, grx3 compensated the absence of grx1, grx2, grx5, or glr under oxidative stress while its absence was compensated only by undeletable grx4 under normal conditions but by most of other undeleted grx and glr genes in response to menadione. Consequently, the redox state was disturbed in Δglr more than in Δgrx3 but not in Δgrx1/2/5. Superoxide dismutases were more active in normal Δgrx1-3 cultures but less in Δgrx5 or Δglr response to menadione. Total catalase activity increased differentially in all the mutant cultures stressed with or without H2O2 while total peroxidase activity decreased more in the normal or H2O2-stressed culture of Δglr than of Δgrx3. Among the mutants, Δgrx3 showed slightly increased sensitivity to menadione or H2O2; Δglr exhibited greater sensitivity to thiol-oxidizing diamide than thiol-reducing 1-chloro-2,4-dinitrobenzene as well as increased sensitivity to the two oxidants. Intriguingly, all the mutants grew slower in a Fe(3+)-inclusive medium perhaps due to elevated transcripts of two Fe(3+) transporter genes. More or fewer phenotypes linked with biocontrol potential were altered in four deletion mutants excluding Δgrx5. All the changes were restored by targeted gene complementation. Overall, Grx3 played more critical role than other Grx homologues in the Glr-dependent redox system of the fungal entomopathogen.

  2. Discovery of pinoresinol reductase genes in sphingomonads.

    Fukuhara, Y; Kamimura, N; Nakajima, M; Hishiyama, S; Hara, H; Kasai, D; Tsuji, Y; Narita-Yamada, S; Nakamura, S; Katano, Y; Fujita, N; Katayama, Y; Fukuda, M; Kajita, S; Masai, E


    Bacterial genes for the degradation of major dilignols produced in lignifying xylem are expected to be useful tools for the structural modification of lignin in plants. For this purpose, we isolated pinZ involved in the conversion of pinoresinol from Sphingobium sp. strain SYK-6. pinZ showed 43-77% identity at amino acid level with bacterial NmrA-like proteins of unknown function, a subgroup of atypical short chain dehydrogenases/reductases, but revealed only 15-21% identity with plant pinoresinol/lariciresinol reductases. PinZ completely converted racemic pinoresinol to lariciresinol, showing a specific activity of 46±3 U/mg in the presence of NADPH at 30°C. In contrast, the activity for lariciresinol was negligible. This substrate preference is similar to a pinoresinol reductase, AtPrR1, of Arabidopsis thaliana; however, the specific activity of PinZ toward (±)-pinoresinol was significantly higher than that of AtPrR1. The role of pinZ and a pinZ ortholog of Novosphingobium aromaticivorans DSM 12444 were also characterized.

  3. A mycothiol synthase mutant of Mycobacterium smegmatis produces novel thiols and has an altered thiol redox status.

    Newton, Gerald L; Ta, Philong; Fahey, Robert C


    Mycobacteria and other actinomycetes do not produce glutathione but make mycothiol (MSH; AcCys-GlcN-Ins) that has functions similar to those of glutathione and is essential for growth of Mycobacterium tuberculosis. Mycothiol synthase (MshD) catalyzes N acetylation of Cys-GlcN-Ins to produce MSH in Mycobacterium smegmatis mc2155, and Cys-GlcN-Ins is maintained at a low level. The mycothiol synthase mutant, the mshD::Tn5 mutant, produces high levels of Cys-GlcN-Ins along with two novel thiols, N-formyl-Cys-GlcN-Ins and N-succinyl-Cys-GlcN-Ins, and a small amount of MSH. The nonenzymatic reaction of acyl-coenzyme A (CoA) with Cys-GlcN-Ins to produce acyl-Cys-GlcN-Ins is a facile reaction under physiologic conditions, with succinyl-CoA being an order of magnitude more reactive than acetyl-CoA. The uncatalyzed reaction rates are adequate to account for the observed production of N-succinyl-Cys-GlcN-Ins and MSH under physiologic conditions. It was shown that the N-acyl-Cys-GlcN-Ins compounds are maintained in a substantially reduced state in the mutant but that Cys-GlcN-Ins exists in disulfide forms at 5 to 40% at different stages of growth. MSH was able to facilitate reduction of N-succinyl-Cys-GlcN-Ins disulfide through thiol-disulfide exchange, but N-formyl-Cys-GlcN-Ins was ineffective. The oxidized state of Cys-GlcN-Ins in cells appears to result from a high susceptibility to autoxidation and a low capacity of the cell to reduce its disulfide forms. The mutant exhibited no enhanced sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, or cumene hydroperoxide relative to the parent strain, suggesting that the most abundant thiol, N-formyl-Cys-GlcN-Ins, functions as a substitute for MSH.

  4. Molecular modeling, structural analysis and identification of ligand binding sites of trypanothione reductase from Leishmania mexicana

    Ozal Mutlu


    Full Text Available Background & objectives: Trypanothione reductase (TR is a member of FAD-dependent NADPH oxidoreductase protein family and it is a key enzyme which connects the NADPH and the thiol-based redox system. Inhibition studies indicate that TR is an essential enzyme for parasite survival. Therefore, it is an attractive target enzyme for novel drug candidates. There is no structural model for TR of Leishmania mexicana (LmTR in the protein databases. In this work, 3D structure of TR from L. mexicana was identified by template-based in silico homology modeling method, resultant model was validated, structurally analyzed and possible ligand binding pockets were identified. Methods: For computational molecular modeling study, firstly, template was identified by BLAST search against PDB database. Multiple alignments were achieved by ClustalW2. Molecular modeling of LmTR was done and possible drug targeting sites were identified. Refinement of the model was done by performing local energy minimization for backbone, hydrogen and side chains. Model was validated by web-based servers. Results: A reliable 3D model for TR from L. mexicana was modeled by using L. infantum trypanothione reductase (LiTR as a template. RMSD results according to C-alpha, visible atoms and backbone were 0.809 Å, 0.732 Å and 0.728 Å respectively. Ramachandran plot indicates that model shows an acceptable stereochemistry. Conclusion: Modeled structure of LmTR shows high similarity with LiTR based on overall structural features like domains and folding patterns. Predicted structure will provide a source for the further docking studies of various peptide-based inhibitors.

  5. The protonation state of thiols in self-assembled monolayers on roughened Ag/Au surfaces and nanoparticles.

    Bandyopadhyay, Sabyasachi; Chattopadhyay, Samir; Dey, Abhishek


    The protonation state of thiols in self-assembled monolayers (SAMs) on Ag and Au surfaces and nanoparticles (NPs) has been an issue of contestation. It has been recently demonstrated that deuterating the thiol proton produces ostentatious changes in the Raman spectra of thiols and can be used to detect the presence of the thiol functional group. Surface enhanced Raman spectroscopy (SERS) of H/D substituted aliphatic thiols on Ag surfaces clearly shows the presence of S-H vibration between 2150-2200 cm(-1) which shifts by 400 cm(-1) upon deuteration and a simultaneous >20 cm(-1) shift in the C-S vibration of thiol deuteration. Large shifts (>15 cm(-1)) in the C-S vibration are also observed for alkyl thiol SAMs on Au surfaces. Alternatively, neither the S-H vibration nor the H/D isotope effect on the C-S vibration is observed for alkyl thiol SAMs on Ag/Au NPs. XPS data on Ag/Au surfaces bearing aliphatic thiol SAMs show the presence of both protonated and deprotonated thiols while on Ag/Au NPs only deprotonated thiols are detected. These data suggest that aliphatic thiol SAMs on Au/Ag surfaces are partially protonated whereas they are totally deprotonated on Au/Ag NPs. Aromatic PhSH SAMs on Ag/Au surfaces and Ag/Au NPs do not show these vibrations or H/D shifts as well indicating that the thiols are deprotonated at these interfaces.

  6. Novel Thiol-Ene Hybrid Coating for Metal Protection

    Mona Taghavikish


    Full Text Available A novel hybrid anticorrosion coating with dual network of inorganic (Si–O–Si and organic bonds (C–S–C was prepared on metal through an in situ sol-gel and thiol-ene click reaction. This novel interfacial thin film coating incorporates (3-mercaptopropyl trimethoxysilane (MPTS and 1,4-di(vinylimidazolium butane bisbromide based polymerizable ionic liquid (PIL to form a thiol-ene based photo-polymerized film, which on subsequent sol-gel reaction forms a thin hybrid interfacial layer on metal surface. On top of this PIL hybrid film, a self-assembled nanophase particle (SNAP coating was employed to prepare a multilayer thin film coating for better corrosion protection and barrier performance. The novel PIL hybrid film was characterised for structure and properties using Fourier transform infrared spectroscopy (FTIR, differential scanning calorimetry (DSC, and thermogravimetric analysis (TGA. The corrosion protection performance of the multilayer coating was examined using potentiodynamic polarization and electrochemical impedance spectroscopy (EIS. The results reveal that this novel double layer coating on metal offers excellent protection against corrosion and has remarkably improved the barrier effect of the coating.

  7. Electrodeposition of gold templated by patterned thiol monolayers

    She, Zhe; Di Falco, Andrea; Hähner, Georg; Buck, Manfred


    The electrochemical deposition of Au onto Au substrates modified by self-assembled monolayers (SAMs) was studied by linear sweep voltammetry (LSV), atomic force microscopy (AFM) and scanning electron microscopy (SEM). Patterned SAMs exhibiting electrochemical contrast were prepared by two different methods. One used microcontact printing (μCP) to generate a binary SAM of ω-(4‧-methyl-biphenyl-4-yl)-propane thiol (CH3-C6H4-C6H4-(CH2)3-SH, MBP3) and octadecane thiol (CH3(CH2)17SH, ODT). Templated by the SAM, a gold microelectrode structure was electrodeposited featuring a line 15 μm wide and 3 mm long. After transfer to an epoxy substrate the structure proved to be electrically conductive across the full length. The other patterning method applied electron beam lithography (EBL) where electrochemical contrast was achieved by crosslinking molecules in a single component SAM of MBP3. An electron dose above 250 mC/cm2 results in a high deposition contrast. The choice of parameters for the deposition/lift-off process is found to be more critical for Au compared to Cu studied previously. The origin of the differences and implications for nanoscale patterning are discussed.

  8. Purification and characterization of a pineapple crown leaf thiol protease.

    Singh, L Rupachandra; Devi, Th Premila; Devi, S Kunjeshwori


    A thiol protease was isolated and purified from the crown leaf of pineapple, Ananas comosus (L.) Merr. cv. Queen, by an immunoaffinity procedure. After the purification to electrophoretic homogeneity, the enzyme was characterized with respect to some of its physico-chemical and kinetic properties. The molecular weight of the protease (22.4-22.9 kDa), Km (97 microM) and kcat (8.8 s(-1)) for its esterolytic cleavage of the synthetic protease substrate N(alpha)-CBZ-L-lysine p-nitrophenyl ester, the concentration of its thiol activator L-cysteine required for half maximal activation A0.5 (9.9 microM), optimum pH (6.5) for its proteolytic action on azocasein, T(1/2) (60 degrees C) for inactivation by heating the enzyme (35.5 microg protein/mL) in citrate buffer pH 6.0 for 15 min, and SH-group content (0.98 mol/mol enzyme) were determined. Most of these physicochemical and kinetic properties were found to be similar to those of the already well-characterized stem bromelain (EC Thus, the immunoaffinity purified crown leaf protease appeared to be closely related to stem bromelain.

  9. Activity assays of mammalian thioredoxin and thioredoxin reductase: fluorescent disulfide substrates, mechanisms, and use with tissue samples.

    Montano, Sergio J; Lu, Jun; Gustafsson, Tomas N; Holmgren, Arne


    Thioredoxin (Trx) is a protein disulfide reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin-glutathione disulfide (Di-E-GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC-insulin), which displayed higher fluorescence on disulfide reduction. Di-E-GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC-insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.

  10. Purification and characterization of Taenia crassiceps cysticerci thioredoxin: insight into thioredoxin-glutathione-reductase (TGR) substrate recognition.

    Martínez-González, J J; Guevara-Flores, A; Rendón, J L; Sosa-Peinado, A; Del Arenal Mena, I P


    Thioredoxin (Trx) is an oxidoreductase central to redox homeostasis in cells and is involved in the regulation of protein activity through thiol/disulfide exchanges. Based on these facts, our goal was to purify and characterize cytosolic thioredoxin from Taenia crassiceps cysticerci, as well as to study its behavior as a substrate of thioredoxin-glutathione reductase (TGR). The enzyme was purified >133-fold with a total yield of 9.7%. A molecular mass of 11.7kDa and a pI of 4.84 were measured. Native electrophoresis was used to identify the oxidized and reduced forms of the monomer as well as the presence of a homodimer. In addition to the catalytic site cysteines, cysticerci thioredoxin contains Cys28 and Cys65 residues conserved in previously sequenced cestode thioredoxins. The following kinetic parameters were obtained for the substrate of TGR: a Km of 3.1μM, a kcat of 10s(-1) and a catalytic efficiency of 3.2×10(6)M(-1)s(-1). The negative patch around the α3-helix of Trx is involved in the interaction with TGR and suggests variable specificity and catalytic efficiency of the reductase toward thioredoxins of different origins.

  11. The first peripherally masked thiol dendrimers: a facile and highly efficient functionalization strategy of polyester dendrimers via one-pot xanthate deprotection/thiol-acrylate Michael addition reactions.

    Auty, Sam E R; Andrén, Oliver; Malkoch, Michael; Rannard, Steven P


    Introducing multiple reactive functional groups at the periphery of dendrimer materials presents considerable challenges if the functionality is able to self-react. An efficient and facile approach to introducing masked thiols at the surface of polyester dendrimers is presented. One-pot, deprotection/thiol-acrylate Michael addition from the xanthate-functional dendritic substrates (generation zero to two) has been achieved for the first time, with high efficiency demonstrated using three acrylates of varying chemistry and avoiding disulfide formation.


    BUWALDA, F; DE KOK, LJ; Stulen, I.


    Exposure of crop plants to H2S resulted in an increase in thiol level and a change in the composition of the thiol pool. Non-leguminous species accumulated cysteine and glutathione in the light, whereas in the dark, substantial amounts of gamma-glutamyl-cysteine were also detected. In leguminous spe

  13. Rapid photochemical surface patterning of proteins in thiol-ene based microfluidic devices

    Lafleur, Josiane P.; Kwapiszewski, Radoslaw; Jensen, Thomas Glasdam;


    ” and “ene” monomers present in the microfluidic chip bulk material provides a simple and efficient way of tuning the chip’s surface chemistry. Here, thiol-ene chips displaying an excess of functional thiol groups at their surfaces are functionalized with biotin and streptavidin in a controlled fashion using...

  14. A fluorescent probe which allows highly specific thiol labeling at low pH

    Nielsen, Jonas W.; Jensen, Kristine Steen; Hansen, Rosa E.;


    Determination of the thiol-disulfide status in biological systems is challenging as redox pools are easily perturbed during sample preparation. This is particularly pertinent under neutral to mildly alkaline conditions typically required for alkylation of thiols. Here we describe the synthesis...

  15. Resonance Light-Scattering Spectroscopy Study on Interaction between Gold Colloid and Thiol Containing Pharmaceutical

    Liu Xiao-ling; Cai Ru-xiu; Yuan Hong


    In this paper, we used resonance light-scattering (RLS) spectroscopy to study the interaction between thiol-containing pharmaceutical and gold colloid. And for the first time, we proposed that this highly sensitive, gold colloid-based assay using RLS technique may have potential application in detecting thiol-containing substances.




    Ephedrine and pseudoephedrine are converted by means of a Mitsunobu reaction to respectively trans- and cis-aziridines, which can be ring-opened at the benzylic center with inversion of configuration by thiols and thiol acids. The trans-aziridine from ephedrine reacts also with H2S in acetone under

  17. Spectrophotometric Determination of Phenolic Antioxidants in the Presence of Thiols and Proteins

    Aslı Neslihan Avan


    Full Text Available Development of easy, practical, and low-cost spectrophotometric methods is required for the selective determination of phenolic antioxidants in the presence of other similar substances. As electron transfer (ET-based total antioxidant capacity (TAC assays generally measure the reducing ability of antioxidant compounds, thiols and phenols cannot be differentiated since they are both responsive to the probe reagent. In this study, three of the most common TAC determination methods, namely cupric ion reducing antioxidant capacity (CUPRAC, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt/trolox equivalent antioxidant capacity (ABTS/TEAC, and ferric reducing antioxidant power (FRAP, were tested for the assay of phenolics in the presence of selected thiol and protein compounds. Although the FRAP method is almost non-responsive to thiol compounds individually, surprising overoxidations with large positive deviations from additivity were observed when using this method for (phenols + thiols mixtures. Among the tested TAC methods, CUPRAC gave the most additive results for all studied (phenol + thiol and (phenol + protein mixtures with minimal relative error. As ABTS/TEAC and FRAP methods gave small and large deviations, respectively, from additivity of absorbances arising from these components in mixtures, mercury(II compounds were added to stabilize the thiol components in the form of Hg(II-thiol complexes so as to enable selective spectrophotometric determination of phenolic components. This error compensation was most efficient for the FRAP method in testing (thiols + phenols mixtures.

  18. Isothiazolones; thiol-reactive inhibitors of cysteine protease cathepsin B and histone acetyltransferase PCAF

    Wisastra, Rosalina; Ghizzoni, Massimo; Maarsingh, Harm; Minnaard, Adriaan J.; Haisma, Hidde J.; Dekker, Frank J.


    Isothiazolones and 5-chloroisothiazolones react chemoselectively with thiols by cleavage of the weak nitrogen-sulfur bond to form disulfides. They show selectivity for inhibition of the thiol-dependent cysteine protease cathepsin B and the histone acetyltransferase p300/CBP associated factor (PCAF)

  19. Spectrophotometric Determination of Phenolic Antioxidants in the Presence of Thiols and Proteins

    Avan, Aslı Neslihan; Demirci Çekiç, Sema; Uzunboy, Seda; Apak, Reşat


    Development of easy, practical, and low-cost spectrophotometric methods is required for the selective determination of phenolic antioxidants in the presence of other similar substances. As electron transfer (ET)-based total antioxidant capacity (TAC) assays generally measure the reducing ability of antioxidant compounds, thiols and phenols cannot be differentiated since they are both responsive to the probe reagent. In this study, three of the most common TAC determination methods, namely cupric ion reducing antioxidant capacity (CUPRAC), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt/trolox equivalent antioxidant capacity (ABTS/TEAC), and ferric reducing antioxidant power (FRAP), were tested for the assay of phenolics in the presence of selected thiol and protein compounds. Although the FRAP method is almost non-responsive to thiol compounds individually, surprising overoxidations with large positive deviations from additivity were observed when using this method for (phenols + thiols) mixtures. Among the tested TAC methods, CUPRAC gave the most additive results for all studied (phenol + thiol) and (phenol + protein) mixtures with minimal relative error. As ABTS/TEAC and FRAP methods gave small and large deviations, respectively, from additivity of absorbances arising from these components in mixtures, mercury(II) compounds were added to stabilize the thiol components in the form of Hg(II)-thiol complexes so as to enable selective spectrophotometric determination of phenolic components. This error compensation was most efficient for the FRAP method in testing (thiols + phenols) mixtures. PMID:27529232


    Upadhyaya, Bijaya; Tian, Xiaoli; Wu, Hongli; Lou, Marjorie F.


    Glutaredoxin2 (Grx2) is a mitochondrial isozyme of the cytosolic glutaredoxin1 (thioltransferase or TTase). Both belong to the large oxidoreductase family and play an important role in maintaining thiol/disulfide redox homeostasis in the cells. Grx2 is recently found in the lens where its activities of disulfide reductase and peroxidase, similar to TTase, can protect the lens against oxidative stress. Since other eye tissues are also highly sensitive to oxidative stress, and TTase’s distribution in the eye is known, we focused on this study by investigating the Grx2 distribution in the ocular tissues in comparison to the lens. Fresh porcine eyes were dissected into cornea, iris, ciliary body, the lens, vitreous humor, retina, and optic nerve. Each tissue (pooled from three eyes) was homogenized and processed for mitochondrial isolation. The mitochondrial fraction was analyzed for Grx2 protein using Western blotting with anti-Grx2 antibody, and Grx2 activity using the published procedure. The eye tissues were also measured for Grx2 mRNA expression by RT-PCR with GAPDH as the control. Grx2-rich mouse liver and purified recombinant mouse Grx2 were used as positive controls for the above analyses. It was found that Grx2 was present in all the tested ocular tissues, except vitreous humor. In comparison with the mouse liver, the protein levels of Grx2 in porcine ciliary body and the lens were 27-fold and 0.75-fold, respectively. Comparing to the lens, Grx2 protein was highest in the ciliary body (13.5-fold), followed by retina (9.2-fold), iris and optic nerve (2-fold), and cornea (1.2-fold). Enzyme activity assays showed that the retina had the highest Grx2 specific activity (3.9 mU/mg protein), followed by ciliary body (3.1 mU/mg), the lens (0.58 mU/mg), and optic nerve (0.32 mU/mg). Grx2 gene expression in these ocular tissues was further confirmed by RT-PCR analysis. Grx2 mRNA expression showed the highest in ciliary body, followed by retina, optic nerve, cornea

  1. Ester-free Thiol-ene Dental Restoratives – Part A: Resin Development

    Podgórski, Maciej; Becka, Eftalda; Claudino, Mauro; Flores, Alexander; Shah, Parag K.; Stansbury, Jeffrey W.; Bowman, Christopher N.


    Objectives To detail the development of ester-free thiol-ene dental resins with enhanced mechanical performance, limited potential for water uptake/leachables/degradation and low polymerization shrinkage stress. Methods Thiol-terminated oligomers were prepared via a thiol-Michael reaction and a bulky tetra-allyl monomer containing urethane linkages was synthesized. The experimental oligomers and/or monomers were photopolymerized using visible light activation. Several thiol-ene formulations were investigated and their performance ranked by comparisons of the thermo-mechanical properties, polymerization shrinkage stress, water sorption/solubility, and reactivity with respect to a control comprising a conventional BisGMA/TEGDMA dental resin. Results The ester-free thiol-ene formulations had significantly lower viscosities, water sorption and solubility than the BisGMA/TEGDMA control. Depending on the resin, the limiting functional conversions were equivalent to or greater than that of BisGMA/TEGDMA. At comparable conversions, lower shrinkage stress values were achieved by the thiol-ene systems. The polymerization shrinkage stress was dramatically reduced when the tetra-allyl monomer was used as the ene in ester-free thiol-ene mixtures. Although exhibiting lower Young’s modulus, flexural strength, and glass transition temperatures, the toughness values associated with thiol-ene resins were greater than that of the BisGMA/TEGDMA control. In addition, the thiol-ene polymerization resulted in highly uniform polymer networks as indicated by the narrow tan delta peak widths. Significance Employing the developed thiol-ene resins in dental composites will reduce shrinkage stress and moisture absorption and form tougher materials. Furthermore, their low viscosities are expected to enable higher loadings of functionalized micro/nano-scale filler particles relevant for practical dental systems. PMID:26360013

  2. Two mutations of dihydropteridine reductase deficiency.

    Ponzone, A; Guardamagna, O; Ferraris, S; Bracco, G; Niederwieser, A; Cotton, R G


    Two patients with dihydropteridine reductase (DHPR) deficiency, in one case due to the absence of any enzyme protein (DHPR- cross reactive material (CRM)-) and in the other case due to the production of a mutant type devoid of catalytic activity (DHPR- CRM+) were examined. This latter form of malignant phenylketonuria, whose relative frequency seems to be higher in the Italian population, possibly has a worse prognosis. The earlier onset and the greater severity of clinical symptoms are associated with a more pronounced hydroxylation defect, as shown by higher degree of neonatal hyperphenylalaninaemia, unresponsiveness to an oral tetrahydrobiopterin load, lower concentrations of neurotransmitter metabolites, and reduced tyrosine production after an oral phenylalanine load.

  3. Functional thioredoxin reductase from pathogenic and free-living Leptospira spp.

    Sasoni, Natalia; Iglesias, Alberto A; Guerrero, Sergio A; Arias, Diego G


    Low molecular mass thiols and antioxidant enzymes have essential functions to detoxify reactive oxygen and nitrogen species maintaining cellular redox balance. The metabolic pathways for redox homeostasis in pathogenic (Leptospira interrogans) and free-living (Leptospira biflexa) leptospires species were not functionally characterized. We performed biochemical studies on recombinantly produced proteins to in depth analyze kinetic and structural properties of thioredoxin reductase (LinTrxR) and thioredoxin (LinTrx) from L. interrogans, and two TrxRs (LbiTrxR1 and LbiTrxR2) from L. biflexa. All the TrxRs were characterized as homodimeric flavoproteins, with LinTrxR and LbiTrxR1 catalyzing the NADPH dependent reduction of LinTrx and DTNB. The thioredoxin system from L. interrogans was able to use glutathione disulfide, lipoamide disulfide, cystine and bis-γ-glutamyl cysteine and homologous peroxiredoxin as substrates. Classic TrxR activity of LinTrxR2 had not been evidenced in vitro, but recombinant Escherichia coli cells overexpressing LbiTrxR2 showed high tolerance to oxidative stress. The enzymatic systems herein characterized could play a key role for the maintenance of redox homeostasis and the function of defense mechanisms against reactive oxidant species in Leptospira spp. Our results contribute to the general knowledge about redox biochemistry in these bacteria, positioning TrxR as a critical molecular target for the development of new anti-leptospiral drugs.

  4. HPLC analysis of nonprotein thiols in planktonic diatoms: Pool size, redox state and response to copper and cadmium exposure

    Rijstenbil, J.W.; Wijnholds, J.A.


    A sensitive method was developed to analyze low molecular weight thiols involved in metal homeostasis and detoxification in phytoplankton. The aims of this study were to (1) separate and measure all relevant thiols in a single HPLC run; (2) measure redox states of the thiols and (3) identify specifi

  5. Bacillithiol: a key protective thiol in Staphylococcus aureus.

    Perera, Varahenage R; Newton, Gerald L; Pogliano, Kit


    Bacillithiol is a low-molecular-weight thiol analogous to glutathione and is found in several Firmicutes, including Staphylococcus aureus. Since its discovery in 2009, bacillithiol has been a topic of interest because it has been found to contribute to resistance during oxidative stress and detoxification of electrophiles, such as the antibiotic fosfomycin, in S. aureus. The rapid increase in resistance of methicillin-resistant Staphylococcus aureus (MRSA) to available therapeutic agents is a great health concern, and many research efforts are focused on identifying new drugs and targets to combat this organism. This review describes the discovery of bacillithiol, studies that have elucidated the physiological roles of this molecule in S. aureus and other Bacilli, and the contribution of bacillithiol to S. aureus fitness during pathogenesis. Additionally, the bacillithiol biosynthesis pathway is evaluated as a novel drug target that can be utilized in combination with existing therapies to treat S. aureus infections.

  6. Biliverdin Reductase: a Target for Cancer Therapy?

    Peter eGibbs


    Full Text Available Biliverdin reductase (BVR is a multifunctional protein that is the primary source of the potent antioxidant, bilirubin. BVR regulates activities/functions in the insulin/IGF-1/IRK/PI3K/MAPK pathways. Activation of certain kinases in these pathways is/are hallmark(s of cancerous cells. The protein is a scaffold/bridge and intracellular transporter of kinases that regulate growth and proliferation of cells, including PKCs, ERK and Akt, and their targets including NF-κB, Elk1, HO-1 and iNOS. The scaffold and transport functions enable activated BVR to relocate from the cytosol to the nucleus or to the plasma membrane, depending on the activating stimulus. This enables the reductase to function in diverse signaling pathways. And, its expression at the transcript and protein levels are increased in human tumors and the infiltrating T-cells, monocytes and circulating lymphocytes, as well as the circulating and infiltrating macrophages. These functions suggest that the cytoprotective role of BVR may be permissive for cancer/tumor growth. In this review, we summarize the recent developments that define the pro-growth activities of BVR, particularly with respect to its input into the MAPK signaling pathway and present evidence that BVR-based peptides inhibit activation of protein kinases, including MEK, PKCδ and ERK as well as downstream targets including Elk1 and iNOS, and thus offers a credible novel approach to reduce cancer cell proliferation.

  7. Iron-mediated effects on nitrate reductase in marine phytoplankton

    Timmermans, K.R.; Stolte, W.; Baar, H.J.W. de


    The potential activity of nitrate reductase was determined in uni-algal cultures in the laboratory and in natural marine phytoplankton assemblages. In the laboratory bioassays, distinct differences in nitrate reductase activity were observed in iron replete versus depleted cultures for Emiliania hux

  8. Thiol compounds from a free-living pathogenic opportunistic amoeba, Acanthamoeba polyphaga.

    Ondarza, Raúl N; Iturbe, Angélica; Hernández, Eva; Hurtado, Gerardo


    New bimane-reacting compounds from perchloric acid extracts have been detected by HPLC from Acanthamoeba polyphaga. The main compounds detected are cysteine, glutathione and other novel thiol compounds. All of these compounds must be thiols, since they disappear or decrease substantially when treated by N -ethylmaleimide prior to acetonitrile/bimane derivatization. Cysteine and glutathione increase in quantity when dithiothreitol reduction is applied to the fresh extract. This means that they are likely to be present in their oxidized and reduced form and indicates the possible presence of a corresponding thiol/disulphide enzymic system. There are other compounds that have a different behaviour, since although they can react with bimane, they do not disappear if treated previously by N -ethylmaleimide. This shows that they are not thiols but can react with bimane. The main thiol compounds found to be present, in both the parasite and the host lymphocyte cells, were cysteine and glutathione. We were unable to detect ovothiol A in Acanthamoeba but instead we found another thiol compound that could be structurally related to trypanothione. The new thiol compounds unique to this parasite and not present in lymphocytes will permit the study of disulphide-reducing enzymes as potential drug targets.

  9. Studies of Aqueous U(IV) Complexation under Thiol-rich Conditions

    Cha, Wansik; Cho, Hyeryun; Jung, Euo Chang [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)


    Organic thiol compounds and hydrogen sulfide (H{sub 2}S) are electron donors and metabolic products of sulfate reducing bacteria. In addition, they are among redox potential (Eh) determinants of groundwater systems due to their redox characteristics. The low values of acid dissociation constants for .SH (pK{sub a}, 7-9) compared to those of aliphatic or phenolic .OH, impart greater anionic and metal-binding properties to the molecules. Recently, we demonstrated that a thiol compound (i. e., thiosalicylate) enhances the solubility of U(VI) at higher pH levels (< ∼9). In this study, to have a better knowledge of the behaviors of U(IV) species under anaerobic conditions, the U(IV)-OH complex formation in the presence of thiol was examined using UV-Vis spectrophotometry and TRLFS (time-resolved laser-induced fluorescence spectroscopy). A TRLFS-based U(IV) quantification methodology developed earlier was applied to examine the effects of thiol species on the dissolution behaviors. Based on UV-Vis absorption monitoring, the presence of thiol does not result in a significant changes in the low-pH hydrolysis behaviors of U(IV). However, the concentration of U(IV) dissolved in bulk phase of aqueous solutions increased with the increase of thiol concentration. The formation of soluble thiol complexes or the stabilization of UO{sub 2} nanoparticles may explain the observed solubility increase.

  10. Redox regulation of sperm surface thiols modulates adhesion to the fallopian tube epithelium.

    Talevi, Riccardo; Zagami, Maria; Castaldo, Marianna; Gualtieri, Roberto


    Sperm that adhere to the fallopian tube epithelium are of superior quality and adhesion extends their fertile life. It has been postulated that periovulatory signals, as yet undefined, promote sperm release. In the in vitro studies described here, we examined the effects of several antioxidants, reportedly present within oviductal fluid, on the modulation of sperm-oviduct adhesion in bovine species. Results showed that 1) the cell-permeant thiols (penicillamine, beta mercaptoethanol, cysteine, and dithiotreitol), as well as the nonpermeant thiol, reduced glutathione, cause adhering spermatozoa to release from the epithelium; 2) thiol action is exerted on spermatozoa; and 3) oxidized glutathione, as well as the non-thiol antioxidants (dimethylthiourea, trolox, superoxide dismutase, and catalase) have no effect. Sperm surface sulfhydryls labeled with iodoacetamide fluorescein showed that spermatozoa devoid of sulfhydryls on the head surface adhered to the fallopian epithelium in vitro, whereas thiol-induced release increased the exposure of sulfhydryls on the sperm head surface. Finally, analysis of capacitation status demonstrated that uncapacitated spermatozoa adhered to the oviduct, and that thiol-induced release of spermatozoa was accompanied by capacitation. In conclusion, thiol-reducing agents in the oviductal fluid may modulate the redox status of sperm surface proteins, leading to the release of spermatozoa selected and stored through adhesion to the fallopian tube epithelium in the bovine species.

  11. 3-Methyl-2-butene-1-thiol: identification, analysis, occurrence and sensory role of an uncommon thiol in wine.

    San-Juan, Felipe; Cacho, Juan; Ferreira, Vicente; Escudero, Ana


    A highly uncommon odorant, 3-methyl-2-butene-1-thiol was detected by using Gas Chromatography-Olfactometry (GC-O) and unequivocally identified for the first time in wine. A purge and trap sampling technique which provides highly representative extracts for olfactometric analysis was used for the extraction of the volatile fraction of a Spanish red wine made from Prieto Picudo grapes. The identification of the odorant was achieved by multidimensional gas chromatography analysis of the same purge and trap extract. Mass spectrum and retention indices in both polar and non-polar columns allowed knowing unequivocally the identity. To obtain quantitative data a method was validated for the analysis of the compound at ng L(-1) level with acceptable precision. This powerful odorant presented an odor threshold in wine of 0.5-1 ng L(-1) and it has been detected in several Prieto Picudo wines at concentrations slightly above the odor threshold.

  12. Beer thiol-containing compounds and redox stability: kinetic study of 1-hydroxyethyl radical scavenging ability.

    de Almeida, Natália E C; Lund, Marianne N; Andersen, Mogens L; Cardoso, Daniel R


    The 1-hydroxyethyl radical is a central intermediate in oxidative reactions occurring in beer. The reactivity of thiol-containing compounds toward 1-hydroxyethyl radical was evaluated in beer model solutions using a competitive kinetic approach, employing the spin-trap 4-POBN as a probe and by using electron paramagnetic resonance to detect the generated 1-hydroxyethyl/4-POBN spin adduct. Thiol-containing compounds were highly reactive toward the 1-hydroxyethyl radical with apparent second-order rate constants close to the diffusion limit in water and ranging from 0.5 × 10⁹ L mol⁻¹ s⁻¹ for the His-Cys-Lys-Phe-Trp-Trp peptide to 6.1 × 10⁹ L mol⁻¹ s⁻¹ for the reduced lipid transfer protein 1 (LTP1) isolated from beer. The reactions gave rise to a moderate kinetic isotope effect (k(H)/k(D) = 2.3) suggesting that reduction of the 1-hydroxyethyl radical by thiol-containing compounds takes place by hydrogen atom abstraction from the RSH group rather than electron transfer. The content of reduced thiols in different beers was determined using a previously established method based on ThioGlo-1 as the thiol derivatization reagent and detection of the derivatized thiols by reverse-phase liquid chromatography coupled to a fluorescence detector. The total level of thiol in beer (oxidized and reduced) was determined after a reduction step employing 3,3',3″-phosphanetriyltripropanoic acid (TCEP) as the disulfide reductant. A good correlation among total protein and total thiol content in different beers was observed. The results suggest a similar ratio between reduced thiols and disulfides in all of the tested beers, which indicates a similar redox state.

  13. Quinoline-2-thiol Derivatives as Fluorescent Sensors for Metals, pH and HNO

    Naphtali A. O’Connor


    Full Text Available A tautomeric equilibrium exists for quinoline-2-thiol and quinoline-2(1H-thione. Quantum mechanical calculations predict the thione is the major tautomer and this is confirmed by the absorption spectra. The utility of quinolone-2-thiol/quinoline-2(1H-thione as a chromophore for developing fluorescent sensors is explored. No fluorescence is observed when excited at absorption maxima, however a fluorescence increase is observed when exposed to HNO, a molecule of import as a cardiovascular therapeutic. Alkylated quinoline-2-thiol derivatives are found to be fluorescent and show a reduction in fluorescence when exposed to metals and changes in pH.

  14. Readout fidelity of coaxial holographic digital data page recording in nanoparticle-(thiol-ene) polymer composites

    Nagaya, Kohta; Hata, Eiji; Tomita, Yasuo


    We report on an experimental investigation of nanoparticle-concentration and thiol-to-ene stoichiometric ratio dependences of symbol error rates (SERs) and signal-to-noise ratios (SNRs) of digital data pages recorded at a wavelength of 532 nm in thiol-ene based nanoparticle-polymer composite (NPC) films by using a coaxial holographic digital data storage method. We show that SERs and SNRs at the optimized material condition can be lower than 1 × 10-4 and higher than 10, respectively, without error correction coding. These results show the usefulness of thiol-ene based NPCs as coaxial holographic data storage media.

  15. Preparation and Preliminary Dielectric Characterization of Structured C60-Thiol-Ene Polymer Nanocomposites Assembled Using the Thiol-Ene Click Reaction

    Hanaa M. Ahmed


    Full Text Available Fullerene-containing materials have the ability to store and release electrical energy. Therefore, fullerenes may ultimately find use in high-voltage equipment devices or as super capacitors for high electric energy storage due to this ease of manipulating their excellent dielectric properties and their high volume resistivity. A series of structured fullerene (C60 polymer nanocomposites were assembled using the thiol-ene click reaction, between alkyl thiols and allyl functionalized C60 derivatives. The resulting high-density C60-urethane-thiol-ene (C60-Thiol-Ene networks possessed excellent mechanical properties. These novel networks were characterized using standard techniques, including infrared spectroscopy (FTIR, differential scanning calorimetry (DSC, dynamic mechanical analysis (DMA, and thermal gravimetric analysis (TGA. The dielectric spectra for the prepared samples were determined over a broad frequency range at room temperature using a broadband dielectric spectrometer and a semiconductor characterization system. The changes in thermo-mechanical and electrical properties of these novel fullerene-thiol-ene composite films were measured as a function of the C60 content, and samples characterized by high dielectric permittivity and low dielectric loss were produced. In this process, variations in chemical composition of the networks were correlated to performance characteristics.

  16. Novel pyrazoline-based fluorescent probe for detecting thiols and its application in cells

    Zhang, Rong-Rong; Zhang, Jin-Feng; Wang, Sheng-Qing; Cheng, Yan-Long; Miao, Jun-Ying; Zhao, Bao-Xiang


    A new compound, N-(4-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-acrylamide (probe L), was designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting thiol from other amino acids. On being mixed with thiol in buffered DMSO:HEPES = 1:1 solution at pH 7.4, the probe exhibited the blue emission at 474 nm. This probe is very sensitive and displayed a linear fluorescence off-on response to thiol. The fluorescence emission of the probe is pH independent in the physiological pH range. Living cell imaging of HeLa cells confirmed its cell permeability and its ability to selectively detect thiol in cells. The structure of the probe was characterized by IR, NMR and HRMS spectroscopy analysis.

  17. Controlling Topological Entanglement in Engineered Protein Hydrogels with a Variety of Thiol Coupling Chemistries

    Shengchang eTang


    Full Text Available Topological entanglements between polymer chains are achieved in associating protein hydrogels through the synthesis of high molecular weight proteins via chain extension using a variety of thiol coupling chemistries, including disulfide formation, thiol-maleimide, thiol-bromomaleimide and thiol-ene. Coupling of cysteines via disulfide formation results in the most pronounced entanglement effect in hydrogels, while other chemistries provide versatile means of changing the extent of entanglement, achieving faster chain extension, and providing a facile method of controlling the network hierarchy and incorporating stimuli responsivities. The addition of trifunctional coupling agents causes incomplete crosslinking and introduces branching architecture to the protein molecules. The high-frequency plateau modulus and the entanglement plateau modulus can be tuned by changing the ratio of difunctional chain extender to the trifunctional branching unit. Therefore, these chain extension reactions show promise in delicately controlling the relaxation and mechanical properties of engineered protein hydrogels in ways that complement their design through genetic engineering.

  18. AFM-assisted fabrication of thiol SAM pattern with alternating quantified surface potential

    Simons Janet


    Full Text Available Abstract Thiol self-assembled monolayers (SAMs are widely used in many nano- and bio-technology applications. We report a new approach to create and characterize a thiol SAMs micropattern with alternating charges on a flat gold-coated substrate using atomic force microscopy (AFM and Kelvin probe force microscopy (KPFM. We produced SAMs-patterns made of alternating positively charged, negatively charged, and hydrophobic-terminated thiols by an automated AFM-assisted manipulation, or nanografting. We show that these thiol patterns possess only small topographical differences as revealed by AFM, and distinguished differences in surface potential (20-50 mV, revealed by KPFM. The pattern can be helpful in the development of biosensor technologies, specifically for selective binding of biomolecules based on charge and hydrophobicity, and serve as a model for creating surfaces with quantified alternating surface potential distribution.

  19. Preparation of reactive three-dimensional microstructures via direct laser writing and thiol-ene chemistry.

    Quick, Alexander S; Fischer, Joachim; Richter, Benjamin; Pauloehrl, Thomas; Trouillet, Vanessa; Wegener, Martin; Barner-Kowollik, Christopher


    Three-dimensional microstructures are fabricated employing the direct laser writing process and radical thiol-ene polymerization. The resin system consists of a two-photon photoinitiator and multifunctional thiols and olefins. Woodpile photonic crystals with 22 layers and a rod distance of 2 μm are fabricated. The structures are characterized via scanning electron microscopy and focused ion beam milling. The thiol-ene polymerization during fabrication is verified via infrared spectroscopy. The structures are grafted in a subsequent thiol-Michael addition reaction with different functional maleimides. The success of the grafting reaction is evaluated via laser scanning microscopy and X-ray photoelectron spectroscopy. The grafting density is calculated to be close to 200 molecules μm(-2) .

  20. Acid Catalyzed Condensation of Phenylethanal Enol or Thiol Enol Ether to 2-Phenylnaphthalene

    CHANG, Yu-An


    Treatment of enol ether or thiol enol ether of phenylethanals with sulfuric or polyphosphoric acid in toluene or xylene gave 2-phenylnaphthalene in good yield. More importantly, a one-pot reaction has been developed.

  1. Tip-enhanced Raman spectroscopic imaging of patterned thiol monolayers

    Johannes Stadler


    Full Text Available Full spectroscopic imaging by means of tip-enhanced Raman spectroscopy (TERS was used to measure the distribution of two isomeric thiols (2-mercaptopyridine (2-PySH and 4-mercaptopyridine (4-PySH in a self-assembled monolayer (SAM on a gold surface. From a patterned sample created by microcontact printing, an image with full spectral information in every pixel was acquired. The spectroscopic data is in good agreement with the expected molecular distribution on the sample surface due to the microcontact printing process. Using specific marker bands at 1000 cm−1 for 2-PySH and 1100 cm−1 for 4-PySH, both isomers could be localized on the surface and semi-quantitative information was deduced from the band intensities. Even though nanometer size resolution information was not required, the large signal enhancement of TERS was employed here to detect a monolayer coverage of weakly scattering analytes that were not detectable with normal Raman spectroscopy, emphasizing the usefulness of TERS.

  2. Cytokinin Determines Thiol-Mediated Arsenic Tolerance and Accumulation.

    Mohan, Thotegowdanapalya C; Castrillo, Gabriel; Navarro, Cristina; Zarco-Fernández, Sonia; Ramireddy, Eswarayya; Mateo, Cristian; Zamarreño, Angel M; Paz-Ares, Javier; Muñoz, Riansares; García-Mina, Jose M; Hernández, Luis E; Schmülling, Thomas; Leyva, Antonio


    The presence of arsenic in soil and water is a constant threat to plant growth in many regions of the world. Phytohormones act in the integration of growth control and stress response, but their role in plant responses to arsenic remains to be elucidated. Here, we show that arsenate [As(V)], the most prevalent arsenic chemical species in nature, causes severe depletion of endogenous cytokinins (CKs) in the model plant Arabidopsis (Arabidopsis thaliana). We found that CK signaling mutants and transgenic plants with reduced endogenous CK levels showed an As(V)-tolerant phenotype. Our data indicate that in CK-depleted plants exposed to As(V), transcript levels of As(V)/phosphate-transporters were similar or even higher than in wild-type plants. In contrast, CK depletion provoked the coordinated activation of As(V) tolerance mechanisms, leading to the accumulation of thiol compounds such as phytochelatins and glutathione, which are essential for arsenic sequestration. Transgenic CK-deficient Arabidopsis and tobacco lines show a marked increase in arsenic accumulation. Our findings indicate that CK is an important regulatory factor in plant adaptation to arsenic stress.

  3. Thiol passivation of MWIR type II superlattice photodetectors

    Salihoglu, O.; Muti, A.; Aydinli, A.


    Poor passivation on photodetectors can result in catastrophic failure of the device. Abrupt termination of mesa side walls during pixel definition generates dangling bonds that lead to inversion layers and surface traps leading to surface leakage currents that short circuit diode action. Good passivation, therefore, is critical in the fabrication of high performance devices. Silicondioxide has been the main stay of passivation for commercial photodetectors, deposited at high temperatures and high RF powers using plasma deposition techniques. In photodetectors based on III-V compounds, sulphur passivation has been shown to replace oxygen and saturate the dangling bonds. Despite its effectiveness, it degrades over time. More effort is required to create passivation layers which eliminate surface leakage current. In this work, we propose the use of sulphur based octadecanethiol (ODT), CH3(CH2)17SH, as a passivation layer for the InAs/GaSb superlattice photodetectors that acts as a self assembled monolayer (SAM). ODT SAMs consist of a chain of 18 carbon atoms with a sulphur atom at its head. ODT Thiol coating is a simple process that consist of dipping the sample into the solution for a prescribed time. Excellent electrical performance of diodes tested confirm the effectiveness of the sulphur head stabilized by the intermolecular interaction due to van der Walls forces between the long chains of ODT SAM which results in highly stable ultrathin hydrocarbon layers without long term degradation.

  4. Revisiting the reactions of superoxide with glutathione and other thiols.

    Winterbourn, Christine C


    The reaction between GSH and superoxide has long been of interest in the free radical biology. Early studies were confusing, as some reports suggested that the reaction could be a major pathway for superoxide removal whereas others questioned whether it happened at all. Further research by several investigators, including Helmut Sies, was required to clarify this complex reaction. We now know that superoxide does react with GSH, but the reaction is relatively slow and occurs mostly by a chain reaction that consumes oxygen and regenerates superoxide. Most of the GSH is converted to GSSG, with a small amount of sulfonic acid. As shown by Sies and colleagues, singlet oxygen is a by-product. Although removal of superoxide by GSH may be a minor pathway, GSH and superoxide have a strong physiological connection. GSH is an efficient free radical scavenger, and when it does so, thiyl radicals are generated. These further react to generate superoxide. Therefore, radical scavenging by GSH and other thiols is a source of superoxide and hydrogen peroxide, and to be an antioxidant pathway, there must be efficient removal of these species.

  5. Processing and targeting of the thiol protease, aleurain

    Rogers, J.C.


    We have identified a cDNA clone from barley aleurone mRNA that encodes a protein with unusual homologies: the C-terminal portion, about 270 amino acids, is 65% identical to the mammalian thiol protease, cathepsin H. This degree of sequence conservation indicates that the enzyme must have some specific function in both plants and mammals that cannot tolerate further divergence. The N-terminal 1/3 of the protein, about 140 amino acids, has no detectable homologies to other known protein sequences; its function is unknown. In aleurone tissue, the mRNA level is increased by gibberellic acid and decreased by abscisic acid, but is expressed apparently constitutively at high levels in leaf and root tissues. The amino acid sequence and cathepsin H homology suggest that the protein will be both secreted into the endoplasmic reticulum and glycosylated. Using our cDNA clone in a bacterial expression system, we have made a fusion protein containing the protease domain of aleurain, and have used it to raise specific antisera in rabbits. These antibodies identify a 32 kd protein in extracts of aleurone layers that is induced with GA treatment but not secreted; a similarly sized protein is specifically identified in extracts of leaf tissue. Experiments are underway to characterize the pattern of expression in different tissues, to identify the subcellular locations of the protein, to characterize processing of the precursor to the 32 kd mature form, and to purify the enzyme from barley. 2 figs.

  6. (Processing and targeting of the thiol protease aleurain)

    Rogers, J.C.


    Our goal for work during the past two years under this Grant was to characterize the barley thiol protease, aleurain, to determine if it is secreted or retained intracellularly in aleurone cells, and to begin to elucidate structural features that might control targeting of the protein to its final destination. We have shown that aleurain is synthesized as a proenzyme with two N-linked oligosaccharide chains, one high mannose-type and one complex-type. Aleurain undergoes processing to mature form by removal of an Nterminal prosegment, and is retained intracellularly; it cannot be detected among proteins secreted from aleurone cells. Treatment of aleurone cells with tunicamycin to prevent glycosylation of aleurain does not prevent processing of the unglycosylated form. The N-terminal portion of aleurain's prosegment is homologous to the comparable region in two yeast vacuolar proteases, where that region is known to contain the signal necessary for targeting the proteases to the vacuole. 18 refs., 7 figs.

  7. Oligomerization of Indole Derivatives with Incorporation of Thiols

    Jarl E.S. Wikberg


    Full Text Available Abstract: Two molecules of indole derivative, e.g. indole-5-carboxylic acid, reacted with one molecule of thiol, e.g. 1,2-ethanedithiol, in the presence of trifluoroacetic acid to yield adducts such as 3-[2-(2-amino-5-carboxyphenyl-1-(2-mercaptoethylthioethyl]-1Hindole-5-carboxylic acid. Parallel formation of dimers, such as 2,3-dihydro-1H,1'H-2,3'-biindole-5,5'-dicarboxylic acid and trimers, such as 3,3'-[2-(2-amino-5-carboxyphenyl ethane-1,1-diyl]bis(1H-indole-5-carboxylic acid of the indole derivatives was also observed. Reaction of a mixture of indole and indole-5-carboxylic acid with 2-phenylethanethiol proceeded in a regioselective way, affording 3-[2-(2-aminophenyl-1-(phenethylthioethyl]-1H-indole-5-carboxylic acid. An additional product of this reaction was 3-[2-(2-aminophenyl-1-(phenethylthioethyl]-2,3-dihydro-1H,1'H-2,3'-biindole-5'-carboxylic acid, which upon standing in DMSO-d6 solution gave 3-[2-(2-aminophenyl-1-(phenethylthioethyl]-1H,1'H-2,3'-biindole-5'-carboxylic acid. Structures of all compounds were elucidated by NMR, and a mechanism for their formation was suggested.

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

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


    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.

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

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


    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.

  10. The role of the thiol group in protein modification with methylglyoxal



    Full Text Available Methylglyoxal is a highly reactive α-oxoaldehyde with elevated production in hyperglycemia. It reacts with nucleophilic Lys and Arg side-chains and N-terminal amino groups causing protein modification. In the present study, the importance of the reaction of the Cys thiol group with methylglyoxal in protein modification, the competitiveness of this reaction with those of amino and guanidine groups, the time course of these reactions and their role and contribution to protein cross-linking were investigated. Human and bovine serum albumins were used as model systems. It was found that despite the very low levels of thiol groups on the surface of the examined protein molecules (approx. 80 times lower than those of amino and guanidino groups, a very high percentage of it reacts (25–85 %. The amount of reacted thiol groups and the rate of the reaction, the time for the reaction to reach equilibrium, the formation of a stable product and the contribution of thiol groups to protein cross-linking depend on the methylglyoxal concentration. The product formed in the reaction of thiol and an insufficient quantity of methylglyoxal (compared to the concentrations of the groups accessible for modification participates to a significant extent (4 % to protein cross-linking. Metformin applied in equimolar concentration with methylglyoxal prevents its reaction with amino and guanidino groups but, however, not with thiol groups.

  11. Cyclic Voltammetric Responses of Nitrate Reductase on Chemical Modified Electrodes

    YaRuSONG; HuiBoSHAO; 等


    Electrochemistry of nitrate reductases (NR) incorporated into 2-aminoethanethiol self-assembled on the gold electrode and polyacrylamide cast on the pyrolytic graphite electrode was examined. NR on chemical modified electrode showed electrochemical cyclic voltammetric responses in phosphate buffers.

  12. Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

    Nestoras, Konstantinos; Mohammed, Asma Hadi; Schreurs, Ann-Sofie


    The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and...

  13. Purification and characterization of assimilatory nitrite reductase from Candida utilis.

    Sengupta, S; Shaila, M S; Rao, G R


    Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC and nitrite reductase (EC They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.

  14. Thiol-ene immobilisation of carbohydrates onto glass slides as a simple alternative to gold-thiol monolayers, amines or lipid binding.

    Biggs, Caroline I; Edmondson, Steve; Gibson, Matthew I


    Carbohydrate arrays are a vital tool in studying infection, probing the mechanisms of bacterial, viral and toxin adhesion and the development of new treatments, by mimicking the structure of the glycocalyx. Current methods rely on the formation of monolayers of carbohydrates that have been chemically modified with a linker to enable interaction with a functionalised surface. This includes amines, biotin, lipids or thiols. Thiol-addition to gold to form self-assembled monolayers is perhaps the simplest method for immobilisation as thiolated glycans are readily accessible from reducing carbohydrates in a single step, but are limited to gold surfaces. Here we have developed a quick and versatile methodology which enables the use of thiolated carbohydrates to be immobilised as monolayers directly onto acrylate-functional glass slides via a 'thiol-ene'/Michael-type reaction. By combining the ease of thiol chemistry with glass slides, which are compatible with microarray scanners this offers a cost effective, but also useful method to assemble arrays.

  15. A Study of Functional Polymer Colloids Prepared Using Thiol-Ene/Yne Click Chemistry

    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

  16. Distribution of Prx-linked hydroperoxide reductase activity among microorganisms.

    Takeda, Kouji; Nishiyama, Yoshitaka; Yoda, Koji; Watanabe, Toshihiro; Nimura-Matsune, Kaori; Mura, Kiyoshi; Tokue, Chiyoko; Katoh, Tetzuya; Kawasaki, Shinji; Niimura, Youichi


    Peroxiredoxin (Prx) constitutes a large family of enzymes found in microorganisms, animals, and plants, but the detection of the activities of Prx-linked hydroperoxide reductases (peroxiredoxin reductases) in cell extracts, and the purification based on peroxide reductase activity, have only been done in bacteria and Trypanosomatidae. A peroxiredoxin reductase (NADH oxidase) from a bacterium, Amphibacillus, displayed only poor activities in the presence of purified Prx from Saccharomyces or Synechocystis, while it is highly active in the presence of bacterial Prx. These results suggested that an enzyme system different from that in bacteria might exist for the reduction of Prx in yeast and cyanobacteria. Prx-linked hydroperoxide reductase activities were detected in cell extracts of Saccharomyces, Synechocystis, and Chlorella, and the enzyme activities of Saccharomyces and Chlorella were induced under vigorously aerated culture conditions and intensive light exposure conditions, respectively. Partial purification of Prx-linked peroxidase from the induced yeast cells indicated that the Prx-linked peroxidase system consists of two protein components, namely, thioredoxin and thioredoxin reductase. This finding is consistent with the previous report on its purification based on its protein protection activity against oxidation [Chae et al., J. Biol. Chem., 269, 27670-27678 (1994)]. In this study we have confirmed that Prx-linked peroxidase activity are widely distributed, not only in bacteria species and Trypanosomatidae, but also in yeast and photosynthetic microorganisms, and showed reconstitution of the activity from partially purified interspecies components.

  17. Infection free titanium alloys by stabile thiol based nanocoating.

    Cökeliler, Dilek; Göktaş, Hilal; Tosun, Pinar Deniz; Mutlu, Selma


    As biomedical materials, titanium and titanium alloys (Ti-6Al-4V) are superior to many materials in terms of mechanical properties and biocompatibility. However, they are still not sufficient for prolonged clinical use because the biocompatibility of these materials must be improved. In this study, the prevention of the attachment of test microorganism on the Ti alloy surfaces by thiol (-SH) and hydroxyl (-OH) functional group containing monomer in plasma based electron beam generator was reported in order to prepare anti-fouling surfaces. The precursor, 11-mercaptoundecanoic acid is used as plasma source to create nano-film with 30-60 nm approximately. The surface chemistry and topology of uncoated and coated samples are characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Atomic Force Microscopy (AFM). Static contact angle measurements are performed to state the change of surface hydrophilicity. All coated samples are tested in-vitro environment with Staphylococcus epidermidis that is chosen as the test bacteria strain in view of its significance for the pathogenesis of medical-device-related infections. This test is repeated after certain period of times and samples are waited in dynamic fluid media in order to investigate the stability of nano-coating. Plasma polymerized 11-mercaptoundecanoic acid film (PP MUA) with 42 +/- 4 nm is found alternative, stabile and simple method to create bacterial anti-fouling surfaces. The static contact angle of the coated surface is 34 +/- 80 whereas the uncoated surface is 57 +/- 50. For the coated surface, the presence of C-OH and C==O groups in infrared spectra defining the PP MUA is achieved by the plasma polymerization. The attachment of the model microorganism on the biomaterial surface prepared by PP MUA is reduced 85.3% if compared to unmodified control surface.

  18. Uptake of mercury by thiol-grafted chitosan gel beads.

    Merrifield, John D; Davids, William G; MacRae, Jean D; Amirbahman, Aria


    This study describes the synthesis and characterization of thiol-grafted chitosan beads for use as mercury (Hg) adsorbents. Chitosan flakes were dissolved and formed into spherical beads using a phase inversion technique, then crosslinked to improve their porosity and chemical stability. Cysteine was grafted onto the beads in order to improve the adsorption affinity of Hg to the beads. The beads possessed an average diameter of 3.2 mm, porosity of 0.9, specific surface area of approximately 100 m2/g, average pore size of approximately 120 angstroms, and specific gravity of 2.0. Equilibrium and kinetic uptake experiments were conducted to study the uptake of Hg by the beads. The adsorption capacity was approximately 8.0 mmol-Hg/g-dry beads at pH 7, and decreased with decreasing pH. Hg adsorption kinetics was modeled as radial pore diffusion into a spherical bead with nonlinear adsorption. Use of the nonlinear Freundlich isotherm in the diffusion equation allowed modeling of the uptake kinetics with a single tortuosity factor of 1.5 +/- 0.3 as the fitting parameter for all initial Hg concentrations, chitosan loadings, and agitation rates. At agitation rates of 50 and 75 rpm, where uptake rate was reduced significantly due to the boundary layer effect, the mass transfer coefficient at the outside boundary was also used as a fitting parameter to model the kinetic data. At agitation rates higher than 150 rpm, pore diffusion was the rate-limiting step. The beads exhibited a high initial uptake rate followed by a slower uptake rate suggesting pore diffusion as the rate-determining step especially at high agitation rates. Higher uptake rates observed in this study compared to those in a previous study of chitosan-based crab shells indicate that dissolution and gel formation increase the porosity and pore accessibility of chitosan.

  19. Aldose reductase mediates retinal microglia activation.

    Chang, Kun-Che; Shieh, Biehuoy; Petrash, J Mark


    Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1(GFP) mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR(WT) background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy.

  20. Methylenetetrahydrofolate Reductase Activity and Folate Metabolism

    Nursen Keser


    Full Text Available Folate is a vital B vitamin which is easily water-soluble. It is a natural source which is found in the herbal and animal foods. Folate has important duties in the human metabolism, one of them is the adjustment of the level of plasma homocysteine. Reduction in MTHFR (methylenetetrahydrofolate reductase,which is in charge of the metabolism of homocysteine activity affects the level of homocysteine. Therefore MTHFR is an important enzyme in folate metabolism. Some of the mutations occurring in the MTHFR gene is a risk factor for various diseases and may be caused the hyperhomocysteinemia or the homocystinuria, and they also may lead to metabolic problems. MTHFR is effective in the important pathways such as DNA synthesis, methylation reactions and synthesis of RNA. C677T and A1298C are the most commonly occurring polymorphisms in the gene of MTHFR. The frequency of these polymorphisms show differences in the populations. MTHFR, folate distribution, metabolism of homocysteine and S-adenosylmethionine, by the MTHFR methylation the genetic defects have the potential of affecting the risk of disease in the negative or positive way.

  1. Aldose reductase, oxidative stress and diabetic mellitus

    Waiho eTang


    Full Text Available Diabetes mellitus (DM is a complex metabolic disorder arising from lack of insulin production or insulin resistance 1. DM is a leading cause of morbidity and mortality in the developed world, particularly from vascular complications such as atherothrombosis in the coronary vessels. Aldose reductase (AR [ALR2; EC], a key enzyme in the polyol pathway, catalyzes NADPH-dependent reduction of glucose to sorbitol, leading to excessive accumulation of intracellular reactive oxygen species (ROS in various tissues of DM including the heart, vasculature, neurons, eyes and kidneys. As an example, hyperglycemia through such polyol pathway induced oxidative stress, may have dual heart actions, on coronary blood vessel (atherothrombosis and myocardium (heart failure leading to severe morbidity and mortality (reviewed in 2. In cells cultured under high glucose conditions, many studies have demonstrated similar AR-dependent increases in ROS production, confirming AR as an important factor for the pathogenesis of many diabetic complications. Moreover, recent studies have shown that AR inhibitors may be able to prevent or delay the onset of cardiovascular complications such as ischemia/reperfusion injury, atherosclerosis and atherothrombosis. In this review, we will focus on describing pivotal roles of AR in the pathogenesis of cardiovascular diseases as well as other diabetic complications, and the potential use of AR inhibitors as an emerging therapeutic strategy in preventing DM complications.

  2. Comparative evaluation of antioxidant capacities of thiol-based antioxidants measured by different in vitro methods.

    Güngör, Nilay; Ozyürek, Mustafa; Güçlü, Kubilay; Cekiç, Sema Demirci; Apak, Reşat


    Thiol-type compounds are an important class of strong antioxidants and main determinants of total antioxidant capacity (TAC) of cellular homogenates. The TAC of thiol mixtures and the corresponding TEAC (trolox equivalent antioxidant capacity) values of individual thiols were determined by the CUPRAC (CUPric Reducing Antioxidant Capacity) method, and the results were compared with those found by reference assays for method validation. Synthetic mixtures of thiols were prepared, and the expected and found TAC values (in mM trolox (TR) equivalents) of these mixtures showed a good agreement. The technique of standard additions was performed for thiol mixtures and human serum, and the absorbance results confirmed that apparent chemical deviations from Beer's law were absent in the system. The CUPRAC results were compared with those of reference methods, namely 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)/persulphate and Ferric Reducing Antioxidant Power (FRAP). As being a most important thiol (-SH) peptide at in vivo conditions, glutathione (GSH) showed a TEAC value of 0.57 in the CUPRAC method, as opposed to the corresponding value (1.51) in the ABTS/persulphate method. The ABTS/persulphate result was not in accordance with the reversible 1-e oxidation of GSH to the corresponding disulfide that is expected to occur under physiological conditions. FRAP did not give consistent results, and even at relatively high concentrations of GSH, the TEAC(FRAP) value was only 0.07. The thiol-type antioxidant-bearing pharmaceuticals of Brunac eye drop, Trom and Mentopin effervescent tablets containing N-acetyl-L-cysteine (NAC) were assayed with HPLC for comparison, and the obtained results for NAC were in accordance with those found with CUPRAC.

  3. Electrochemistry behavior of endogenous thiols on fluorine doped tin oxide electrodes

    Rojas, Luciana; Molero, Leonard; Tapia, Ricardo A.; Rio, Rodrigo del; Valle, M. Angelica del; Antilen, Monica [Departamento de Quimica Inorganica, Facultad de Quimica, Pontificia Universidad Catolica de Chile, Av Vicuna Mackenna 4860, Casilla 306, Correo 22, Macul, Santiago (Chile); Armijo, Francisco, E-mail: [Departamento de Quimica Inorganica, Facultad de Quimica, Pontificia Universidad Catolica de Chile, Av Vicuna Mackenna 4860, Casilla 306, Correo 22, Macul, Santiago (Chile)


    Highlights: > The first time that fluorine doped tin oxide electrodes are used for the electrooxidation of endogenous thiols. > Low potentials of electrooxidation were obtained for the different thiols. > The electrochemical behavior of thiols depends on the pH and the ionic electroactive species, the electrooxidation proceeds for a process of adsorption of electroactive species on FTO and high values the heterogeneous electron tranfer rate constant of the reaction were obtained. - Abstract: In this work the electrochemical behavior of different thiols on fluorine doped tin oxide (FTO) electrodes is reported. To this end, the mechanism of electrochemical oxidation of glutathione (GSH), cysteine (Cys), homocysteine (HCys) and acetyl-cysteine (ACys) at different pH was investigated. FTO showed electroactivity for the oxidation of the first three thiols at pH between 2.0 and 4.0, but under these conditions no acetyl-cysteine oxidation was observed on FTO. Voltammetric studies of the electro-oxidation of GSH, Cys and HCys showed peaks at about 0.35, 0.29, and 0.28 V at optimum pH 2.4, 2.8 and 3.4, respectively. In addition, this study demonstrated that GSH, Cys and HCys oxidation occurs when the zwitterion is the electro-active species that interact by adsorption on FTO electrodes. The overall reaction involves 4e{sup -}/4H{sup +} and 2e{sup -}/2H{sup +}, respectively, for HCys and for GSH and Cys and high heterogeneous electron transfer rate constants. Besides, the use of FTO for the determination of different thiols was evaluated. Experimental square wave voltammetry shows a linear current vs. concentrations response between 0.1 and 1.0 mM was found for HCys and GSH, indicating that these FTO electrodes are promising candidates for the efficient electrochemical determination of these endogenous thiols.

  4. Thioredoxin-related protein of 14 kDa is an efficient L-cystine reductase and S-denitrosylase.

    Pader, Irina; Sengupta, Rajib; Cebula, Marcus; Xu, Jianqiang; Lundberg, Jon O; Holmgren, Arne; Johansson, Katarina; Arnér, Elias S J


    Thioredoxin-related protein of 14 kDa (TRP14, also called TXNDC17 for thioredoxin domain containing 17, or TXNL5 for thioredoxin-like 5) is an evolutionarily well-conserved member of the thioredoxin (Trx)-fold protein family that lacks activity with classical Trx1 substrates. However, we discovered here that human TRP14 has a high enzymatic activity in reduction of l-cystine, where the catalytic efficiency (2,217 min(-1)⋅µM(-1)) coupled to Trx reductase 1 (TrxR1) using NADPH was fivefold higher compared with Trx1 (418 min(-1)⋅µM(-1)). Moreover, the l-cystine reduction with TRP14 was in contrast to that of Trx1 fully maintained in the presence of a protein disulfide substrate of Trx1 such as insulin, suggesting that TRP14 is a more dedicated l-cystine reductase compared with Trx1. We also found that TRP14 is an efficient S-denitrosylase with similar efficiency as Trx1 in catalyzing TrxR1-dependent denitrosylation of S-nitrosylated glutathione or of HEK293 cell-derived S-nitrosoproteins. Consequently, nitrosylated and thereby inactivated caspase 3 or cathepsin B could be reactivated through either Trx1- or TRP14-catalyzed denitrosylation reactions. TRP14 was also, in contrast to Trx1, completely resistant to inactivation by high concentrations of hydrogen peroxide. The oxidoreductase activities of TRP14 thereby complement those of Trx1 and must therefore be considered for the full understanding of enzymatic control of cellular thiols and nitrosothiols.

  5. Catalysis by methyl-coenzyme M reductase: a theoretical study for heterodisulfide product formation.

    Pelmenschikov, Vladimir; Siegbahn, Per E M


    Hybrid density functional theory has been used to investigate the catalytic mechanism of methyl-coenzyme M reductase (MCR), an essential enzyme in methanogenesis. In a previous study of methane formation, a scheme was suggested involving oxidation of Ni(I) in the starting square-planar coordination to the high-spin Ni(II) form in the CoM-S-Ni(II)F(430) octahedral intermediate. The methyl radical, concomitantly released by methyl-coenzyme M (CoM), is rapidly quenched by hydrogen atom transfer from the coenzyme B (CoB) thiol group, yielding methane as the first product of the reaction. The present investigation primarily concerns the second and final step of the reaction: oxidation of CoB and CoM to the CoB-S-S-CoM heterodisulfide product and reduction of nickel back to the Ni(I) square-planar form. The activation energy for the second step is found to be around 10 kcal/mol, implying that the first step of methane formation with an activation energy of 20 kcal/mol should be rate-limiting. An oxygen of the Gln147 residue, occupying the rear axial position in the oxidized Ni(II) state, is shown to stabilize the intermediate by 6 kcal/mol, thereby slightly decreasing the barrier for the preceding rate-limiting transition state. The mechanism suggested is discussed in the context of available experimental data. An analysis of the flexibility of the F(430) cofactor during the reaction cycle is also given.

  6. Evaluation of Thiol Raman Activities and pKa Values Using Internally Referenced Raman-Based pH Titration.

    Suwandaratne, Nuwanthi; Hu, Juan; Siriwardana, Kumudu; Gadogbe, Manuel; Zhang, Dongmao


    Thiols, including organothiol and thiol-containing biomolecules, are among the most important classes of chemicals that are used broadly in organic synthesis, biological chemistry, and nanosciences. Thiol pKa values are key indicators of thiol reactivity and functionality. Reported herein is an internally referenced Raman-based pH titration method that enables reliable quantification of thiol pKa values for both mono- and dithiols in water. The degree of thiol ionization is monitored directly using the peak intensity of the S-H stretching feature in the 2600 cm(-1) region relative to an internal reference peak as a function of the titration solution's pH. The thiol pKa values and Raman activity relative to its internal reference were then determined by curve fitting the experimental data with equations derived on the basis of the Henderson-Hasselbalch equation. Using this Raman titration method, we determined for the first time the first and second thiol pKa values for 1,2-benzenedithiol in water. This Raman-based method is convenient to implement, and its underlying theory is easy to follow. It should therefore have broad application for thiol pKa determinations and verification.

  7. Transcripts of Anthocyanidin Reductase and Leucoanthocyanidin Reductase and Measurement of Catechin and Epicatechin in Tartary Buckwheat

    Yeon Bok Kim


    Full Text Available Anthocyanidin reductase (ANR and leucoanthocyanidin reductase (LAR play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions.

  8. Isolation and characterization of cDNAs encoding leucoanthocyanidin reductase and anthocyanidin reductase from Populus trichocarpa.

    Lijun Wang

    Full Text Available Proanthocyanidins (PAs contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR and leucoanthocyanidin reductase (LAR are two key enzymes of the PA biosynthesis that produce the main subunits: (+-catechin and (--epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05 in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus.

  9. Thiol-yne adsorbates for stable, low-density, self-assembled monolayers on gold.

    Stevens, Christopher A; Safazadeh, Leila; Berron, Brad J


    We present a novel approach toward carboxylate-terminated, low-density monolayers on gold, which provides exceptional adsorbate stability and conformational freedom of interfacial functional groups. Adsorbates are synthesized through the thiol-yne addition of two thiol-containing head groups to an alkyne-containing tail group. The resulting monolayers have two distinct phases: a highly crystalline head phase adjacent to the gold substrate, and a reduced density tail phase, which is in contact with the environment. The ellipsometric thickness of 27 Å is consistent with the proposed structure, where a densely packed decanedithiol monolayer is capped with an 11 carbon long, second layer at 50% lateral chain density. The Fourier transform infrared peak at 1710 cm(-1) supports the presence of the carbonyl group. Further, the peaks associated with asymmetric and symmetric methylene stretching are shifted toward higher wavenumbers compared to those of well-packed self-assembled monolayers (SAMs), which shows a lower average crystallinity of the thiol-yne monolayers compared to a typical monolayer. Contact angle measurements indicate an intermediate surface energy for the thiol-yne monolayer surface, owing to the contribution of exposed methylene functionality at the surface in addition to the carbonyl terminal group. The conformational freedom at the surface was demonstrated through remodeling the thiol-yne surface under an applied potential. Changes in the receding contact angle in response to an external potential support the capacity for reorientation of the surface presenting groups. Despite the low packing at the solution interface, thiol-yne monolayers are resistant to water and ion transport (R(f) ~ 10(5)), supporting the presence of a densely structured layer at the gold surface. Further, the electrochemical stability of the thiol-yne adsorbates exceeded that of well-packed SAMs, requiring a more reductive potential to desorb the thiol-yne monolayers from the

  10. Thiol groups controls on arsenite binding by organic matter: new experimental and modeling evidence.

    Catrouillet, Charlotte; Davranche, Mélanie; Dia, Aline; Bouhnik-Le Coz, Martine; Pédrot, Mathieu; Marsac, Rémi; Gruau, Gérard


    Although it has been suggested that several mechanisms can describe the direct binding of As(III) to organic matter (OM), more recently, the thiol functional group of humic acid (HA) was shown to be an important potential binding site for As(III). Isotherm experiments on As(III) sorption to HAs, that have either been grafted with thiol or not, were thus conducted to investigate the preferential As(III) binding sites. There was a low level of binding of As(III) to HA, which was strongly dependent on the abundance of the thiols. Experimental datasets were used to develop a new model (the modified PHREEQC-Model VI), which defines HA as a group of discrete carboxylic, phenolic and thiol sites. Protonation/deprotonation constants were determined for each group of sites (pKA=4.28±0.03; ΔpKA=2.13±0.10; pKB=7.11±0.26; ΔpKB=3.52±0.49; pKS=5.82±0.052; ΔpKS=6.12±0.12 for the carboxylic, phenolic and thiols sites, respectively) from HAs that were either grafted with thiol or not. The pKS value corresponds to that of single thiol-containing organic ligands. Two binding models were tested: the Mono model, which considered that As(III) is bound to the HA thiol site as monodentate complexes, and the Tri model, which considered that As(III) is bound as tridentate complexes. A simulation of the available literature datasets was used to validate the Mono model, with logKMS=2.91±0.04, i.e. the monodentate hypothesis. This study highlighted the importance of thiol groups in OM reactivity and, notably, determined the As(III) concentration bound to OM (considering that Fe is lacking or at least negligible) and was used to develop a model that is able to determine the As(III) concentrations bound to OM.

  11. Self-Healing Photocurable Epoxy/thiol-ene Systems Using an Aromatic Epoxy Resin

    Ricardo Acosta Ortiz


    Full Text Available A rapid and efficient method to obtain self-healing epoxy resins is discussed. This method is based on the use of a thiol-disulfide oligomer obtained by partial oxidation of a multifunctional thiol using a hypervalent iodine (III compound as oxidant. The oligomer was characterized by Fourier transform infrared spectroscopy (FTIR, Raman and nuclear magnetic resonance spectroscopies, and gel permeation chromatography (GPC. The oligomer was a joint component of the thiol-ene system along with a tetra-allyl-functionalized curing agent. The kinetics of the photopolymerization of diglycidylether of bisphenol A (DGEBA revealed that conversions of the epoxy groups as high as 80% were achieved in only 15 minutes by increasing the concentration of the thiol-ene system in the formulation. The disulfide bonds introduced in the copolymer using the thiol-disulfide oligomer allowed the repairing of the test specimens in as little as 10 minutes when the specimens were heated at 80°C or for 500 minutes at room temperature. The analysis of the mechanical properties using dynamic mechanical analysis (DMA showed that the specimens displayed a healing efficiency up to 111% compared with the unhealed specimens, depending on the amount of polythioethers present in the copolymer.

  12. Superoxide dismutase and media dependence of far-UV radiation resistance in thiol-treated cells

    Claycamp, H.G.; McCormick, M.L.; DeRose, C.M.; Elwell, J.H.; Oberley, L.W. (Iowa Univ., Iowa City, IA (USA). Radiation Research Lab.)


    Pretreatment of wild-type Escherichia coli K12 cells with dithiothreitol (DTT) induces far-UV radiation resistance after the thiol is removed (Claycamp 1988). The present study shows that a 1 h treatment of cells with DTT in minimal medium followed by a 0.5 h incubation in buffer (37{sup 0}C) results in a dose reduction factor (DRF) calculated at F{sub 37} of 1.81. When the thio pretreatment was in rich medium, sensitization occurs with DRF = 0.729. This could be reversed to protection by inhibiting extracellular thiol oxidation in rich medium with the chelator, DETAPAC, such that thiol oxidation rate was equivalent to that of DTT in minimal medium. Both thiol-induced resistance and sensitization produced changes predominantly in the shoulders of survival curves. For either protection or sensitization, at least one form of endogenous superoxide dismutase (SOD) was required. These results suggest that different targets are involved in thiol-induced UV protection and sensitization: DNA and extracellular targets (e.g. the membrane), respectively. (author).

  13. Facile and Efficient Synthesis of Carbosiloxane Dendrimers via Orthogonal Click Chemistry Between Thiol and Ene.

    Zhang, Zhida; Feng, Shengyu; Zhang, Jie


    A combination of a thiol-Michael addition reaction and a free radical mediated thiol-ene reaction is employed as a facile and efficient approach to carbosiloxane dendrimer synthesis. For the first time, carbosiloxane dendrimers are constructed rapidly by an orthogonal click strategy without protection/deprotection procedures. The chemoselectivity of these two thiol-ene click reactions leads to a design of a new monomer containing both electron-deficient carbon-carbon double bonds and unconjugated carbon-carbon double bonds. Siloxane bonds are introduced as the linker between these two kinds of carbon-carbon double bonds. Starting from a bifunctional thiol core, the dendrimers are constructed by iterative thiol-ene click reactions under different but both mild reaction conditions. After simple purification steps the fifth dendrimer with 54 peripheral functional groups is obtained with an excellent overall yield in a single day. Furthermore, a strong blue glow is observed when the dendrimer is excited by a UV lamp.

  14. Selective Adsorption of Thiols Using Gold Nanoparticles Supported on Metal Oxides.

    Sui, Ruohong; Lesage, Kevin L; Carefoot, Sarah K; Fürstenhaupt, Tobias; Rose, Chelsea J; Marriott, Robert A


    Selective capture of thiols from a synthetic hydrogen sulfide containing mixture using supported nanogold materials has been explored for the potential removal of thiols from sour gas production fluids. In this research, TiO2-, Al2O3-, SiO2-, and ZnO-supported gold nanoparticles have been studied for their usage as regeneratable adsorbents to capture CH3SH, C2H5SH, and i-C3H7SH. Au/TiO2 and Au/Al2O3 showed promising properties for removing the thiols efficiently from a gas-phase mixture; however, Au/Al2O3 did catalyze some undesirable side reactions, e.g., carbonyl sulfide formation. It was found that a mild temperature of T = 200 °C was sufficient for regeneration of either Au/TiO2 or Au/Al2O3 adsorbent. The metal oxide mesopores played an important role for accommodating gold particles and chemisorption of the thiols, where smaller pore sizes were found to inhibit the agglomeration/growth of gold particles. The nature of thiol adsorption and the impact of multiple adsorption-desorption cycles on the adsorbents have been studied using electron microscopy, XPS, XRD, GC, and physi/chemiadsorption analyses.

  15. Quantification of protein thiols using ThioGlo 1 fluorescent derivatives and HPLC separation.

    Hoff, Signe; Larsen, Flemming H; Andersen, Mogens L; Lund, Marianne N


    A method for quantification of total soluble protein-derived thiols in beer was developed based on the formation of fluorescent adducts with the maleimide compound ThioGlo 1. The problem of interference from fluorescent adducts of sulfite and ThioGlo 1 was solved by HPLC separation of the adducts followed by fluorescence detection. Using standard addition of GSH, a detection limit of 0.028 μM thiols was achieved. The application and validation of the method was demonstrated for beers with different color intensities, and the application range is in principle for any biological system containing thiols. However, the quantification of cysteine was complicated by a lower fluorescence response of its ThioGlo 1 adducts. Based on the studies of the responses of a series of cysteine-derived thiols and (1)H NMR studies of the structures of ThioGlo 1 adducts with GSH and cysteine, it was concluded that thiols with a neighboring free amino group yield ThioGlo 1 adducts with a reduced fluorescence intensity.

  16. Thiol-disulfide exchange in peptides derived from human growth hormone.

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


    Disulfide bonds stabilize proteins by cross-linking distant regions into a compact three-dimensional structure. They can also participate in hydrolytic and oxidative pathways to form nonnative 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 was monitored to investigate the effect of pH (6.0-10.0), temperature (4-50°C), oxidation suppressants [ethylenediaminetetraacetic acid (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 reverse-phase HPLC and liquid chromatography-mass spectrometry. Concentration versus time data were fitted to a mathematical model using nonlinear least squares regression analysis. At all pH values, the model was able to fit the data with R(2) ≥ 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.

  17. Pore surface engineering in a zirconium metal–organic framework via thiol-ene reaction

    Gui, Bo; Hu, Guiping; Zhou, Tailin; Wang, Cheng, E-mail:


    A porous olefin-functionalized Zr(IV)-based metal–organic framework, denoted as UiO-68-allyl, has been constructed. Our results clearly demonstrated that the surface of UiO-68-allyl could be decorated with organic molecule (ethanethiol) via thiol-ene reaction. More importantly, the crystallinity of the framework were maintained during the post-synthetic modification process. However, the microporosity of the framework is retained but the surface area decreased, due to the grafting of ethylthio groups into the pores. From our studies, we can conclude that the strategy of post-synthetic modification of UiO-68-allyl via thiol-ene reaction may be general. Furthermore, we may anchor other desired functional group onto the pore walls in Zr-MOFs via thiol-ene reaction, enabling more potential applications. - graphical abstract: In this manuscript, we reported the post-synthetic modification of an olefin-functionalized Zr(IV)-based metal–organic framework via thiol-ene reaction. - Highlights: • A porous olefin-functionalized Zr(IV)-based metal–organic framework has been constructed. • The surface of olefin-functionalized Zr-MOF could be decorated with organic molecules via thiol-ene reaction. • The crystallinity and permanent porosity of the framework were maintained during the post-synthetic modification process.

  18. A Central Role for Thiols in Plant Tolerance to Abiotic Stress

    Lyuben Zagorchev


    Full Text Available Abiotic stress poses major problems to agriculture and increasing efforts are being made to understand plant stress response and tolerance mechanisms and to develop new tools that underpin successful agriculture. However, the molecular mechanisms of plant stress tolerance are not fully understood, and the data available is incomplete and sometimes contradictory. Here, we review the significance of protein and non-protein thiol compounds in relation to plant tolerance of abiotic stress. First, the roles of the amino acids cysteine and methionine, are discussed, followed by an extensive discussion of the low-molecular-weight tripeptide, thiol glutathione, which plays a central part in plant stress response and oxidative signalling and of glutathione-related enzymes, including those involved in the biosynthesis of non-protein thiol compounds. Special attention is given to the glutathione redox state, to phytochelatins and to the role of glutathione in the regulation of the cell cycle. The protein thiol section focuses on glutaredoxins and thioredoxins, proteins with oxidoreductase activity, which are involved in protein glutathionylation. The review concludes with a brief overview of and future perspectives for the involvement of plant thiols in abiotic stress tolerance.

  19. Near-Edge X-ray Absorption Fine Structure Spectroscopy of Diamondoid Thiol Monolayers on Gold

    Willey, T M; Fabbri, J; Lee, J I; Schreiner, P; Fokin, A A; Tkachenko, B A; Fokina, N A; Dahl, J; Carlson, B; Vance, A L; Yang, W; Terminello, L J; van Buuren, T; Melosh, N


    Diamondoids, hydrocarbon molecules with cubic-diamond-cage structures, have unique properties with potential value for nanotechnology. The availability and ability to selectively functionalize this special class of nanodiamond materials opens new possibilities for surface-modification, for high-efficiency field emitters in molecular electronics, as seed crystals for diamond growth, or as robust mechanical coatings. The properties of self-assembled monolayers (SAMs) of diamondoids are thus of fundamental interest for a variety of emerging applications. This paper presents the effects of thiol substitution position and polymantane order on diamondoid SAMs on gold using near-edge X-ray absorption fine structure spectroscopy (NEXAFS) and X-ray photoelectron spectroscopy (XPS). A framework to determine both molecular tilt and twist through NEXAFS is presented and reveals highly ordered diamondoid SAMs, with the molecular orientation controlled by the thiol location. C 1s and S 2p binding energies are lower in adamantane thiol than alkane thiols on gold by 0.67 {+-} 0.05 eV and 0.16 {+-} 0.04 eV respectively. These binding energies vary with diamondoid monolayer structure and thiol substitution position, consistent with different amounts of steric strain and electronic interaction with the substrate. This work demonstrates control over the assembly, in particular the orientational and electronic structure, providing a flexible design of surface properties with this exciting new class of diamond clusters.

  20. Detection of biological thiols based on a colorimetric method

    Yuan-yuan XU; Yang-yang SUN; Yu-juan ZHANG; Chen-he LU; Jin-feng MIAO‡


    Biological thiols (biothiols), an important kind of functional biomolecules, such as cysteine (Cys) and glutathione (GSH), play vital roles in maintaining the stability of the intracellular environment. In past decades, studies have demonstrated that metabolic disorder of biothiols is related to many serious disease processes and wil lead to extreme damage in human and numerous animals. We carried out a series of experiments to detect biothiols in bi-osamples, including bovine plasma and cel lysates of seven different cel lines based on a simple colorimetric method. In a typical test, the color of the test solution could gradualy change from blue to colorless after the addition of biothiols. Based on the color change displayed, experimental results reveal that the percentage of biothiols in the embryonic fibroblast cell line is significantly higher than those in the other six cell lines, which provides the basis for the following biothiols-related study.%中文概要题目:生物巯化物的可视化检测目的:通过简单可靠的可视化检测方法评估牛血清及各细胞系中生物巯化物的含量。创新点:基于银纳米颗粒形成的比色变化过程对牛血清及细胞中生物巯化物进行了检测。方法:将6组不同的细胞系培养后进行裂解,其裂解产物分别与3,3',5,5'-四甲基联苯胺(TMB)和硝酸银(AgNO3)的混合液室温孵育后,用紫外可见分光光度计测量细胞中生物巯化物的含量。结论:通过不同细胞系中生物巯化物含量的比对,证实胚胎成纤维细胞中生物巯化物的含量明显高于其他细胞。

  1. Strategies for creating antifouling surfaces using selfassembled poly(ethylene glycol) thiol molecules

    Lokanathan, Arcot R.


    conditions for the reversible, initial attachment of microbial cells. This effect can be obtained by grafting hydrophilic polymeric chains onto surfaces and thereby provide a steric barrier between the substrate surface and the microbial cell. Poly (ethylene glycol) (PEG) is one of the most widely used....... The work focuses on novel strategies to self assemble PEG thiol monolayers with high graft density. One of the strategies investigated involved backfilling a self assembled layer of 2000 Da PEG thiol with shorter oligo (ethylene glycol) (OEG) thiol molecules to form a mixed monolayer. Detailed quantitative......Microorganisms are one of the most important parts of our ecosystem influencing the sustenance of human society. The beneficial microbes are of high relevance to food industry, development of antibiotics and processing of many raw materials. Mankind has indeed benefitted a lot from large number...

  2. Electrical resistivity of nanoporous gold modified with thiol self-assembled monolayers

    Hakamada, Masataka; Kato, Naoki; Mabuchi, Mamoru


    The electrical resistivity of nanoporous gold (NPG) modified with thiol self-assembled monolayers (SAMs) has been measured at 298 K using a four-probe method. We found that the adsorption of thiol SAMs increases the electrical resistivity of NPG by up to 22.2%. Dependence of the electrical resistivity on the atmosphere (air or water) was also observed in SAMs-modified NPG, suggesting that the electronic states of the tail groups affect the electrons of the binding sulfur and adjacent surface gold atoms. The present results suggest that adsorption of thiol molecules can influence the behavior of the conducting electrons in NPG and that modification of NPG with SAMs may be useful for environmental sensing.

  3. Thiolated pyrimidine nucleotides may interfere thiol groups concentrated at lipid rafts of HIV-1 infected cells.

    Kanizsai, Szilvia; Ongrádi, Joseph; Aradi, János; Nagy, Károly


    Upon HIV infection, cells become activated and cell surface thiols are present in increased number. Earlier we demonstrated in vitro anti-HIV effect of thiolated pyrimidine nucleotide UD29, which interferes thiol function. To further analyse the redox processes required for HIV-1 entry and infection, toxicity assays were performed using HIV-1 infected monolayer HeLaCD4-LTR/ β-gal cells and suspension H9 T cells treated with several thiolated nucleotide derivatives of UD29. Selective cytotoxicity of thiolated pyrimidines on HIV-1 infected cells were observed. Results indicate that thiolated pyrimidine derivates may interfere with -SH (thiol) groups concentrated in lipid rafts of cell membrane and interacts HIV-1 infected (activated) cells resulting in a selective cytotoxicity of HIV-1 infected cells, and reducing HIV-1 entry.

  4. Influence of volatile thiols in the development of blackcurrant aroma in red wine.

    Rigou, Peggy; Triay, Aurélie; Razungles, Alain


    A strong blackcurrant aroma was recently perceived in some red wines originating from the same appellation. Varietal thiols such as 4-mercapto-4-methyl-2-pentanone (4MMP), 3-(mercapto)hexyl acetate (3MHA) and 3-mercapto-1-hexanol (3MH) are compounds potentially responsible for the development of this aroma. In order to demonstrate the correlation between thiols concentrations in red wines and blackcurrant aroma intensity, a multiple variable analysis was realised with thiols concentrations obtained by chemical analysis and blackcurrant aroma intensities obtained by descriptive sensory analysis. The 4MMP concentration was very well correlated to the blackcurrant aroma, and 3MHA and 3MH present at high concentrations act as enhancers of the perception of this aroma. This correlation was further supported after performing a sensory comparison by classification test. The different factors that could impact on the development of blackcurrant aroma in red wine were discussed.

  5. Enhancement in the Glass Transition Temperature in Latent Thiol-Epoxy Click Cured Thermosets

    Dailyn Guzmán


    Full Text Available Tri and tetrafunctional thiol were used as curing agent for diglycidyl ether of bisphenol A (DGEBA catalyzed by a commercially available amine precursor, LC-80. Triglycidyl isocianurate (TGIC was added in different proportions to the mixture to increase rigidity and glass transition temperature (Tg. The cooperative effect of increasing functionality of thiol and the presence of TGIC in the formulation leads to an increased Tg without affecting thermal stability. The kinetics of the curing of mixtures was studied by calorimetry under isothermal and non-isothermal conditions. The latent characteristics of the formulations containing amine precursors were investigated by rheometry and calorimetry. The increase in the functionality of the thiol produces a slight decrease in the storage lifetime of the mixture. The materials obtained with tetrathiol as curing agent showed the highest values of Young’s modulus and Tg.

  6. Hybrid thiol-ene network nanocomposites based on multi(meth)acrylate POSS.

    Li, Liguo; Liang, Rendong; Li, Yajie; Liu, Hongzhi; Feng, Shengyu


    First, multi(meth)acrylate functionalized POSS monomers were synthesized in this paper. Secondly, FTIR was used to evaluate the homopolymerization behaviors of multi(meth)acrylate POSS and their copolymerization behaviors in the thiol-ene reactions with octa(3-mercaptopropyl) POSS in the presence of photoinitiator. Results showed that the photopolymerization rate of multimethacrylate POSS was faster than that of multiacrylate POSS. The FTIR results also showed that the copolymerizations were dominant in the thiol-ene reactions with octa(3-mercaptopropyl) POSS, different from traditional (meth)acrylate-thiol system, in which homopolymerizations were predominant. Finally, the resulted hybrid networks based on POSS were characterized by XRD, FE-SEM, DSC, and TGA. The characterization results showed that hybrid networks based on POSS were homogeneous and exhibited high thermal stability.

  7. Thiol peptides induction in the seagrass Thalassia testudinum (Banks ex Koenig) in response to cadmium exposure

    Alvarez-Legorreta, Teresa [Departamento de Recursos del Mar, CINVESTAV-IPN, Unidad Merida, Apdo. Postal 73-Cordemex, Merida, Yucatan 97310 (Mexico); Mendoza-Cozatl, David; Moreno-Sanchez, Rafael [Departamento de Bioquimica, Instituto Nacional de Cardiologia, Mexico D.F. 14080 (Mexico); Gold-Bouchot, Gerardo [Departamento de Recursos del Mar, CINVESTAV-IPN, Unidad Merida, Apdo. Postal 73-Cordemex, Merida, Yucatan 97310 (Mexico)], E-mail:


    Trace metal accumulation and thiol compounds synthesis as induced by cadmium exposure was studied in the seagrass Thalassia testudinum. Shoots were exposed for 24, 48, 96 and 144 h to several CdCl{sub 2} concentrations (0, 30, 50 and 70 {mu}M). Levels of cadmium, cysteine, glutathione (GSH), {gamma}-glutamylcysteine ({gamma}-EC), and phytochelatin-like peptides were determined in green blades, live sheaths and root/rhizomes tissues. Metal accumulation was dependent on Cd concentration and type of tissue, with green blades showing the highest content followed by live sheaths and root/rhizomes. All tissues experienced an increase in thiol-containing compounds as a response to cadmium exposure. Live sheaths showed the highest levels of cysteine, GSH and {gamma}-EC. This is the first report of induction of thiol peptides, presumably phytochelatins, by a trace metal in a sea grass species.

  8. Serum paraoxonase activity, total thiols levels, and oxidative status in patients with acute brucellosis.

    Esen, Ramazan; Aslan, Mehmet; Kucukoglu, Mehmet Emin; Cıkman, Aytekin; Yakan, Umit; Sunnetcioglu, Mahmut; Selek, Sahbettin


    It is well known that paraoxonase-1 (PON1) activity may decrease during the course of infection and inflammation. The aim of this study was to investigate serum PON1 activity, oxidative status, and thiols levels in patients with acute brucellosis. In addition, we investigated the PON1 phenotype in patients with acute brucellosis. Thirty patients with acute brucellosis and 35 healthy controls were enrolled. Serum paraoxonase and arylesterase activities, thiols levels, lipid hydroperoxide levels, total antioxidant capacity (TAC), total oxidant status (TOS) and oxidative stress index (OSI) were determined. Serum basal and salt-stimulated paraoxonase-arylesterase activities, TAC levels and thiols levels were significantly lower in patients with acute brucellosis than controls (for all, p brucellosis. These results indicate that lower PON1 activity is associated with oxidant-antioxidant imbalance.

  9. Stretching of BDT-gold molecular junctions: Thiol or thiolate termination?

    Souza, Amaury De Melo


    It is often assumed that the hydrogen atoms in the thiol groups of a benzene-1,4-dithiol dissociate when Au-benzene-1,4-dithiol-Au junctions are formed. We demonstrate, by stability and transport property calculations, that this assumption cannot be made. We show that the dissociative adsorption of methanethiol and benzene-1,4-dithiol molecules on a flat Au(111) surface is energetically unfavorable and that the activation barrier for this reaction is as high as 1 eV. For the molecule in the junction, our results show, for all electrode geometries studied, that the thiol junctions are energetically more stable than their thiolate counterparts. Due to the fact that density functional theory (DFT) within the local density approximation (LDA) underestimates the energy difference between the lowest unoccupied molecular orbital and the highest occupied molecular orbital by several electron-volts, and that it does not capture the renormalization of the energy levels due to the image charge effect, the conductance of the Au-benzene-1,4-dithiol-Au junctions is overestimated. After taking into account corrections due to image charge effects by means of constrained-DFT calculations and electrostatic classical models, we apply a scissor operator to correct the DFT energy level positions, and calculate the transport properties of the thiol and thiolate molecular junctions as a function of the electrode separation. For the thiol junctions, we show that the conductance decreases as the electrode separation increases, whereas the opposite trend is found for the thiolate junctions. Both behaviors have been observed in experiments, therefore pointing to the possible coexistence of both thiol and thiolate junctions. Moreover, the corrected conductance values, for both thiol and thiolate, are up to two orders of magnitude smaller than those calculated with DFT-LDA. This brings the theoretical results in quantitatively good agreement with experimental data.

  10. Production of Thiol Species From An Exponential Growth Diatom Under Copper Exposure

    Tang, D.; Shafer, M. M.; Karner, D. A.; Armstrong, D. E.; Schauer, J.


    The intracellar induction of phytochelatins is a well documented response of eukaryotic microorganisms to aqueous metal exposure. The extracellular release of thiolic compounds from algal species has been observed; and in some cases, this release can contribute a significant fraction of the uncharacterized metal-complexing ligands. Glutathione (GSH) or cysteine is among the detectable thiols excreted. A quantitative assessment of the excretion of thiols from algae cells into growth media is needed to assess the significance of biogenic-thiols as a source of strong ligands in natural waters and as a "forgotten" route in sulfur biogeochemical cycle. Unbuffered growth media (e.g., without adding complexing ligand such as EDTA) have only rarely been used to study the possible release of metal-complexing ligands from algal species, and the ligand titration techniques used varied considerably. The majority of culture studies have applied metal-buffered media. A direct comparison of released ligands under buffered and unbuffered conditions is lacking, partially due to the inherent difficulties of the titration methods applied. Using HPLC with fluorescence detection of thiol-monobromobimane derivatives, we were able to follow the dynamic change of GSH released in both media types during algal growth: (1) the cell quotas for thiols and pigments varied (mostly decreases) with growth time. Therefore, pigment-normalized cellular thiol concentrations were more or less conservative. (2) GSH was released into both the EDTA-buffered and -unbuffered growth media at similar concentrations. (3) at similar available Cu concentrations, EDTA possibly enhanced, rather than hindered, the release of GSH.

  11. Surface modification of cyclomatrix polyphosphazene microsphere by thiol-ene chemistry and lectin recognition

    Chen, Chen; Zhu, Xue-yan; Gao, Qiao-ling; Fang, Fei; Huang, Xiao-jun, E-mail:


    Graphical abstract: A new synthetic route leading to polyphosphazene cyclomatrix microsphere with various functional groups has achieved via thiol-ene click modification. Herein, hexacholorocyclophosphazene (HCCP) crosslinked with bisphenol-S and 4,4′-diallyl bisphenol-S to generate broadly dispersed microspheres. Thiol-ene modification under UV irradiation not only presented high efficiency and flexibility for post-functionalization, but also imposed no harm on global morphology and crosslinked skeleton of such microspheres. - Highlights: • Functional polyphosphazene microspheres with high chemical flexibility were synthesized by thiol-ene modification. • Polyphosphazene microspheres possessed high thermal stability. • Glycosylated polyphosphazene microspheres showed affinity to lectin Con-A, which inferred potential application in biomedicine. - Abstract: A new synthetic route leading to functional polyphosphazene cyclomatrix microsphere has been developed via thiol-ene click modification. Hexacholorocyclophosphazene (HCCP) was crosslinked with both bisphenol-S and 4,4′-diallyl bisphenol-S to obtain vinyl polyphosphazene microspheres (VPZM) in order to ensure high crosslinking degree and introduce vinyl moieties. Compared to the microspheres obtained by HCCP and bisphenol-S, the size of VPZM was broadly dispersed from 400 nm to 1.40 μm. Thiol-ene click reactions were carried out to attach functional groups, such as glucosyl, carboxyl, ester and dodecyl groups onto polyphosphazene microspheres, which demonstrated no change in morphology and size after modification. Solid state NMR (SSNMR) and Fourier transform infrared spectoscopy (FT-IR) results showed that the vinyl moieties were introduced in the period of crosslinking and functionalization was also successful via click reactions. Moreover, the microspheres presented a little difference in thermal properties after modification. Concanavalin A (Con-A) fluorescent adsorption was also observed for

  12. Mutagenesis of the redox-active disulfide in mercuric ion reductase: Catalysis by mutant enzymes restricted to flavin redox chemistry

    Distefano, M.D.; Au, K.G.; Walsh, C.T. (Harvard Medical School, Boston, MA (USA))


    Mercuric reductase, a flavoenzyme that possesses a redox-active cystine, Cys{sub 135}Cys{sub 140}, catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, the authors have constructed mutants lacking a redox-active disulfide by eliminating Cys{sub 135} (Ala{sub 135}Cys{sub 140}), Cys{sub 14} (Cys{sub 135}Ala{sub 140}), or both (Ala{sub 135}Ala{sub 140}). Additionally, they have made double mutants that lack Cys{sub 135} (Ala{sub 135}Cys{sub 139}Cys{sub 140}) or Cys{sub 140} (Cys{sub 135}Cys{sub 139}Ala{sub 140}) but introduce a new Cys in place of Gly{sub 139} with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH{sub 2} redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. Preliminary evidence for the Ala{sub 135}Cys{sub 139}Cys{sub 14} mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala{sub 135}Cys{sub 140} protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. They conclude that the Cys{sub 135} and Cys{sub 140} thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate.

  13. Endothelium-dependent vasorelaxation in inhibited by in vivo depletion of vascular thiol levels

    Laursen, J B; Boesgaard, S; Trautner, S;


    Thiols like glutathione may serve as reducing cofactors in the production of nitric oxide (NO) and protect NO from inactivation by radical oxygen species. Depletion of thiol compounds reduces NO-mediated vascular effects in vitro and in vivo. The mechanisms underlying these actions are not clear......, but may involve decreased synthesis of NO and/or increased degradation of NO. This study investigates the effect of glutathione depletion on the response to NO-mediated vasodilation induced by acetylcholine (Ach, 10 micrograms/kg), endothelial NO synthase (eNOS) activity and potential markers of vascular...

  14. Characterization of self-assembled monolayers of porphyrins bearing multiple-thiol and photoelectric response

    Jian Dong Yang; Xiao Quan Lu


    Self-assembled monolayers (SAMs) of thiol-derivatized porphyrin molecules on Au substrate have attracted extensively interest for use in sensing,optoelectronic devices and molecular electronics.In this paper,tetra-[p-(3-mercaptopropyloxy)-phenyl]-porphyrin was synthesized and self-assembled with thiol on Au substrate for porphyrin SAMs (PPS4).The electrochemical results demonstrated that PPS4 could form excellent SAMs on gold surface.Self-assembled nanojunctions of PPS4 were fabricated by using gold nanogap electrodes (gap width:ca.100 nm).With the light on/off,the nanojunctions showed current high/low as nanometer scaled photo switch.

  15. The Nox/Ferric reductase/Ferric reductase-like families of Eumycetes.

    Grissa, Ibtissem; Bidard, Frédérique; Grognet, Pierre; Grossetete, Sandrine; Silar, Philippe


    Reactive Oxygen Species (ROS) are involved in plant biomass degradation by fungi and development of fungal structures. While the ROS-generating NADPH oxidases from filamentous fungi are under strong scrutiny, much less is known about the related integral Membrane (or Ferric) Reductases (IMRs). Here, we present a survey of these enzymes in 29 fungal genomes covering the entire available range of fungal diversity. IMRs are present in all fungal genomes. They can be classified into at least 24 families, underscoring the high diversity of these enzymes. Some are differentially regulated during colony or fruiting body development, as well as by the nature of the carbon source of the growth medium. Importantly, functional characterization of IMRs has been made on proteins belonging to only two families, while nothing or very little is known about the proteins of the other 22 families.

  16. Methylphosphonic Dichloride as Reagent for the Determination of the Enantiomeric Excess of Chiral Thiols. Scope and Limitations

    Strijtveen, Bert; Kellogg, Richard M.; Feringa, Bernard


    Methylphosphonic dichloride, CH3P(=O)Cl2, reacts cleanly and quantitatively with thiols to form dialkylthiophosphonates, CH3P(=O)(SR)2. From the ratio of the integrations of the 31P absorptions in the NMR spectra, the enantiomeric excesses of the thiols can be obtained for the cases that R is chiral

  17. Functionalization of embedded thiol-ene waveguides for evanescent wave-induced fluorescence detection in a microfluidic device

    Feidenhans, Nikolaj A.; Jensen, Thomas Glasdam; Lafleur, Josiane P.;


    We demonstrate the use of functional surface groups inherently present on off-stoichiometric thiol-ene polymers, for site-specific immobilization of biomolecules and detection by evanescent wave-induced fluorescence. An optofluidic chip featuring an embedded thiol-ene waveguide was selectively fu...

  18. Rapid and simple preparation of thiol-ene emulsion-templated monoliths and their application as enzymatic microreactors

    Lafleur, Josiane P; Senkbeil, Silja; Novotny, Jakub;


    electron microscopy showed that the methanol-based emulsion templating process resulted in a network of highly interconnected and regular thiol-ene beads anchored solidly inside thiol-ene microchannels. Surface area measurements indicate that the monoliths are macroporous, with no or little micro...

  19. 4-Dimethylaminoazobenzenes: carcinogenicities and reductive cleavage by microsomal azo reductase.

    Lambooy, J P; Koffman, B M


    Twenty-four 4-dimethylaminoazobenzenes (DABs) in which systematic structural modifications have been made in the prime ring have been studied for substrate specificity for microsomal azo reductase. The DABs were also evaluated for carcinogenicity and it was found that there was no correlation between carcinogenicity and extent of azo bond cleavage by azo reductase. While any substituent in the prime ring reduces the rate of cleavage of the azo bond relative to the unsubstituted dye, there is a correlation between substituent size and susceptibility to the enzyme. Substituent size was also found to be a significant factor in the induction of hepatomas by the dyes. Preliminary studies have shown that there appears to be a positive correlation between microsomal riboflavin content and the activity of the azo reductase.

  20. Preparation of a novel carboxyl stationary phase by "thiol-ene" click chemistry for hydrophilic interaction chromatography.

    Peng, Xi-Tian; Liu, Tao; Ji, Shu-Xian; Feng, Yu-Qi


    A novel carboxyl-bonded silica stationary phase was prepared by "thiol-ene" click chemistry. The resultant Thiol-Click-COOH phase was evaluated under hydrophilic interaction liquid chromatography (HILIC) mobile phase conditions. A comparison of the chromatographic performance of Thiol-Click-COOH and pure silica columns was performed according to the retention behaviors of analytes and the charged state of the stationary phases. The results indicated that the newly developed Thiol-Click-COOH column has a higher surface charge and stronger hydrophilicity than the pure silica column. Furthermore, the chromatographic behaviors of five nucleosides on the Thiol-Click-COOH phase were investigated in detail. Finally, a good separation of 13 nucleosides and bases, and four water-soluble vitamins was achieved.

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

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


    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.


    Klimenko S.B.


    Full Text Available Nitrates are the basic source of nitrogen for the majority of plants. Absorption and transformation of nitrates in plants are determined by external conditions and, first of all, temperature and light intensity. The influence of the temperature increasing till +40 0С on activity of nitrate reductase was studied. It is shown, that the rise of temperature was accompanied by sharp decrease of activity nitrate reductase in leaves of winter wheat, what, apparently, occurred for the account deactivations of enzyme and due to its dissociation.

  3. Determination of low molecular weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography

    Fahey, Robert C.; Newton, Gerald L.


    Methods are described for the preparation and high-performance liquid chromatography (HPLC) analysis of monobromobimane derivatives of low molecular weight thiols in extracts of biological samples. Typical problems encountered in the development and application of these methods are discussed. Analysis of mung bean extract is used as an example.

  4. Gold Nanoparticles Protected with Thiol-Derivatized Amphiphilic Poly( -caprolactone)-b-poly(acrylic acid)

    Javakhishvili, Irakli; Hvilsted, Søren


    chromatography (SEC), nuclear magnetic resonance eR NMR) and infrared (FT IR) spectroscopy. The capacity of the resulting block copolymer in preparation of monolayer-protected gold nanoparticles has been examined by reduction of a gold salt in the presence of this macroligand under thiol-deficient conditions...

  5. A New Pyrene-Spacer-Maleimide Dyad for Sensing Molecules with One or Two Thiol Groups

    WANG,Zhuo; ZHANG,De-Qing; ZHU,Dao-Ben


    A new pyrene-spacer-maleimide dyad 1 was used to selectively detect cysteine in the presence of other amino acids, and sequentially react with dithiols to generate the molecule with two pyrene units showing typical excimer fluorescence. Accordingly, dyad 1 was able to differentiate molecules with one or two thiol groups.

  6. Association of bilirubin and protein thiols in relation to copper and ceruloplasmin in hyperbilirubinemic patients

    Mungli Prakash; Jeevan K Shetty; Roshan D'Souza; Suhasa Upadhya; Vijay Kumar


    Objective:Bilirubin is a double edged sword in biological system,acting as a toxic molecule and cytoprotecrant.Unconjugated bilirubin is proved to show antioxidant activity in vitro and in vivo.In the current work we tried to know the relationship between both conjugated and unconjugated bilirubin with copper and protein thiols in patients with hyperbilirnbinemia.Methods:Study was conducted on 56 hyperbilirubinemic cases and 56 healthy controls.Serum copper,ceruloplasmin,protein thiols,total bilirubin,conjugated and unconjugated bilirubin,unconjugated bilimbin/albumin ratio,total protein,albumin,AST,ALT and ALP were estimated.Results:There was significant increase in serum copper,total bilirubin,conjugated and unconjugated bilimbin.uriconjugated bilirubin/albumin ratio,AST,ALT,and ALP,and decrease in serum ceruloplasmin,protein thiols,total protein,and albumin in hyperbilimbinemic cases when compared to healthy controls.Conjugated bilimbin correlated positively with liver enzymes AST and ALP,and negatively with protein thials,total protein and albumin.Unconjugated bilirubin correlated positively with ALT.Protein thiols correlated negatively with copper and positively with ceruloplasmin,and also correlated negativelv with liver enzymes like AST,ALT and ALP,and positively with total protein and albumin.Conclusion:Combination of elevated levels of trace elements like copper and availability of reducing agent like bilimbin may prove deleterious by generating free radicals.

  7. Synthesis and Microstructural Investigations of Organometallic Pd(II Thiol-Gold Nanoparticles Hybrids

    Cervellino Antonio


    Full Text Available Abstract In this work the synthesis and characterization of gold nanoparticles functionalized by a novel thiol-organometallic complex containing Pd(II centers is presented. Pd(II thiol,trans, trans-[dithiolate-dibis(tributylphosphinedipalladium(II-4,4′-diethynylbiphenyl] was synthesized and linked to Au nanoparticles by the chemical reduction of a metal salt precursor. The new hybrid made of organometallic Pd(II thiol-gold nanoparticles, shows through a single S bridge a direct link between Pd(II and Au nanoparticles. The size-control of the Au nanoparticles (diameter range 2–10 nm was achieved by choosing the suitable AuCl4 −/thiol molar ratio. The size, strain, shape, and crystalline structure of these functionalized nanoparticles were determined by a full-pattern X-ray powder diffraction analysis, high-resolution TEM, and X-ray photoelectron spectroscopy. Photoluminescence spectroscopy measurements of the hybrid system show emission peaks at 418 and 440 nm. The hybrid was exposed to gaseous NO x with the aim to evaluate the suitability for applications in sensor devices; XPS measurements permitted to ascertain and investigate the hybrid –gas interaction.

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

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


    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.

  9. Synthesis of Novel Fluorescence reagents and Their Application in Determination of Thiol Compounds

    LIANG Shu-Cai; WANG Hong; ZHANG Hua-Shan; HU Xian-Ming


    @@ The identification and determination of thiol compounds is essential both for the clinical diagnosis and the con trol of diseases because alteration of their concentrations in biological systems are somehow responsive for some diseases such as myocardial infarction, diabetes, peripheral vascular and so on. [1

  10. Sytematic Study of the Adsorption of Thiol Molecules on Noble-Metal Nanoparticles

    Barron, H.; Hidalgo, F.; Fernandez-Seivane, L.; Noguez, C.; Lopez-Lozano, X.


    The study of the interaction between nanoparticles and different types of ligands has been intensively investigated in the last years due to the potential contribution of their properties to the nanotechnology device design. These properties have opened new research fields like plasmonics, with interesting applications in optics, electronics, biophysics, medicine, pharmacology and materials science. Self-assembly monolayers have been thoroughly studied at experimental and theoretical level on extended (111) gold and silver surfaces. However, nanoparticle and molecule properties after the adsorption are still not well understood due to the different factors involved in this process such as the adsorption sites, size and element type of the nanoparticle. In this work we have performed a systematic study of the adsorption of methyl-thiol molecules on Au55 and Ag55 clusters through density functional theory calculations with the SIESTA code. Different adsorption modes of the methyl-thiol molecule on Au55 and Ag55 were considered. In general, for both type of nanoparticles, the methyl-thiol molecule prefers to be adsorbed on the Bridge sites. These results provide valuable information of the structural and electronic properties of methyl-thiol passivated Au and Ag nanoparticles.


    The removal of mercury(II) from wastewaters (coal-fired utility plant scrubber solutions) using a thiol functional organoceramic composite (SOL-AD-IV) is investigated. A simulant is employed as a surrogate to demonstrate the removal of mercury from real waste solutions. Equilibri...

  12. Differential Labeling of Free and Disulfide-Bound Thiol Functions in Proteins

    Seiwert, Bettina; Hayen, Heiko; Karst, Uwe


    A method for the simultaneous determination of the number of free cysteine groups and disulfide-bound cysteine groups in proteins has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. Liquid chromatography/electrochem

  13. An electrophoretic profiling method for thiol-rich phytochelatins and metallothioneins.

    Fan, Teresa W M; Lane, Andrew N; Higashi, Richard M


    Thiol-rich peptides such as phytochelatins (PCs) and metallothioneins (MTs) are important cellular chelating agents which function in metal detoxification and/or homeostasis. The variations in molecular sizes and lack of chromophores of these peptides make their analysis difficult. This paper reports an electrophoresis-based method for a broad screen of thiol-rich peptides and proteins. The method uses the thiol-selective fluorescent tag, monobromobimane, coupled with Tricine--sodium dodecyl sulphate--urea polyacrylamide gel electrophoresis for a sensitive determination of both PCs and MTs. Results for PCs were confirmed by two-dimensional NMR and HPLC-tandem MS analyses. Sample throughput is substantially improved over chromatography-based methods through parallel sample analysis in 1 h of electrophoretic separation. The method is versatile in that peptides ranging from glutathione to large proteins can be analysed by simple modification(s) of the extraction and electrophoretic conditions, and the nature of the method supports serendipitous detection of unexpected or novel thiol metabolites.

  14. Thiol Redox Transitions in Cell Signaling: a Lesson from N-Acetylcysteine

    Tiziana Parasassi


    Full Text Available The functional status of cells is under the control of external stimuli affecting the function of critical proteins and eventually gene expression. Signal sensing and transduction by messengers to specific effectors operate by post-translational modification of proteins, among which thiol redox switches play a fundamental role that is just beginning to be understood. The maintenance of the redox status is, indeed, crucial for cellular homeostasis and its dysregulation towards a more oxidized intracellular environment is associated with aberrant proliferation, ultimately related to diseases such as cancer, cardiovascular disease, and diabetes. Redox transitions occur in sensitive cysteine residues of regulatory proteins relevant to signaling, their evolution to metastable disulfides accounting for the functional redox switch. N-acetylcysteine (NAC is a thiol-containing compound that is able to interfere with redox transitions of thiols and, thus, in principle, able to modulate redox signaling. We here review the redox chemistry of NAC, then screen possible mechanisms to explain the effects observed in NAC-treated normal and cancer cells; such effects involve a modification of global gene expression, thus of functions and morphology, with a leitmotif of a switch from proliferation to terminal differentiation. The regulation of thiol redox transitions in cell signaling is, therefore, proposed as a new tool, holding promise not only for a deeper explanation of mechanisms, but indeed for innovative pharmacological interventions.

  15. Development of ionic gels using thiol-based monomers in ionic liquid

    Ahmed, Kumkum; Naga, Naofumi; Kawakami, Masaru; Furukawa, Hidemitsu


    Ionic gels (IGs) using ionic liquids (ILs) can propose diverse applications in the field of optics, sensors and separation have opened wide prospects in materials science. ILs have attracted remarkable interest for gel polymer electrolytes and batteries based on their useful properties such as non-volatility, non-flammability, a wide electrochemical window, high thermal stability and a high ionic conductivity. The formation of gel in IL media makes it possible to immobilize ILs within organic or inorganic matrices and to take advantage of their unique properties in the solid state, thus eliminating some shortcomings related to shaping and risk of leakage. In this work for the first time we used multifunctional thiol monomers having uniform structure and good compatibility with the IL of our interest. Therefore we focused on developing thiol monomer-based IGs using multifunctional thiol monomers and acrylate crosslinkers utilizing thiol-ene reaction between monomer and crosslinking molecules in an IL medium and characterize their physico-chemical properties like thermal, conductive, mechanical properties etc.. This work has been focused mainly to improve the mechanical strength of IGs and make prospects of IGs in tribology and lubricants.

  16. Aroma extraction dilution analysis of Sauternes wines. Key role of polyfunctional thiols.

    Bailly, Sabine; Jerkovic, Vesna; Marchand-Brynaert, Jacqueline; Collin, Sonia


    The aim of the present work was to investigate Sauternes wine aromas. In all wine extracts, polyfunctional thiols were revealed to have a huge impact. A very strong bacon-petroleum odor emerged at RI = 845 from a CP-Sil5-CB column. Two thiols proved to participate in this perception: 3-methyl-3-sulfanylbutanal and 2-methylfuran-3-thiol. A strong synergetic effect was evidenced between the two compounds. The former, never mentioned before in wines, and not found in the musts of this study, is most probably synthesized during fermentation. 3-Methylbut-2-ene-1-thiol, 3-sulfanylpropyl acetate, 3-sulfanylhexan-1-ol, and 3-sulfanylheptanal also contribute to the global aromas of Sauternes wines. Among other key odorants, the presence of a varietal aroma (alpha-terpineol), sotolon, fermentation alcohols (3-methylbutan-1-ol and 2-phenylethanol) and esters (ethyl butyrate, ethyl hexanoate, and ethyl isovalerate), carbonyls (trans-non-2-enal and beta-damascenone), and wood flavors (guaiacol, vanillin, eugenol, beta-methyl-gamma-octalactone, and Furaneol) is worth stressing.

  17. Solvent effects in adsorption of alkyl thiols on gold structures: A molecular simulation study

    Pool, R.; Schapotschnikow, P.Z.; Vlugt, T.J.H.


    We carried out Monte Carlo simulations of gold nanocrystals (NCs) and (111) slabs covered with alkyl thiols, with and without explicit solvent (n-hexane), at T ) 300 K. Adsorption isotherms for propane- and octanethiol showed a phase behavior measured previously in experiments. Comparison of the ads

  18. Quantitative interpretation of the transition voltages in gold-poly(phenylene) thiol-gold molecular junctions

    Wu, Kunlin


    The transition voltage of three different asymmetric Au/poly(phenylene) thiol/Au molecular junctions in which the central molecule is either benzene thiol, biphenyl thiol, or terphenyl thiol is investigated by first-principles quantum transport simulations. For all the junctions, the calculated transition voltage at positive polarity is in quantitative agreement with the experimental values and shows weak dependence on alterations of the Au-phenyl contact. When compared to the strong coupling at the Au-S contact, which dominates the alignment of various molecular orbitals with respect to the electrode Fermi level, the coupling at the Au-phenyl contact produces only a weak perturbation. Therefore, variations of the Au-phenyl contact can only have a minor influence on the transition voltage. These findings not only provide an explanation to the uniformity in the transition voltages found for π-conjugated molecules measured with different experimental methods, but also demonstrate the advantage of transition voltage spectroscopy as a tool for determining the positions of molecular levels in molecular devices. © 2013 AIP Publishing LLC.

  19. Thiol-ene click chemistry: computational and kinetic analysis of the influence of alkene functionality.

    Northrop, Brian H; Coffey, Roderick N


    The influence of alkene functionality on the energetics and kinetics of radical initiated thiol-ene click chemistry has been studied computationally at the CBS-QB3 level. Relative energetics (ΔH°, ΔH(++), ΔG°, ΔG(++)) have been determined for all stationary points along the step-growth mechanism of thiol-ene reactions between methyl mercaptan and a series of 12 alkenes: propene, methyl vinyl ether, methyl allyl ether, norbornene, acrylonitrile, methyl acrylate, butadiene, methyl(vinyl)silanediamine, methyl crotonate, dimethyl fumarate, styrene, and maleimide. Electronic structure calculations reveal the underlying factors that control activation barriers for propagation and chain-transfer processes of the step-growth mechanism. Results are further extended to predict rate constants for forward and reverse propagation and chain-transfer steps (k(P), k(-P), k(CT), k(-CT)) and used to model overall reaction kinetics. A relationship between alkene structure and reactivity in thiol-ene reactions is derived from the results of kinetic modeling and can be directly related to the relative energetics of stationary points obtained from electronic structure calculations. The results predict the order of reactivity of alkenes and have broad implications for the use and applications of thiol-ene click chemistry.

  20. Two-Dimensional Structure of Disulfides and Thiols on Gold(111)

    Nelles, Gabriele; Schönherr, Holger; Jaschke, Manfred; Wolf, Heiko; Schaub, Matthias; Kuther, Jörg; Tremel, Wolfgang; Bamberg, Ernst; Ringsdorf, Helmut; Butt, Hans-Jürgen


    In order to find factors which determine the two-dimensional structure of self-assembled monolayers (SAMs), several classes of thiols and disulfides on gold (111) have been investigated by atomic force microscopy (AFM). SAMs were formed from a series of symmetrical and asymmetrical diethylalkanoate

  1. Thimerosal Exposure and the Role of Sulfation Chemistry and Thiol Availability in Autism

    Mark R. Geier


    Full Text Available Autism spectrum disorder (ASD is a neurological disorder in which a significant number of the children experience a developmental regression characterized by a loss of previously acquired skills and abilities. Typically reported are losses of verbal, nonverbal, and social abilities. Several recent studies suggest that children diagnosed with an ASD have abnormal sulfation chemistry, limited thiol availability, and decreased glutathione (GSH reserve capacity, resulting in a compromised oxidation/reduction (redox and detoxification capacity. Research indicates that the availability of thiols, particularly GSH, can influence the effects of thimerosal (TM and other mercury (Hg compounds. TM is an organomercurial compound (49.55% Hg by weight that has been, and continues to be, used as a preservative in many childhood vaccines, particularly in developing countries. Thiol-modulating mechanisms affecting the cytotoxicity of TM have been identified. Importantly, the emergence of ASD symptoms post-6 months of age temporally follows the administration of many childhood vaccines. The purpose of the present critical review is provide mechanistic insight regarding how limited thiol availability, abnormal sulfation chemistry, and decreased GSH reserve capacity in children with an ASD could make them more susceptible to the toxic effects of TM routinely administered as part of mandated childhood immunization schedules.

  2. Roll-to-plate fabrication of microfluidic devices with rheology-modified thiol-ene resins

    Senkbeil, Silja; Aho, Johanna; Yde, Leif


    In this paper, the replication possibilities of microfluidic channels by UV-roll-to-plate fabrication were investigated and a study of rheology-modified thiol-ene for the application in such a UV-roll-to-plate setup was conducted. The system allows the manufacture of channels with aspect ratios...

  3. Gold Nanoparticles Protected with Thiol-Derivatized Amphiphilic Poly(epsilon-caprolactone)-b-poly(acrylic acid)

    Javakhishvili, Irakli; Hvilsted, Søren


    Amphiphilic poly(epsilon-caprolactone)-b-poly(acrylic acid) (HS-PCL-b-PAA) with a thiol functionality in the PCL terminal has been prepared in a novel synthetic cascade. Initially, living anionic ring-opening polymerization (ROP) of epsilon-caprolactone (epsilon-CL) employing the difunctional...

  4. Influence cadmium ions on the synthesis of thiol compounds for flax

    Olga Krystofova


    Full Text Available Evaluation of the effectiveness of phytoremediation technologies isvery difficult. One way to quickly and inexpensively identifyphytoremediation potential of plants is found easily detectablemarker. In our study, we examined the content of thiol compoundsin plants, of Flax effects of various concentrations of cadmium ions.

  5. Inhibition of Albendazole and Oxfendazole on the Activity of Fumaric Reductase in Cysticercus cellulosae

    GAO Xue-jun; LI Qing-zhang; LI Xia


    The activity of fumaric reductase in Cysticercus cellulosae tissue homogenate with albendazole and oxfendazole individually was detected. Results showed that the two kinds of drugs both could inhabite the activity of fumaric reductase. The results indicate that the mechanism of action of benzimidazole carbamate drugs is probably inhabiting the complex of fumaric reductase noncompetently, thus lead to the exhaostion of energy and death.

  6. Transdermal thiol-acrylate polyethylene glycol hydrogel synthesis using near infrared light

    Chung, Solchan; Lee, Hwangjae; Kim, Hyung-Seok; Kim, Min-Gon; Lee, Luke P.; Lee, Jae Young


    Light-induced polymerization has been widely applied for hydrogel synthesis, which conventionally involves the use of ultraviolet or visible light to activate a photoinitiator for polymerization. However, with these light sources, transdermal gelation is not efficient and feasible due to their substantial interactions with biological systems, and thus a high power is required. In this study, we used biocompatible and tissue-penetrating near infrared (NIR) light to remotely trigger a thiol-acrylate reaction for efficient in vivo gelation with good controllability. Our gelation system includes gold nanorods as a photothermal agent, a thermal initiator, diacrylate polyethylene glycol (PEG), and thiolated PEG. Irradiation with a low-power NIR laser (0.3 W cm-2) could induce gelation via a mixed-mode reaction with a small increase in temperature (~5 °C) under the optimized conditions. We also achieved successful transdermal gelation via the NIR-assisted photothermal thiol-acryl reactions. This new type of NIR-assisted thiol-acrylate polymerization provides new opportunities for in situ hydrogel formation for injectable hydrogels and delivery of drugs/cells for various biomedical applications.Light-induced polymerization has been widely applied for hydrogel synthesis, which conventionally involves the use of ultraviolet or visible light to activate a photoinitiator for polymerization. However, with these light sources, transdermal gelation is not efficient and feasible due to their substantial interactions with biological systems, and thus a high power is required. In this study, we used biocompatible and tissue-penetrating near infrared (NIR) light to remotely trigger a thiol-acrylate reaction for efficient in vivo gelation with good controllability. Our gelation system includes gold nanorods as a photothermal agent, a thermal initiator, diacrylate polyethylene glycol (PEG), and thiolated PEG. Irradiation with a low-power NIR laser (0.3 W cm-2) could induce gelation

  7. Thiol-disulfide redox equilibria of glutathione metaboloma compounds investigated by tandem mass spectrometry.

    Rubino, Federico M; Pitton, Marco; Caneva, Enrico; Pappini, Marco; Colombi, Antonio


    The thiol group of cysteine plays a pivotal role in structural and functional biology. We use mass spectrometry to study glutathione-related homo- and heterodimeric disulfides, aiming at understanding the factors affecting the redox potentials of different disulfide/thiol pairs. Several electrospray ionization (ESI)-protonated disulfides of cysteamine, cysteine, penicillamine, N-acetylcysteine, N-acetylpenicillamine, gammaGluCySH, HSCyGly, and glutathione were analyzed on a triple quadrupole instrument to measure their energy-resolved tandem mass spectra. Fission of the disulfide bond yields RSH*H(+) and RS(+) ions. The logarithm of the intensity ratio of the RS(+)/RSH*H(+) fragments in homodimeric disulfides is proportional to the normal reduction potential of their RSSR/RSH pairs determined by nuclear magnetic resonance (NMR) in solution, the more reducing ones yielding the higher ratios. Also in some R(1)S-SR(2) disulfides, the ratio of the intensities of the RSH + H(+) and RS(+) ions of each participating thiol shows a linear relationship with the Nernst equation potential difference of the corresponding redox pairs. This behavior allows us to measure the redox potentials of some disulfide/thiol pairs by using different thiol-reducing probes of known oxidoreductive potential as reference. To assist understanding of the fission mechanism of the disulfide bond, the fragments tentatively identified as 'sulfenium' were themselves fragmented; accurate mass measurement of the resulting second-generation fragments demonstrated a loss of thioformaldehyde, thus supporting the assigned structure of this elusive intermediate of the oxidative stress pathway. Understanding this fragmentation process allows us to employ this technique with larger molecules to measure by mass spectrometry the micro-redox properties of different disulfide bonds in peptides with catalytic and signaling biological activity.

  8. Aerobic nitric oxide-induced thiol nitrosation in the presence and absence of magnesium cations.

    Kolesnik, Bernd; Heine, Christian L; Schmidt, Renate; Schmidt, Kurt; Mayer, Bernd; Gorren, Antonius C F


    Although different routes for the S-nitrosation of cysteinyl residues have been proposed, the main in vivo pathway is unknown. We recently demonstrated that direct (as opposed to autoxidation-mediated) aerobic nitrosation of glutathione is surprisingly efficient, especially in the presence of Mg(2+). In the present study we investigated this reaction in greater detail. From the rates of NO decay and the yields of nitrosoglutathione (GSNO) we estimated values for the apparent rate constants of 8.9 ± 0.4 and 0.55 ± 0.06 M(-1)s(-1) in the presence and absence of Mg(2+). The maximum yield of GSNO was close to 100% in the presence of Mg(2+) but only about half as high in its absence. From this observation we conclude that, in the absence of Mg(2+), nitrosation starts by formation of a complex between NO and O2, which then reacts with the thiol. Omission of superoxide dismutase (SOD) reduced by half the GSNO yield in the absence of Mg(2+), demonstrating O2(-) formation. The reaction in the presence of Mg(2+) seems to involve formation of a Mg(2+)•glutathione (GSH) complex. SOD did not affect Mg(2+)-stimulated nitrosation, suggesting that no O2(-) is formed in that reaction. Replacing GSH with other thiols revealed that reaction rates increased with the pKa of the thiol, suggesting that the nucleophilicity of the thiol is crucial for the reaction, but that the thiol need not be deprotonated. We propose that in cells Mg(2+)-stimulated NO/O2-induced nitrosothiol formation may be a physiologically relevant reaction.

  9. Synthesis of hyperbranched polypeptide and PEO block copolymer by consecutive thiol-yne chemistry.

    Chang, Xiao; Dong, Chang-Ming


    Hyperbranched poly(ε-benzyloxycarbonyl-L-lysine) (HPlys) with multiple alkyne peripheries was synthesized through the click polycondensation of an AB2 type Plys macromonomer with α-thiol and ω-alkyne terminal groups (thiol is the A unit, and each π bond in alkyne is the B unit), and the resulting HPlys was further conjugated with thiol-termined poly(ethylene oxide) (PEO) to generate HPlys-b-PEO block copolymer by consecutive thiol-yne chemistry. Their molecular structures and physical properties were characterized in detail by FT-IR, (1)H NMR, gel permeation chromatography, differential scanning calorimetry, wide-angle X-ray diffraction, and polarized optical microscopy. HPlys and HPlys-b-PEO mainly assumed an α-helix conformation similar to the linear precursors, while the liquid crystalline phase transition of Plys segment disappeared within HPlys and HPlys-b-PEO. HPlys-b-PEO self-assembled into nearly spherical micelles in aqueous solution, while it gave a 5-fold lower critical aggregation concentration (8.9 × 10(-3) mg/mL) than a linear counterpart (4.5 × 10(-2) mg/mL), demonstrating a dendritic topology effect. Compared with a linear counterpart, HPlys-b-PEO gave a higher drug-loading capacity and efficiency for the anticancer drug doxorubicin (DOX) and a slower drug-release rate with an improved burst-release profile, enabling them useful for drug delivery systems. Importantly, this work provides a versatile strategy for the synthesis of hyperbranched polypeptides and related block copolymers by utilizing thiol-yne chemistry.

  10. Cooperative functions of manganese and thiol redox system against oxidative stress in human spermatozoa

    Amrit Kaur Bansal


    Full Text Available Aims: In this study, the effects of 0.1 mM Mn 2+ on thiol components (total thiols [TSH], glutathione reduced [GSH], glutathione oxidized [GSSG] and redox ratio [GSH/ GSSG] have been determined in human spermatozoa. Settings and Design: The subjects of the study were healthy males having more than 75% motility and 80 x 10 6 sperms/mL. Materials and Methods: Fresh semen was suspended in phosphate-buffered saline (PBS (pH 7.2 and this suspension was divided into eight equal fractions. All fractions, control (containing PBS and experimental (treated/untreated with [ferrous ascorbate, FeAA - 200 FeSO 4 μM, 1000 μM ascorbic acid, nicotine (0.5 mM and FeAA + nicotine], supplemented/unsupplemented with Mn 2+ [0.1 mM], were incubated for 2 h at 378C. These fractions were assessed for determining the thiol components. Statistical Analysis: The data were statistically analyzed by Students " t" test. Results and Conclusions: Ferrous ascorbate, nicotine and ferrous ascorbate + nicotine induced oxidative stress and decreased GSH and redox ratio (GSH/GSSG ratio but increased the TSH and GSSG levels. Mn 2+ supplementation improved TSH, GSH and redox ratio (GSH/GSSG but decreased the GSSG level under normal and oxidative stress conditions. Thiol groups serve as defense mechanisms of sperm cells to fight against oxidative stress induced by stress inducers such as ferrous ascorbate, nicotine and their combination (ferrous ascorbate + nicotine. In addition, Mn 2+ supplementation maintains the thiol level by reducing oxidative stress.

  11. Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones.

    Clemente, Maria R; Bustos-Sanmamed, Pilar; Loscos, Jorge; James, Euan K; Pérez-Rontomé, Carmen; Navascués, Joaquín; Gay, Marina; Becana, Manuel


    In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1-48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24-48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses.

  12. Piezo dispensed microarray of multivalent chelating thiols for dissecting complex protein-protein interactions.

    Klenkar, Goran; Valiokas, Ramûnas; Lundström, Ingemar; Tinazli, Ali; Tampé, Robert; Piehler, Jacob; Liedberg, Bo


    The fabrication of a novel biochip, designed for dissection of multiprotein complex formation, is reported. An array of metal chelators has been produced by piezo dispensing of a bis-nitrilotriacetic acid (bis-NTA) thiol on evaporated gold thin films, prestructured with a microcontact printed grid of eicosanethiols. The bis-NTA thiol is mixed in various proportions with an inert, tri(ethylene glycol) hexadecane thiol, and the thickness and morphological homogeneity of the dispensed layers are characterized by imaging ellipsometry before and after back-filling with the same inert thiol and subsequent rinsing. It is found that the dispensed areas display a monotonic increase in thickness with increasing molar fraction of bis-NTA in the dispensing solution, and they are consistently a few Angströms thicker than those prepared at the same molar fraction by solution self-assembly under equilibrium-like conditions. The bulkiness of the bis-NTA tail group and the short period of time available for chemisorption and in-plane organization of the dispensed thiols are most likely responsible for the observed difference in thickness. Moreover, the functional properties of this biochip are demonstrated by studying multiple protein-protein interactions using imaging surface plasmon resonance. The subunits of the type I interferon receptor are immobilized as a composition array determined by the surface concentration of bis-NTA in the array elements. Ligand dissociation kinetics depends on the receptor surface concentration, which is ascribed to the formation of a ternary complex by simultaneous interaction of the ligand with the two receptor subunits. Thus, multiplexed monitoring of binding phenomena at various compositions (receptor densities) offers a powerful tool to dissect protein-protein interactions.

  13. Direct Electrochemistry With Nitrate Reductase in Chitosan Films

    Xiao Xia CHEN; Jing Bo HU; Hong WU; Hui Bo SHAO


    Stable films made from chitosan(CS)on pyrolytic graphite electrode(PGE)gave direct electrochemistry for incorporated enzyme nitrate reductase(NR).Cyclic voltammetry of CS/NR films showed a pair of well-defined and nearly reversible redox peaks at about-0.430 V vs.SCE at pH 7.0 phosphate buffers.

  14. Plasmid-encoded diacetyl (acetoin) reductase in Leuconostoc pseudomesenteroides

    Rattray, Fergal P; Myling-Petersen, Dorte; Larsen, Dianna;


    ) reductases reported previously. Downstream of the butA gene of L. pseudomesenteroides, but coding in the opposite orientation, a putative DNA recombinase was identified. A two-step PCR approach was used to construct FPR02, a butA mutant of the wild-type strain, CHCC2114. FPR02 had significantly reduced...

  15. Bidirectional catalysis by copper-containing nitrite reductase

    Wijma, HJ; Canters, GW; de Vries, S; Verbeet, MP


    The copper-containing nitrite reductase from Alcaligenes faecalis S-6 was found to catalyze the oxidation of nitric oxide to nitrite, the reverse of its physiological reaction. Thermodynamic and kinetic constants with the physiological electron donor pseudoazurin were determined for both directions

  16. Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene

    Edman, J C; Edman, U; Cao, Mi-Mi;


    Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinii is a member of the Fungi...

  17. The effect of copper on human erythrocyte glutathione reductase

    Flikweert, J.P.; Hoorn, R.K.J.; Staal, Gerard E.J.


    1. 1. The influence of copper on purified human erythrocyte glutathione reductase (E.C. was studied. The holoenzyme was inhibited at low oxidized glutathione (GSSG) concentrations. At a glutathione concentration of 1 mM and higher no inhibition at all was found. The inhibition was independe

  18. The intramolecular electron transfer between copper sites of nitrite reductase

    Farver, O; Eady, R R; Abraham, Z H


    The intramolecular electron transfer (ET) between the type 1 Cu(I) and the type 2 Cu(II) sites of Alcaligenes xylosoxidans dissimilatory nitrite reductase (AxNiR) has been studied in order to compare it with the analogous process taking place in ascorbate oxidase (AO). This internal process...

  19. Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells

    Johnson, T. C.; Cao, R. Q.; Kung, J. E.; Buchanan, B. B.


    Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system--NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC more effectively than fructose-1,6-bisphosphatase (EC NADP-thioredoxin reductase (EC was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.

  20. 3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).

    Sheldon, P S; Kekwick, R G; Smith, C G; Sidebottom, C; Slabas, A R


    3-Oxoacyl-[ACP] reductase (E.C., alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.

  1. Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 1: Copper-Catalyzed Oxidation.

    Kreitman, Gal Y; Danilewicz, John C; Jeffery, David W; Elias, Ryan J


    Sulfidic off-odors as a result of hydrogen sulfide (H2S) and low-molecular-weight thiols are commonly encountered in wine production. These odors are usually removed by the process of Cu(II) fining, a process that remains poorly understood. The present study aims to elucidate the underlying mechanisms by which Cu(II) interacts with H2S and thiol compounds (RSH) under wine-like conditions. Copper complex formation was monitored along with H2S, thiol, oxygen, and acetaldehyde concentrations after the addition of Cu(II) (50 or 100 μM) to air-saturated model wine solutions containing H2S, cysteine, 6-sulfanylhexan-1-ol, or 3-sulfanylhexan-1-ol (300 μM each). The presence of H2S and thiols in excess to Cu(II) led to the rapid formation of ∼1.4:1 H2S/Cu and ∼2:1 thiol/Cu complexes, resulting in the oxidation of H2S and thiols and reduction of Cu(II) to Cu(I), which reacted with oxygen. H2S was observed to initially oxidize rather than form insoluble copper sulfide. The proposed reaction mechanisms provide insight into the extent to which H2S can be selectively removed in the presence of thiols in wine.

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

    M. Ryan Smith


    Full Text Available 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 24 h 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 (10 µM 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

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

    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


    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

  4. Crystal structures of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their relationship to isoflavone reductases

    Min, Tongpil; Kasahara, Hiroyuki; Bedgar, Diana L.; Youn, Buhyun; Lawrence, Paulraj K.; Gang, David R.; Halls, Steven C.; Park, HaJeung; Hilsenbeck, Jacqueline L.; Davin, Laurence B.; Lewis, Norman G.; Kang, ChulHee


    Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

  5. (+)-Pinoresinol/(+)-lariciresinol reductase from Forsythia intermedia. Protein purification, cDNA cloning, heterologous expression and comparison to isoflavone reductase.

    Dinkova-Kostova, A T; Gang, D R; Davin, L B; Bedgar, D L; Chu, A; Lewis, N G


    Lignans are a widely distributed class of natural products, whose functions and distribution suggest that they are one of the earliest forms of defense to have evolved in vascular plants; some, such as podophyllotoxin and enterodiol, have important roles in cancer chemotherapy and prevention, respectively. Entry into lignan enzymology has been gained by the approximately 3000-fold purification of two isoforms of (+)-pinoresinol/(+)-lariciresinol reductase, a pivotal branchpoint enzyme in lignan biosynthesis. Both have comparable ( approximately 34.9 kDa) molecular mass and kinetic (Vmax/Km) properties and catalyze sequential, NADPH-dependent, stereospecific, hydride transfers where the incoming hydride takes up the pro-R position. The gene encoding (+)-pinoresinol/(+)-lariciresinol reductase has been cloned and the recombinant protein heterologously expressed as a functional beta-galactosidase fusion protein. Its amino acid sequence reveals a strong homology to isoflavone reductase, a key branchpoint enzyme in isoflavonoid metabolism and primarily found in the Fabaceae (angiosperms). This is of great evolutionary significance since both lignans and isoflavonoids have comparable plant defense properties, as well as similar roles as phytoestrogens. Given that lignans are widespread from primitive plants onwards, whereas the isoflavone reductase-derived isoflavonoids are mainly restricted to the Fabaceae, it is tempting to speculate that this branch of the isoflavonoid pathway arose via evolutionary divergence from that giving the lignans.

  6. Crystal structures of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their relationship to isoflavone reductases.

    Min, Tongpil; Kasahara, Hiroyuki; Bedgar, Diana L; Youn, Buhyun; Lawrence, Paulraj K; Gang, David R; Halls, Steven C; Park, HaJeung; Hilsenbeck, Jacqueline L; Davin, Laurence B; Lewis, Norman G; Kang, ChulHee


    Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

  7. Recominant Pinoresino-Lariciresinol Reductase, Recombinant Dirigent Protein And Methods Of Use

    Lewis, Norman G. (Pullman, WA); Davin, Laurence B. (Pullman, WA); Dinkova-Kostova, Albena T. (Baltimore, MD); Fujita, Masayuki (Kita-gun, JP), Gang; David R. (Ann Arbor, MI), Sarkanen; Simo (Minneapolis, MN), Ford; Joshua D. (Pullman, WA)


    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided from source species Forsythia intermedia, Thuja plicata, Tsuga heterophylla, Eucommia ulmoides, Linum usitatissimum, and Schisandra chinensis, which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  8. Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

    Lewis, Norman G. (Pullman, WA); Davin, Laurence B. (Pullman, WA); Dinkova-Kostova, Albena T. (Baltimore, MD); Fujita, Masayuki (Kagawa, JP); Gang, David R. (Ann Arbor, MI); Sarkanen, Simo (S. Minneapolis, MN); Ford, Joshua D. (Pullman, WA)


    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  9. S-Glutathionyl-(chloro)hydroquinone reductases: a novel class of glutathione transferases.

    Xun, Luying; Belchik, Sara M; Xun, Randy; Huang, Yan; Zhou, Huina; Sanchez, Emiliano; Kang, Chulhee; Board, Philip G


    Sphingobium chlorophenolicum completely mineralizes PCP (pentachlorophenol). Two GSTs (glutathione transferases), PcpC and PcpF, are involved in the degradation. PcpC uses GSH to reduce TeCH (tetrachloro-p-hydroquinone) to TriCH (trichloro-p-hydroquinone) and then to DiCH (dichloro-p-hydroquinone) during PCP degradation. However, oxidatively damaged PcpC produces GS-TriCH (S-glutathionyl-TriCH) and GS-DiCH (S-glutathionyl-TriCH) conjugates. PcpF converts the conjugates into TriCH and DiCH, re-entering the degradation pathway. PcpF was further characterized in the present study. It catalysed GSH-dependent reduction of GS-TriCH via a Ping Pong mechanism. First, PcpF reacted with GS-TriCH to release TriCH and formed disulfide bond between its Cys53 residue and the GS moiety. Then, a GSH came in to regenerate PcpF and release GS-SG. A TBLASTN search revealed that PcpF homologues were widely distributed in bacteria, halobacteria (archaea), fungi and plants, and they belonged to ECM4 (extracellular mutant 4) group COG0435 in the conserved domain database. Phylogenetic analysis grouped PcpF and homologues into a distinct group, separated from Omega class GSTs. The two groups shared conserved amino acid residues, for GSH binding, but had different residues for the binding of the second substrate. Several recombinant PcpF homologues and two human Omega class GSTs were produced in Escherichia coli and purified. They had zero or low activities for transferring GSH to standard substrates, but all had reasonable activities for GSH-dependent reduction of disulfide bond (thiol transfer), dehydroascorbate and dimethylarsinate. All the tested PcpF homologues reduced GS-TriCH, but the two Omega class GSTs did not. Thus PcpF homologues were tentatively named S-glutathionyl-(chloro)hydroquinone reductases for catalysing the GSH-dependent reduction of GS-TriCH.

  10. Pyridine Nucleotide Complexes with Bacillus anthracis Coenzyme A-Disulfide Reductase: A Structural Analysis of Dual NAD(P)H Specificity

    Wallen,J.; Paige, C.; Mallett, T.; Karplus, P.; Claiborne, A.


    We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis, and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 Angstroms resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides. The structures of the NADH and NADPH complexes at ca. 2.3 Angstroms resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2'-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367'-OH and Tyr425'-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367, 425F BACoADR mutants indicate that Tyr425' is the primary proton donor in catalysis, with Tyr367' functioning as a cryptic alternate donor in the absence of Tyr425'.

  11. The interplay between thiol-compounds against chromium (VI) in the freshwater green alga Monoraphidium convolutum: Toxicology, photosynthesis, and oxidative stress at a glance

    Takami, R. [Postgraduate Program in Environmental Chemistry, CBS, Universidade Cruzeiro do Sul, 08060070, Sao Paulo, SP (Brazil); Almeida, J.V. [Department of Biochemistry, Instituto de Quimica, Universidade de Sao Paulo (IQ-USP), Sao Paulo, SP (Brazil); Vardaris, C.V. [Postgraduate Program in Environmental Chemistry, CBS, Universidade Cruzeiro do Sul, 08060070, Sao Paulo, SP (Brazil); Colepicolo, P. [Department of Biochemistry, Instituto de Quimica, Universidade de Sao Paulo (IQ-USP), Sao Paulo, SP (Brazil); Barros, M.P., E-mail: [Postgraduate Program in Environmental Chemistry, CBS, Universidade Cruzeiro do Sul, 08060070, Sao Paulo, SP (Brazil)


    In this paper, the multifaceted Cr(VI) toxicity over the freshwater green alga Monoraphidium convolutum was assessed by concomitantly monitoring thiol-dependent redox balances, photosynthesis activity and growth-survival scores. Control group showed exponential growth rate at (5.78 {+-} 0.29) division/day until 8th day with linear increasing chlorophyll a/protein ratios (CHLa/PROT) throughout the period. Cultures of M. convolutum were exposed for 5 days to Cr(VI) concentrations from 0 up to 100 mg/L showing that CHLa/PROT ratios were sensibly affected, in agreement to the calculated LC{sub 50,48h} (5.38 {+-} 0.72) mg/L from the concentration-response curve of cell mortality after 48 h. Regarding photosynthesis effects, Cr(VI) concentrations >1.0 mg/L showed significant increases in short-term (after 2 h) electron transfer rates (ETR) and quantum yields of photosystem II ({Phi}{sub PSII}), followed by subsequent decline of both parameters after 48 and 72 h. Biochemical analyses showed that maximal GSH concentrations in algal cultures were observed upon 1 mg Cr(VI)/L and higher dichromate concentrations dramatically increased the activity of antioxidant GSH-dependent enzymes ascorbate peroxidase and glutathione reductase. However, no variation was observed in the cellular GSH levels, whereas GSSG and lipid peroxidation indexes abruptly increased upon 10 mg Cr(VI)/L exposure. Altogether, plant physiology, photosynthesis and biochemical data suggest that the GSH-dependent antioxidant system is capable to sustain M. convolutum viability through efficient photosynthesis activity and adequate antioxidant responses up to Cr(VI) concentrations of 1.0 mg/L, when redox unbalances were first evidenced.

  12. Cytoplasmic glutathione redox status determines survival upon exposure to the thiol-oxidant 4,4'-dipyridyl disulfide

    López-Mirabal, H Reynaldo; Thorsen, Michael; Kielland-Brandt, Morten C


    Dipyridyl disulfide (DPS) is a highly reactive thiol oxidant that functions as electron acceptor in thiol-disulfide exchange reactions. DPS is very toxic to yeasts, impairing growth at low micromolar concentrations. The genes TRX2 (thioredoxin), SOD1 (superoxide dismutase), GSH1 (gamma-glutamyl-c......Dipyridyl disulfide (DPS) is a highly reactive thiol oxidant that functions as electron acceptor in thiol-disulfide exchange reactions. DPS is very toxic to yeasts, impairing growth at low micromolar concentrations. The genes TRX2 (thioredoxin), SOD1 (superoxide dismutase), GSH1 (gamma...... antioxidant pools of glutathione (GSH) and thioredoxin are required for resistance to DPS. We found that DPS-sensitive mutants display increases in the disulfide form of GSH (GSSG) during DPS exposure that roughly correlate with their more oxidizing GSH redox potential in the cytosol and their degree of DPS...

  13. Rocket fuel for the quantification of S-nitrosothiols. Highly specific reduction of S-nitrosothiols to thiols by methylhydrazine.

    Wiesweg, M; Berchner-Pfannschmidt, U; Fandrey, J; Petrat, F; de Groot, H; Kirsch, M


    Reduction of S-nitrosothiols to the corresponding thiol function is the key step in analyzing S-nitrosocysteinyl residues in proteins. Though it has been shown to give low yields, ascorbate-dependent reduction is commonly performed in the frequently used biotin-switch technique. We demonstrate that the compound methylhydrazine can act as a specific and efficient reducing agent for S-nitrosothiols. The corresponding thiol function is exclusively generated from low molecular weight and proteinaceous S-nitrosothiols while methylhydrazine failed to reduce disulfides. It was possible to optimize the experimental conditions so that thiol autoxidation is excluded, and high reaction yields (>90%) are obtained for the thiol function. The biotin-switch technique performed with methylhydrazine-dependent reduction shows remarkably improved sensitivity compared to the ascorbate-dependent procedure.

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

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


    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.

  15. Highly stretchable thermoset fibers and nonwovens using thiol-ene photopolymerization.

    Shanmuganathan, Kadhiravan; Elliot, Steven M; Lane, Austin P; Ellison, Christopher J


    In this report, we describe the preparation and characterization of a new class of thermoset fibers with high elongation and elastic recovery. Integrating UV-activated thiol-ene photopolymerization and electrospinning, we demonstrate an environmentally friendly single step approach to convert small monomeric precursor molecules into highly elastic fibers and nonwoven mats. The fibers were derived by in situ photopolymerization of a trifunctional vinyl ether monomer and a tetrafunctional thiol. Although thermosets often offer good chemical and thermal stability, these fibers also have a high average elongation at break of 62%. The elastomeric nature of these vinyl-ether based fibers can be partly attributed to their subambient Tg and partly to the cross-link density, monomer structure, and resulting network homogeneity. Nonwoven mats of these fibers were also stretchable and exhibited a much higher elongation at break of about 85%. These thermoset stretchable fibers could have potential applications as textile, biomedical, hot chemical filtration, and composite materials.

  16. Synthesis and Biological Evaluation of Novel Benzothiazole-2-thiol Derivatives as Potential Anticancer Agents

    Luo-Ting Yu


    Full Text Available A series of novel benzothiazole-2-thiol derivatives were synthesized and their structures determined by 1H-NMR, 13C-NMR and HRMS (ESI. The effects of all compounds on a panel of different types of human cancer cell lines were investigated. Among them, pyridinyl-2-amine linked benzothiazole-2-thiol compounds 7d, 7e, 7f and 7i exhibited potent and broad-spectrum inhibitory activities. Compound 7e displayed the most potent anticancer activity on SKRB-3 (IC50 = 1.2 nM, SW620 (IC50 = 4.3 nM, A549 (IC50 = 44 nM and HepG2 (IC50 = 48 nM and was found to induce apoptosis in HepG2 cancer cells.

  17. Rapid photochemical surface patterning of proteins in thiol-ene based microfluidic devices

    Lafleur, Josiane P.; Kwapiszewski, Radoslaw; Jensen, Thomas Glasdam;


    The suitable optical properties of thiol–ene polymers combined with the ease of modifying their surface for the attachment of recognition molecules make them ideal candidates in many biochip applications. This paper reports the rapid one-step photochemical surface patterning of biomolecules...... in microfluidic thiol–ene chips. This work focuses on thiol–ene substrates featuring an excess of thiol groups at their surface. The thiol–ene stoichiometric composition can be varied to precisely control the number of surface thiol groups available for surface modification up to an average surface density of 136...... ! 17 SH nm"2. Biotin alkyne was patterned directly inside thiol–ene microchannels prior to conjugation with fluorescently labelled streptavidin. The surface bound conjugates were detected by evanescent waveinduced fluorescence (EWIF), demonstrating the success of the grafting procedure and its...

  18. Modification of porous silicon rugate filters through thiol-yne photochemistry

    Soeriyadi, Alexander H., E-mail:; Zhu, Ying, E-mail:; Gooding, J. Justin, E-mail: [Australian Centre for Nanomedicine and School of Chemistry, University of New South Wales, Sydney 2052 (Australia); Reece, Peter [School of Physics, University of New South Wales, Sydney 2052 (Australia)


    Porous silicon (PSi) has a considerable potential as biosensor platform. In particular, the ability to modify the surface chemistry of porous silicon is of interest. Here we present a generic method to modify the surface of porous silicon through thiol-yne photochemistry initiated by a radical initiator. Firstly, a freshly etched porous silicon substrate is modified through thermal hydrosilylation with 1,8-nonadiyne to passivate the surface and introduce alkyne functionalities. The alkyne functional surface could then be further reacted with thiol species in the presence of a radical initiator and UV light. Functionalization of the PSi rugate filter is followed with optical reflectivity measurements as well as high resolution X-ray photoelectron spectroscopy (XPS)

  19. Enhanced removal of Hg(II) from acidic aqueous solution using thiol-functionalized biomass.

    Chai, Liyuan; Wang, Qingwei; Li, Qingzhu; Yang, Zhihui; Wang, Yunyan


    Spent grain, the low-cost and abundant biomass produced in the brewing industry, was functionalized with thiol groups to be used as an adsorbent for Hg(II) removal from acidic aqueous solution. The adsorbents were characterized by the energy-dispersive X-ray analysis (EDAX) and Fourier transform infrared (FTIR) spectroscopy. Optimum pH for Hg(II) adsorption onto the thiol-functionalized spent grain (TFSG) was 2.0. The equilibrium and kinetics of the adsorption of Hg(II) onto TFSG from acidic aqueous solution were investigated. From the Langmuir isotherm model the maximum adsorption capacity of TFSG for Hg(II) was found to be 221.73 mg g(-1), which was higher than that of most various adsorbents reported in literature. Moreover, the adsorption of Hg(II) onto TFSG followed pseudo-second-order kinetic model.

  20. Kandelia obovata (S., L.) Yong tolerance mechanisms to Cadmium: subcellular distribution, chemical forms and thiol pools.

    Weng, Bosen; Xie, Xiangyu; Weiss, Dominik J; Liu, Jingchun; Lu, Haoliang; Yan, Chongling


    In order to explore the detoxification mechanisms adopted by mangrove under cadmium (Cd) stress, we investigated the subcellular distribution and chemical forms of Cd, in addition to the change of the thiol pools in Kandelia obovata (S., L.) Yong, which were cultivated in sandy culture medium treated with sequential Cd solution. We found that Cd addition caused a proportional increase of Cd in the organs of K. obovata. The investigation of subcellular distribution verified that most of the Cd was localized in the cell wall, and the lowest was in the membrane. Results showed sodium chloride and acetic acid extractable Cd fractions were dominant. The contents of non-protein thiol compounds, Glutathione and phytochelatins in K. obovata were enhanced by the increasing strength of Cd treatment. Therefore, K. obovata can be defined as Cd tolerant plant, which base on cell wall compartmentalization, as well as protein and organic acids combination.

  1. Characterization of Helicobacter pylori adhesin thiol peroxidase (HP0390) purified from Escherichia coli

    Huyen Thi Minh Nguyen; Kwang-Ho Nam; Yasar Saleem; Key-Sun Kim


    The antioxidant protein, adhesin thiol peroxidase (HpTpx or HP0390), plays an important role in enabling Helicobacter pylori to survive gastric oxidative stress. The bacterium colonizes the host stomach and produces gastric cancer. However, little information is available about the biochemical characteristics of HpTpx. We expressed recombinant HpTpx in Escherichia coli, purified to homogeneity, and characterized it. The results showed that HpTpx existed in a monomeric hydrodynamic form and the enzyme fully retained its peroxidase and antioxidant activities. The catalytic reaction of the enzyme was similar to an atypical 2-cysteine peroxiredoxin (Prx). The conformation of the enzyme was observed in the presence and absence of dithiothreitol (DTT); similar to other known thiol peroxidases, conformational change was observed in HpTpx by the addition of DTT.

  2. Antioxidant Defense by Thioredoxin Can Occur Independently of Canonical Thiol-Disulfide Oxidoreductase Enzymatic Activity

    Miryoung Song


    Full Text Available The thiol-disulfide oxidoreductase CXXC catalytic domain of thioredoxin contributes to antioxidant defense in phylogenetically diverse organisms. We find that although the oxidoreductase activity of thioredoxin-1 protects Salmonella enterica serovar Typhimurium from hydrogen peroxide in vitro, it does not appear to contribute to Salmonella’s antioxidant defenses in vivo. Nonetheless, thioredoxin-1 defends Salmonella from oxidative stress resulting from NADPH phagocyte oxidase macrophage expression during the innate immune response in mice. Thioredoxin-1 binds to the flexible linker, which connects the receiver and effector domains of SsrB, thereby keeping this response regulator in the soluble fraction. Thioredoxin-1, independently of thiol-disulfide exchange, activates intracellular SPI2 gene transcription required for Salmonella resistance to both reactive species generated by NADPH phagocyte oxidase and oxygen-independent lysosomal host defenses. These findings suggest that the horizontally acquired virulence determinant SsrB is regulated post-translationally by ancestrally present thioredoxin.

  3. Generation of thiols by biotransformation of cysteine-aldehyde conjugates with baker's yeast.

    Huynh-Ba, Tuong; Matthey-Doret, Walter; Fay, Laurent B; Bel Rhlid, Rachid


    Baker's yeast was shown to catalyze the transformation of cysteine-furfural conjugate into 2-furfurylthiol. The biotransformation's yield and kinetics were influenced by the reaction parameters such as pH, incubation mode (aerobic and anaerobic), and substrate concentration. 2-Furfurylthiol was obtained in an optimal 37% yield when cysteine-furfural conjugate at a 20 mM concentration was anaerobically incubated with whole cell baker's yeast at pH 8.0 and 30 degrees C. Similarly to 2-furfurylthiol, 5-methyl-2-furfurylthiol (11%), benzylthiol (8%), 2-thiophenemethanethiol (22%), 3-methyl-2-thiophenemethanethiol (3%), and 2-pyrrolemethanethiol (6%) were obtained from the corresponding cysteine-aldehyde conjugates by incubation with baker's yeast. This work indicates the versatile bioconversion capacity of baker's yeast for the generation of thiols from cysteine-aldehyde conjugates. Thanks to its food-grade character, baker's yeast provides a biochemical tool to produce thiols, which can be used as flavorings in foods and beverages.

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

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


    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.

  5. The respiratory arsenate reductase from Bacillus selenitireducens strain MLS10

    Afkar, E.; Lisak, J.; Saltikov, C.; Basu, P.; Oremland, R.S.; Stolz, J.F.


    The respiratory arsenate reductase from the Gram-positive, haloalkaliphile, Bacillus selenitireducens strain MLS10 was purified and characterized. It is a membrane bound heterodimer (150 kDa) composed of two subunits ArrA (110 kDa) and ArrB (34 kDa), with an apparent Km for arsenate of 34 ??M and Vmax of 2.5 ??mol min-1 mg-1. Optimal activity occurred at pH 9.5 and 150 g l-1 of NaCl. Metal analysis (inductively coupled plasma mass spectrometry) of the holoenzyme and sequence analysis of the catalytic subunit (ArrA; the gene for which was cloned and sequenced) indicate it is a member of the DMSO reductase family of molybdoproteins. ?? 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

  6. Polarographic determination of europium(III) with 3-hydroxypyridine-2-thiol

    Sindhu, R.S.; Katyal, M.; Puri, B.K.


    The polarographic behavior of europium(III) has been studied in 3-hydroxypyridine-2-thiol (HPT) as the supporting electrolyte. The polarographic wave in this electrolyte is diffusion controlled, quasi-reversible, and well defined, and there is no need of a maximum suppressor. The interference of various ions has been studied in detail, and this method has been utilized for the quantitative determination of europium in various synthetic samples

  7. Magnetometry and electron paramagnetic resonance studies of phosphine- and thiol-capped gold nanoparticles

    Guerrero, Estefanía; Muñoz-Márquez, Miguel Ángel; Fernández-Camacho, A.; Crespo, P.; Hernando, Antonio, 1947-; Lucena, R; Conesa Cegarra, José Carlos


    In the last years, the number of studies performed by wholly independent research groups that confirm the permanent magnetism, first observed in our research lab, for thiol-capped Au nanoparticles (NPs) has rapidly increased. Throughout the years, the initial magnetometry studies have been completed with element-specific magnetization measurements based on, for example, the x-ray magnetic circular dichroism technique that have allowed the identification of gold as the magnetic moment carrier....

  8. Ruthenium(III Chloride Catalyzed Acylation of Alcohols, Phenols, and Thiols in Room Temperature Ionic Liquids

    Mingzhong Cai


    Full Text Available Ruthenium(III chloride-catalyzed acylation of a variety of alcohols, phenols, and thiols was achieved in high yields under mild conditions (room temperature in the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF6]. The ionic liquid and ruthenium catalyst can be recycled at least 10 times. Our system not only solves the basic problem of ruthenium catalyst reuse, but also avoids the use of volatile acetonitrile as solvent.

  9. Adaptive aneuploidy protects against thiol peroxidase deficiency by increasing respiration via key mitochondrial proteins.

    Kaya, Alaattin; Gerashchenko, Maxim V; Seim, Inge; Labarre, Jean; Toledano, Michel B; Gladyshev, Vadim N


    Aerobic respiration is a fundamental energy-generating process; however, there is cost associated with living in an oxygen-rich environment, because partially reduced oxygen species can damage cellular components. Organisms evolved enzymes that alleviate this damage and protect the intracellular milieu, most notably thiol peroxidases, which are abundant and conserved enzymes that mediate hydrogen peroxide signaling and act as the first line of defense against oxidants in nearly all living organisms. Deletion of all eight thiol peroxidase genes in yeast (∆8 strain) is not lethal, but results in slow growth and a high mutation rate. Here we characterized mechanisms that allow yeast cells to survive under conditions of thiol peroxidase deficiency. Two independent ∆8 strains increased mitochondrial content, altered mitochondrial distribution, and became dependent on respiration for growth but they were not hypersensitive to H2O2. In addition, both strains independently acquired a second copy of chromosome XI and increased expression of genes encoded by it. Survival of ∆8 cells was dependent on mitochondrial cytochrome-c peroxidase (CCP1) and UTH1, present on chromosome XI. Coexpression of these genes in ∆8 cells led to the elimination of the extra copy of chromosome XI and improved cell growth, whereas deletion of either gene was lethal. Thus, thiol peroxidase deficiency requires dosage compensation of CCP1 and UTH1 via chromosome XI aneuploidy, wherein these proteins support hydroperoxide removal with the reducing equivalents generated by the electron transport chain. To our knowledge, this is the first evidence of adaptive aneuploidy counteracting oxidative stress.

  10. Synthesis of cysteine-rich peptides by native chemical ligation without use of exogenous thiols.

    Tsuda, Shugo; Yoshiya, Taku; Mochizuki, Masayoshi; Nishiuchi, Yuji


    Native chemical ligation (NCL) performed without resorting to the use of thiol additives was demonstrated to be an efficient and effective procedure for synthesizing Cys-rich peptides. This method using tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent facilitates the ligation reaction even at the Thr-Cys or Ile-Cys site and enables one-pot synthesis of Cys-rich peptides throughout NCL and oxidative folding.

  11. A study of oxidative stress, thiol proteins and role of vitamin E supplementation in chronic obstructive pulmonary disease (COPD

    Anita M. Raut


    Full Text Available Background: Lipid peroxide plays an important role in inflammatory lung disease. Increased epithelial permeability produced by cigarette smoke is likely to be mediated through depletion of thiol proteins. Imbalance between oxidants and thiol proteins is also an established fact in these patients. Materials & methods: In the present study 30 healthy non-smokers were served as controls and 20 patients with stable COPD were included. Their base line clinical examination, Malondialdehyde (MDA as an oxidant, alpha tocopherol and erythrocyte superoxide dismutase (SOD as an antioxidants and thiol proteins levels were measured. All above parameters were repeated after 12 weeks of supplementation with 400 IU of vitamin E daily. Results: We observed that the mean malondialdehyde levels in these patients at base line were high (p<0.001 than Control Plasma alpha-tocopherol, SOD and thiol proteins levels were low (p<0.001 in the patients compared to controls. Exogenous vitamin E (400 IU twice daily Supplementation did not bring about any significant change in plasma Erythrocyte Superoxide Dismutase and vitamin E. But slight increase in the plasma thiol proteins levels was seen. The present study shows that initially the plasma lipid peroxide (MDA levels were high antioxidant (alpha- tocopherol, SOD and thiol proteins were low in patients with COPD. Exogenous supplementation with vitamin E increases slightly thiol proteins levels and brings down the levels of MDA showing attenuation of further damage. Conclusion: Our study confirmed the existence of oxidative stress and and the augmentation of antioxidant defenses as shown by slight increase in thiol proteins level. The antioxidant therapy is adjunct in lung disease patients and opens a promising field in prevention of oxidative stress related complications in these patients.

  12. Copper and ceruloplasmin levels in relation to total thiols and GST in type 2 diabetes mellitus patients

    Sarkar, A.; S; Dash; Barik, B. K.; Muttigi, Manjunatha S.; Kedage, V; Shetty, J. K.; Prakash, M.


    Presence of oxidative stress in type 2 diabetes mellitus (DM) is well proved. Current study was undertaken to know the relation between fasting plasma glucose (FPG) and copper along with antioxidants like total thiols and ceruloplasmin, and antioxidant enzyme glutathione S transferase (GST). The study group consisted of a total of 201 subjects which included nondiabetic healthy control subjects (n = 78) and diabetic patients (n = 123). Plasma total thiols, GST, copper and ceruloplasmin levels...

  13. Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase

    Rajib; Sengupta; Arne; Holmgren


    Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas made it an important target for the development ofantiviral and anticancer drugs. Taking account of the re-cent developments in this field of research, this reviewfocuses on the role of thioredoxin and glutaredoxin sys-tems in the redox reactions of the RNR catalysis.

  14. Comparison of photopolymerizable thiol-ene PEG and acrylate-based PEG hydrogels for cartilage development.

    Roberts, Justine J; Bryant, Stephanie J


    When designing hydrogels for tissue regeneration, differences in polymerization mechanism and network structure have the potential to impact cellular behavior. Poly(ethylene glycol) hydrogels were formed by free-radical photopolymerization of acrylates (chain-growth) or thiol-norbornenes (step-growth) to investigate the impact of hydrogel system (polymerization mechanism and network structure) on the development of engineered tissue. Bovine chondrocytes were encapsulated in hydrogels and cultured under free swelling or dynamic compressive loading. In the acrylate system immediately after encapsulation chondrocytes exhibited high levels of intracellular ROS concomitant with a reduction in hydrogel compressive modulus and higher variability in cell deformation upon compressive strain; findings that were not observed in the thiol-norbornene system. Long-term the quantity of sulfated glycosaminoglycans and total collagen was greater in the acrylate system, but the quality resembled that of hypertrophic cartilage with positive staining for aggrecan, collagens I, II, and X and collagen catabolism. The thiol-norbornene system led to hyaline-like cartilage production especially under mechanical loading with positive staining for aggrecan and collagen II and minimal staining for collagens I and X and collagen catabolism. Findings from this study confirm that the polymerization mechanism and network structure have long-term effects on the quality of engineered cartilage, especially under mechanical loading.

  15. Surface modification of cyclomatrix polyphosphazene microsphere by thiol-ene chemistry and lectin recognition

    Chen, Chen; Zhu, Xue-yan; Gao, Qiao-ling; Fang, Fei; Huang, Xiao-jun


    A new synthetic route leading to functional polyphosphazene cyclomatrix microsphere has been developed via thiol-ene click modification. Hexacholorocyclophosphazene (HCCP) was crosslinked with both bisphenol-S and 4,4‧-diallyl bisphenol-S to obtain vinyl polyphosphazene microspheres (VPZM) in order to ensure high crosslinking degree and introduce vinyl moieties. Compared to the microspheres obtained by HCCP and bisphenol-S, the size of VPZM was broadly dispersed from 400 nm to 1.40 μm. Thiol-ene click reactions were carried out to attach functional groups, such as glucosyl, carboxyl, ester and dodecyl groups onto polyphosphazene microspheres, which demonstrated no change in morphology and size after modification. Solid state NMR (SSNMR) and Fourier transform infrared spectoscopy (FT-IR) results showed that the vinyl moieties were introduced in the period of crosslinking and functionalization was also successful via click reactions. Moreover, the microspheres presented a little difference in thermal properties after modification. Concanavalin A (Con-A) fluorescent adsorption was also observed for glucosyl microspheres. Thus, the thiol-ene modified polyphosphazene microspheres displayed chemical flexibility in post-functionalization. These microspheres can be potentially applicated in enzyme immobilization, protein adsorption and chromatographic separation.

  16. Purification and characterization of thiols in an arsenic hyperaccumulator under arsenic exposure.

    Zhang, Weihua; Cai, Yong


    Pteris vittata (Chinese brake fern) is the first reported arsenic hyperaccumulator. To investigate the arsenic tolerance mechanism in this plant, reversed-phase HPLC with postcolumn derivatization was used to analyze the thiols induced under arsenic exposure. A major thiol in the plant leaflets was found to be responsive to arsenic exposure. The arsenic-induced compound was purified on a large scale by combining covalent chromatography and preparative reversed-phase HPLC. About 2 mg of this compound was isolated from 1 kg of fresh leaflets. The purified arsenic-induced compound was characterized using electrospray ionization mass spectrometry. A molecular ion (M + 1) of 540 and fragments were obtained, which indicated that the arsenic-induced thiol was a phytochelatin with two subunits (PC(2)). Compared to the classical methods for purification of phytochelatins, this new method is more specific, simple, and rapid and is suitable for purification of PCs in a large scale as well as sample preparation for mass spectrometry analysis.

  17. The critical role of the cellular thiol homeostasis in cadmium perturbation of the lung extracellular matrix.

    Zhao, Yinzhi; Chen, Lijun; Gao, Song; Toselli, Paul; Stone, Phillip; Li, Wande


    Cadmium (Cd) inhalation can result in emphysema. Cd exposure of rat lung fibroblasts (RFL6) enhanced levels of metal scavenging thiols, e.g., metallothionein (MT) and glutathione (GSH), and the heavy chain of gamma-glutamylcysteine synthetase (gamma-GCS), a key enzyme for GSH biosynthesis, concomitant with downregulation of lysyl oxidase (LO), a copper-dependent enzyme for crosslinking collagen and elastin in the extracellular matrix (ECM). Cd downregulation of LO in treated cells was closely accompanied by suppression of synthesis of collagen, a major structure component of the lung ECM. Using rats intratracheally instilled with cadmium chloride (30 microg, once a week) as an animal model, we further demonstrated that although 2-week Cd instillation induced a non-significant change in the lung LO activity and collagen synthesis, 4- and 6-week Cd instillation resulted in a steady decrease in the lung LO and collagen expression. The lung MT and total GSH levels were both upregulated upon the long-term Cd exposure. Emphysematous lesions were generated in lungs of 6-week Cd-dosed rats. Increases of cellular thiols by transfection of cells with MT-II expression vectors or treatment of cells with GSH monoethyl ester, a GSH delivery system, markedly inhibited LO mRNA levels and catalytic activities in the cell model. Thus, Cd upregulation of cellular thiols may be a critical cellular event facilitating downregulation of LO, a potential mechanism for Cd-induced emphysema.

  18. Improvement of aromatic thiol release through the selection of yeasts with increased β-lyase activity.

    Belda, Ignacio; Ruiz, Javier; Navascués, Eva; Marquina, Domingo; Santos, Antonio


    The development of a selective medium for the rapid differentiation of yeast species with increased aromatic thiol release activity has been achieved. The selective medium was based on the addition of S-methyl-l-cysteine (SMC) as β-lyase substrate. In this study, a panel of 245 strains of Saccharomyces cerevisiae strains was tested for their ability to grow on YCB-SMC medium. Yeast strains with an increased β-lyase activity grew rapidly because of their ability to release ammonium from SMC in comparison to others, and allowed for the easy isolation and differentiation of yeasts with promising properties in oenology, or another field, for aromatic thiol release. The selective medium was also helpful for the discrimination between those S. cerevisiae strains, which present a common 38-bp deletion in the IRC7 sequence (present in around 88% of the wild strains tested and are likely to be less functional for 4-mercapto-4-methylpentan-2-one (4MMP) production), and those S. cerevisiae strains homozygous for the full-length IRC7 allele. The medium was also helpful for the selection of non-Saccharomyces yeasts with increased β-lyase activity. Based on the same medium, a highly sensitive, reproducible and non-expensive GC-MS method for the evaluation of the potential volatile thiol release by different yeast isolates was developed.

  19. Hydrangea-like magneto-fluorescent nanoparticles through thiol-inducing assembly

    Chen, Shun; Zhang, Junjun; Song, Shaokun; Xiong, Chuanxi; Dong, Lijie


    Magneto-fluorescent nanoparticles (NPs), recognized as an emerging class of materials, have drawn much attention because of their potential applications. Due to surface functionalization and thiol-metal bonds, a simple method has been put forward for fabricating hydrangea-like magneto-fluorescent Fe3O4-SH@QD NPs, through assembling thiol-modified Fe3O4 NPs with sub-size multi-layer core/shell CdSe/CdS/ZnS QDs. After a refined but controllable silane hydrolysis process, thiol-modified Fe3O4 was fabricated, resulting in Fe3O4-SH@QD NPs with QDs, while preventing the quenching of the QDs. As a result, the core Fe3O4 NPs were 18 nm in diameter, while the scattered CdSe/CdS/ZnS QDs were 7 nm in diameter. The resultant magneto-fluorescent Fe3O4-SH@QD NPs exhibit efficient fluorescence, superparamagnetism at room temperature, and rapid response to the external field, which make them ideal candidates for difunctional probes in MRI and bio-labels, targeting and photodynamic therapy, and cell tracking and separation.

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

    Ángel Gabriel Díaz-Sánchez


    Full Text Available 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.

  1. Magnetometry and electron paramagnetic resonance studies of phosphine- and thiol-capped gold nanoparticles

    Guerrero, E.; Muñoz-Márquez, M. A.; Fernández, A.; Crespo, P.; Hernando, A.; Lucena, R.; Conesa, J. C.


    In the last years, the number of studies performed by wholly independent research groups that confirm the permanent magnetism, first observed in our research lab, for thiol-capped Au nanoparticles (NPs) has rapidly increased. Throughout the years, the initial magnetometry studies have been completed with element-specific magnetization measurements based on, for example, the x-ray magnetic circular dichroism technique that have allowed the identification of gold as the magnetic moment carrier. In the research work here presented, we have focused our efforts in the evaluation of the magnetic behavior and iron impurities content in the synthesized samples by means of superconducting quantum interference device magnetometry and electron paramagnetic resonance spectrometry, respectively. As a result, hysteresis cycles typical of a ferromagnetic material have been measured from nominally iron-free gold NPs protected with thiol, phosphine, and chlorine ligands. It is also observed that for samples containing both, capped gold NPs and highly diluted iron concentrations, the magnetic behavior of the NPs is not affected by the presence of paramagnetic iron impurities. The hysteresis cycles reported for phosphine-chlorine-capped gold NPs confirm that the magnetic behavior is not exclusively for the metal-thiol system.

  2. Investigation into the Effect of Molds in Grasses on Their Content of Low Molecular Mass Thiols

    Adam Nawrath


    Full Text Available The aim of this study was to investigate the effect of molds on levels of low molecular mass thiols in grasses. For this purpose, the three grass species Lolium perenne, Festulolium pabulare and Festulolium braunii were cultivated and sampled during four months, from June to September. The same species were also grown under controlled conditions. High-performance liquid chromatography with electrochemical detection was used for quantification of cysteine, reduced (GSH and oxidized (GSSG glutathione, and phytochelatins (PC2, PC3, PC4 and PC5. Data were statistically processed and analyzed. Thiols were present in all examined grass species. The effect of fungicide treatments applied under field conditions on the content of the evaluated thiols was shown to be insignificant. Species influenced (p < 0.05 PC3 and GSSG content. F. pabulare, an intergeneric hybrid of drought- and fungi-resistant Festuca arundinacea, was comparable in PC3 content with L. perenne and F. braunii under field conditions. Under controlled conditions, however, F. pabulare had higher (p < 0.05 PC3 content than did L. perenne and F. braunii. Under field conditions, differences between the evaluated species were recorded only in GSSG content, but only sampling in June was significant. F. pabulare had higher (p < 0.05 GSSG content in June than did L. perenne and F. braunii.

  3. Graphene Oxide-Polymer Composite Langmuir Films Constructed by Interfacial Thiol-Ene Photopolymerization

    Luo, Xiaona; Ma, Kai; Jiao, Tifeng; Xing, Ruirui; Zhang, Lexin; Zhou, Jingxin; Li, Bingbing


    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.

  4. Characterization of plasma thiol redox potential in a common marmoset model of aging

    James R. Roede


    Full Text Available Due to its short lifespan, ease of use and age-related pathologies that mirror those observed in humans, the common marmoset (Callithrix jacchus is poised to become a standard nonhuman primate model of aging. Blood and extracellular fluid possess two major thiol-dependent redox nodes involving cysteine (Cys, cystine (CySS, glutathione (GSH and glutathione disulfide (GSSG. Alteration in these plasma redox nodes significantly affects cellular physiology, and oxidation of the plasma Cys/CySS redox potential (EhCySS is associated with aging and disease risk in humans. The purpose of this study was to determine age-related changes in plasma redox metabolites and corresponding redox potentials (Eh to further validate the marmoset as a nonhuman primate model of aging. We measured plasma thiol redox states in marmosets and used existing human data with multivariate adaptive regression splines (MARS to model the relationships between age and redox metabolites. A classification accuracy of 70.2% and an AUC of 0.703 were achieved using the MARS model built from the marmoset redox data to classify the human samples as young or old. These results show that common marmosets provide a useful model for thiol redox biology of aging.

  5. Optimization of Optical Properties of Polycarbonate Film with Thiol Gold-Nanoparticles

    Claudio Larosa


    Full Text Available A new nanostructured composite film based on thiol gold nanoparticles dispersed in polycarbonate and prepared by evaporating a solution of 1-dodecanthiol gold nanoparticles and polycarbonate was developed for applications as optical lenses. Lenses with superior mechanical properties, coloring and UV ray absorption and with the same transparency as the matrix were obtained. The supporting highly transparent polycarbonate matrix and the chloroform solution of thiol gold nanoparticles, 3 nm mean size, was mixed according to a doping protocol employing different concentrations of thiol gold nanoparticles vs. polycarbonate. The presence of nanoparticles in the polymer films was confirmed by the spectrophotometric detection of the characteristic absorbance marker peak at 540–580 nm. The nanostructured films obtained show a better coverage in the UV-vis range (250–450 nm even at very low doping ratios, of the order of 1:1,000. These results offer a very promising approach towards the development of efficient nanostructured materials for applications to optical lenses.

  6. Thiol-Functionalized Mesoporous Silica for Effective Trap of Mercury in Rats

    Wei Zhao


    Full Text Available The chance of exposure to heavy metal for human being rises severely today due to the increasing water contamination and air pollution. Here, we prepared a series of thiol-functionalized mesoporous silica as oral formulation for the prevention and treatment of heavy metal poisoning. The successful incorporation of thiol was verified by the FTIR spectra. SBA15-SH-10 was used for the study as it is of uniform mesopores and fine water dispersibility. In simulated gastrointestinal fluid, the thiol-functionalized mesoporous silica can selectively capture heavy metal, showing a very high affinity for inorganic mercury (II. The blood and urine mercury levels of rats fed with a diet containing Hg (II and material were significantly lower than those of rats fed with the metal-rich diet only. On the contrary, the mercury content in fecal excretion of the treatment group increased more than twice as much as that of the control group. This result indicated that SBA15-SH-10 could effectively remove mercury (II in vivo and the mercury loaded on SBA15-SH-10 would be excreted out. Hence, SBA15-SH-10 has potential application in preventing and treating heavy metal poisoning via digestive system.

  7. Aldo-Keto Reductases 1B in Adrenal Cortex Physiology.

    Pastel, Emilie; Pointud, Jean-Christophe; Martinez, Antoine; Lefrançois-Martinez, A Marie


    Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions.

  8. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D


    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.

  9. Quantum mechanical treatment of As(3+)-thiol model compounds: implication for the core structure of As(III)-metallothionein.

    Garla, Roobee; Kaur, Narinder; Bansal, Mohinder Pal; Garg, Mohan Lal; Mohanty, Biraja Prasad


    Exposure to inorganic arsenic (As) is one of the major health concerns in several regions around the world. Binding of As(III) with thiols is central to the mechanisms related to its toxicity, detoxification, and therapeutic effects. Due to its high thiol content, metallothionein (MT) is presumed to play an important role in case of arsenic toxicity. Consequences of these As-thiol interactions are not yet clear due to various difficulties in the characterization of arsenic bound proteins by spectroscopic techniques. Computational modeling can be a reliable approach in predicting the molecular structures of such complexes. This paper presents the results of a systematic study on different As(III)-thiol model compounds conducted by both ab initio and DFT methods with different Gaussian type basis sets. Proficiency of these theoretical methods has been evaluated in terms of bond lengths, bond angles, free energy, partial atomic charges, computational cost, and comparison with the experimental data. It has been demonstrated that the DFT-B3LYP/6-311+G(3df) functional offers better accuracy in predicting the structure and the UV absorption spectra of As(III)-thiol complexes. The results of the present study also helps in defining the boundaries for the core of arsenic bound MT so that quantum mechanical/molecular mechanical (QM/MM) methods can be employed to predict the structural and functional aspects of the protein. Graphical Abstract Optimized structural parameters of As(3+)-thiol model compounds.

  10. Inelastic Tunneling Spectroscopy of Gold-Thiol and Gold-Thiolate Interfaces in Molecular Junctions: The Role of Hydrogen

    Demir, Firuz


    It is widely believed that when a molecule with thiol (S-H) end groups bridges a pair of gold electrodes, the S atoms bond to the gold and the thiol H atoms detach from the molecule. However, little is known regarding the details of this process, its time scale, and whether molecules with and without thiol hydrogen atoms can coexist in molecular junctions. Here we explore theoretically how inelastic tunneling spectroscopy (IETS) can shed light on these issues. We present calculations of the geometries, low bias conductances and IETS of propanedithiol and propanedithiolate molecular junctions with gold electrodes. We show that IETS can distinguish between junctions with molecules having no, one or two thiol hydrogen atoms. We find that in most cases the single-molecule junctions in the IETS experiment of Hihath et al. [Nano Lett. 8, 1673 (2008)] had no thiol H atoms, but that a molecule with a single thiol H atom may have bridged their junction occasionally. We also consider the evolution of the IETS spectrum ...

  11. Resin-assisted enrichment of thiols as a general strategy for proteomic profiling of cysteine-based reversible modifications

    Guo, Jia [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Gaffrey, Matthew J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Su, Dian [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Genentech Inc., South San Francisco, CA (United States); Liu, Tao [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Camp, David G. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Smith, Richard D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Qian, Weijun [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)


    Reversible modifications on cysteine thiols play a significant role in redox signaling and regulation. A number of reversible redox modifications, including disulfide formation, S-nitrosylation, and S-glutathionylation, have been recognized for their significance in various physiological and pathological processes. Here we describe in detail a resin-assisted thiol-affinity enrichment protocol for both biochemical and proteomics applications. This protocol serves as a general approach for specific isolation of thiol-containing proteins or peptides derived from reversible redox-modified proteins. This approach utilizes thiol-affinity resins to directly capture thiol-containing proteins or peptides through a disulfide exchange reaction followed by on-resin protein digestion and on-resin multiplexed isobaric labeling to facilitate LC-MS/MS based quantitative site-specific analysis of redox modifications. The overall approach requires a much simpler workflow with increased specificity compared to the commonly used biotin switch technique. By coupling different selective reduction strategies, the resin-assisted approach provides the researcher with a useful tool capable of enriching different types of reversible modifications on protein thiols. Procedures for selective enrichment and analyses of S-nitrosylation and total reversible cysteine oxidation are presented to demonstrate the utility of this general strategy.

  12. Methyl-coenzyme M reductase from methanogenic archaea: isotope effects on the formation and anaerobic oxidation of methane.

    Scheller, Silvan; Goenrich, Meike; Thauer, Rudolf K; Jaun, Bernhard


    The nickel enzyme methyl-coenzyme M reductase (MCR) catalyzes two important transformations in the global carbon cycle: methane formation and its reverse, the anaerobic oxidation of methane. MCR uses the methyl thioether methyl-coenzyme M (CH3-S-CH2CH2-SO3(-), Me-S-CoM) and the thiol coenzyme B (CoB-SH) as substrates and converts them reversibly to methane and the corresponding heterodisulfide (CoB-S-S-CoM). The catalytic mechanism is still unknown. Here, we present isotope effects for this reaction in both directions, catalyzed by the enzyme isolated from Methanothermobacter marburgensis . For methane formation, a carbon isotope effect ((12)CH3-S-CoM/(13)CH3-S-CoM) of 1.04 ± 0.01 was measured, showing that breaking of the C-S bond in the substrate Me-S-CoM is the rate-limiting step. A secondary isotope effect of 1.19 ± 0.01 per D in the methyl group of CD3-S-CoM indicates a geometric change of the methyl group from tetrahedral to trigonal planar upon going to the transition state of the rate-limiting step. This finding is consistent with an almost free methyl radical in the highest transition state. Methane activation proceeds with a primary isotope effect of 2.44 ± 0.22 for the C-H vs C-D bond breakage and a secondary isotope effect corresponding to 1.17 ± 0.05 per D. These values are consistent with isotope effects reported for oxidative cleavage/reductive coupling occurring at transition metal centers during C-H activation but are also in the range expected for the radical substitution mechanism proposed by Siegbahn et al. The isotope effects presented here constitute boundary conditions for any suggested or calculated mechanism.

  13. Immunological comparison of the NADH:nitrate reductase from different cucumber tissues

    Jolanta Marciniak


    Full Text Available Soluble nitrate reductase from cucumber roots (Cucumis sativus L. was isolated and purified with blue-Sepharose 4B. Specific antibodies against the NR protein were raised by immunization of a goat. Using polyclonal antibodies anti-NR properties of the nitrate reductase from various cucumber tissues were examined. Experiments showed difference in immuno-logical properties of nitrate reductase (NR from cotyledon roots and leaves.

  14. Comparative studies on the soluble and plasma membrane associated nitrate reductase from Cucumis sativus L.

    Grażyna Kłobus


    Full Text Available The biochemical comparison between two forms of nitrate reductase from cucumber roots: the soluble enzyme and the plasma membrane-associated one was made. Soluble nitrate reductase was purified on the blue-Sepharose 4B. The nitrate reductase bound with plasma membranes was isolated from cucumber roots by partition of microsomes in the 6.5% dextran-PEG two phase system. The molecular weight of native enzyme estimated with HPLC was 240 kDa and 114 kDa for the soluble and membrane bounded enzyme, respectively. Temperature induced phase separation in Triton X-114 indicated a huge difference in hydrophobicity of the plasma membrane associated nitrate reductase and soluble form of enzyme. Small differences were observed in partial activities of plasma membrane nitrate reductase and soluble nitrate reductase. Also experiments with polyclonal antiserum raised against the native nitrate reductase showed some differences in the immunological properties of both forms of the nitrate reductase. The above results indicated that in cucumber roots two different forms of the nitrate reductase are present.

  15. Molecular Characterization of Thiols in Fossil Fuels by Michael Addition Reaction Derivatization and Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

    Wang, Meng; Zhao, Suoqi; Liu, Xuxia; Shi, Quan


    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.

  16. Aldo-keto reductases 1B in adrenal cortex physiology

    Emilie PASTEL


    Full Text Available Aldose reductase proteins are cytosolic monomeric enzymes, belonging to the aldo-keto reductase (AKR superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates such as aliphatic and aromatic aldehydes or ketones. The Aldose reductase subgroup (AKR1B is one of the most characterized because of its involvement in human diseases such as diabetic complications resulting from the ability of its human archetype AKR1B1 to reduce glucose into sorbitol. However the issue of AKR1B function in non pathologic condition remains poorly resolved. Adrenal steroidogenesis is strongly associated with high production of endogenous harmful lipid aldehyde by-products including isocaproaldehyde (4-methylpentanal derived from cholesterol side chain cleavage (the first step of steroid synthesis and 4-hydroxynonenal (4- HNE that can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase activity, suggesting that in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, previous studies have established that the adrenal gland is one of the major site for human and murine AKR1B expression suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms.This review presents the molecular mechanisms accounting for the adrenal specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions.

  17. Terpenoids from Diplophyllum taxifolium with quinone reductase-inducing activity.

    Wang, Xiao; Zhang, Jiao-Zhen; Zhou, Jin-Chuan; Shen, Tao; Lou, Hong-Xiang


    Two new ent-prenylaromadendrane-type diterpenoids, diplotaxifols A (1) and B (2), a new ent-eudesmol, ent-eudesma-4(15),11(13)-dien-6α,12-diol (3), eight new eudesmanolides enantiomers (4-11) of the corresponding compounds from higher plants along with four known ent-eudesmanolides (12-15) were isolated from the 95% EtOH extract of Chinese liverwort Diplophyllum taxifolium. Their structures were elucidated on the basis of MS, NMR and IR spectral data, and confirmed by single-crystal X-ray diffraction analysis. The quinone reductase-inducing activity of the compounds was evaluated.

  18. Applications of Carboxylic Acid Reductases in Oleaginous Microbes

    Resch, Michael G.; Linger, Jeffrey; McGeehan, John; Tyo, Keith; Beckham, Gregg


    Carboxylic acid reductases (CARs) are recently emerging reductive enzymes for the direct production of aldehydes from biologically-produced carboxylic acids. Recent work has demonstrated that these powerful enzymes are able to reduce a very broad range of volatile- to long-chain fatty acids as well as aromatic acids. Here, we express four CAR enzymes from different fungal origins to test their activity against fatty acids commonly produced in oleaginous microbes. These in vitro results will inform metabolic engineering strategies to conduct mild biological reduction of carboxylic acids in situ, which is conventionally done via hydrotreating catalysis at high temperatures and hydrogen pressures.

  19. Methylenetetrahydrofolate reductase (MTHFR) deficiency presenting as a rash.

    Crushell, Ellen


    We report on the case of a 2-year-old girl recently diagnosed with Methylenetetrahydrofolate reductase (MTHFR) deficiency who originally presented in the neonatal period with a distinctive rash. At 11 weeks of age she developed seizures, she had acquired microcephaly and developmental delay. The rash deteriorated dramatically following commencement of phenobarbitone; both rash and seizures abated following empiric introduction of pyridoxine and folinic acid as treatment of possible vitamin responsive seizures. We postulate that phenobarbitone in combination with MTHFR deficiency may have caused her rash to deteriorate and subsequent folinic acid was helpful in treating the rash and preventing further acute neurological decline as commonly associated with this condition.



    In this paper the results of inhibition of the Aldose reductase(AR) activity on Wistar rat lens by Quercetagetin extracted from Tagetes erects Linn and by Patuletin extracted from Tagetes patula Linn are reported.Quercetagetin inhibited AR of the rat lens by 93.9% at 10~(-4)M, 76.0% at 10~(-5)M and 13.3% at 10~(-6)M. Patuletin inhibited AR of the rat lens by 100% at 10~(-1)M, 80% at 10~(-5)M and 22.7% at 10~(-6)M respectively. The results show that these two flavones are lens AR Inhibitors, but further ...

  1. The role of thiol species in the hypertolerance of Aspergillus sp. P37 to arsenic.

    Cánovas, David; Vooijs, Riet; Schat, Henk; de Lorenzo, Víctor


    Aspergillus sp. P37 is an arsenate-hypertolerant fungus isolated from a river in Spain with a long history of contamination with metals. This strain is able to grow in the presence of 0.2 M arsenate, i.e. 20-fold higher than the reference strain, Aspergillus nidulans TS1. Although Aspergillus sp. P37 reduces As(V) to As(III), which is slowly pumped out of the cell, the measured efflux of oxyanions is insufficient to explain the high tolerance levels of this strain. To gain an insight into this paradox, the accumulation of acid-soluble thiol species in Aspergillus sp. P37 when exposed to arsenic was compared with that of the arsenic-sensitive A. nidulans TS1 strain. Increasing levels of arsenic in the medium did not diminish the intracellular pool of reduced glutathione in Aspergillus sp. P37, in sharp contrast with the decline of glutathione in A. nidulans under the same conditions. Furthermore, concentrations of arsenic that were inhibitory for the sensitive A. nidulans strain (e.g. 50 mM and above) provoked a massive formation of vacuoles filled with thiol species. Because the major fraction of the cellular arsenic was present as the glutathione conjugate As(GS)3, it is plausible that the arsenic-hypertolerant phenotype of Aspergillus sp. P37 is in part due to an enhanced capacity to maintain a large intracellular glutathione pool under conditions of arsenic exposure and to sequester As(GS)3 in vacuoles. High pressure liquid chromatography analysis of cell extracts revealed that the contact of Aspergillus sp. P37 (but not A. nidulans) with high arsenic concentrations (> or =150 mM) induced the production of small quantities of a distinct thiol species indistinguishable from plant phytochelatin-2. Yet, we argue that phytochelatins do not explain arsenic resistance in Aspergillus, and we advocate the role of As(GS)3 complexes in arsenic detoxification.

  2. Quantification of Polyfunctional Thiols in Wine by HS-SPME-GC-MS Following Extractive Alkylation

    Lauren E. Musumeci


    Full Text Available Analyses of key odorous polyfunctional volatile thiols in wines (3-mercaptohexanol (3-MH, 3-mercaptohexylacetate (3-MHA, and 4-mercapto-4-methyl-2-pentanone (4-MMP are challenging due to their high reactivity and ultra-trace concentrations, especially when using conventional gas-chromatography electron impact mass spectrometry (GC-EI-MS. We describe a method in which thiols are converted to pentafluorobenzyl (PFB derivatives by extractive alkylation and the organic layer is evaporated prior to headspace solid phase microextraction (HS-SPME and GC-EI-MS analysis. Optimal parameters were determined by response surface area modeling. The addition of NaCl solution to the dried SPME vials prior to extraction resulted in up to less than fivefold improvement in detection limits. Using 40 mL wine samples, limits of detection for 4-MMP, 3-MH, and 3-MHA were 0.9 ng/L, 1 ng/L, and 17 ng/L, respectively. Good recovery (90%–109% and precision (5%–11% RSD were achieved in wine matrices. The new method was used to survey polyfunctional thiol concentrations in 61 commercial California and New York State wines produced from V. vinifera (Riesling, Gewürztraminer, Cabernet Sauvignon, Sauvignon blanc and non-varietal rosé wines, V. labruscana (Niagara, and Vitis spp. (Cayuga White. Mean 4-MMP concentrations in New York Niagara (17 ng/L were not significantly different from concentrations in Sauvignon blanc, but were significantly higher than 4-MMP in other varietal wines.

  3. Advantages and drawbacks of Thiol-ene based resins for 3D-printing

    Leonards, Holger; Engelhardt, Sascha; Hoffmann, Andreas; Pongratz, Ludwig; Schriever, Sascha; Bläsius, Jana; Wehner, Martin; Gillner, Arnold


    The technology of 3D printing is conquering the world and awakens the interest of many users in the most varying of applications. New formulation approaches for photo-sensitive thiol-ene resins in combination with various printing technologies, like stereolithography (SLA), projection based printing/digital light processing (DLP) or two-photon polymerization (TPP) are presented. Thiol-ene polymerizations are known for its fast and quantitative reaction and to form highly homogeneous polymer networks. As the resins are locally and temporally photo-curable the polymerization type is very promising for 3D-printing. By using suitable wavelengths, photoinitiator-free fabrication is feasible for single- and two photon induced polymerization. In this paper divinyl ethers of polyethylene glycols in combination with star-shaped tetrathiols were used to design a simple test-system for photo-curable thiol-ene resins. In order to control and improve curing depth and lateral resolution in 3D-polymerization processes, either additives in chemical formulation or process parameters can be changed. The achieved curing depth and resolution limits depend on the applied fabrication method. While two-/multiphoton induced lithography offers the possibility of micron- to sub-micron resolution it lacks in built-up speed. Hence single-photon polymerization is a fast alternative with optimization potential in sub-10-micron resolution. Absorber- and initiator free compositions were developed in order to avoid aging, yellowing and toxicity of resulting products. They can be cured with UV-laser radiation below 300 nm. The development at Fraunhofer ILT is focusing on new applications in the field of medical products and implants, technical products with respect to mechanical properties or optical properties of 3D-printed objects. Recent process results with model system (polyethylene glycol divinylether/ Pentaerithrytol tetrakis (3-mercaptopropionat), Raman measurements of polymer conversion

  4. Analysis of Phytochelatins and Other Thiol-Containing Compounds by RP-HPLC with Monobromobimane Precolumn Derivatization

    Sun Qin; Ye Zhihong; Wang Xiaorong; Wong Minghong


    In this article,a method for quantitative determination ofphytochelatins(PCn being the classic example)and other thiol-containing compounds in mixed standard solution and plant tissues is presented.Thiols were converted to fluorescent derivatives by precolumn derivatization with monobromobimane.The results showed that PCn and other thiol-containing compounds in standard mixed solutions were rapidly separated within 15 min by using a ACN 0.1% trifluoroacetic acid binary gradient elution.Glutathione was representatively selected to test the precision of this method.The calibration curve was linear in the range of 1.25-160 ng μl-1(regression coefficient r2=0.9999).It was confirmed that this method was rapid,simple,highly sensitive,stable,and had the property of simultaneous determination of PCn and other thiol-containing compounds.This method was applied to determine PCn and other thiol-containing compounds in a Cd hyperaccumulator Sedum alfredii in response to Cd.It was found that no PCn was detected in any tissue at any Cd treatment,suggesting that Cd hyperaccumulation and detoxification in this plant is not based on PC synthesis.

  5. The Synthesis of a Coumarin Carbohydrazide Dinuclear Copper Complex Based Fluorescence Probe and Its Detection of Thiols.

    He, Guangjie; Li, Jing; Yang, Lu; Hou, Chunhua; Ni, Tianjun; Yang, Zhijun; Qian, Xinlai; Li, Changzheng


    Small-molecule thiols, such as cysteine (CYS) and glutathione (GSH), are essential for maintaining the cellular redox environment and play important roles in regulating various cellular physiological functions. A fluorescence probe (compound 1-Cu2+) for thiols based on coumarin carbohydrazide dinuclear copper complex was developed. Compound 1 was synthesized from the reaction of 7-(diethylamino)-2-oxo-2H-chromene-3-carbohydrazide with 4-tert-butyl-2,6- diformylphenol. Accordingly, the copper complex (compound 1-Cu2+) was prepared by mixing compound 1 with 2 equivalents copper ions. Compound 1 had strong fluorescence while compound 1-Cu2+ hardly possessed fluorescence owing to the quenching nature of paramagnetism Cu2+ to the fluorescence molecule excited state. However, the fluorescence intensity of compound 1-Cu2+ was increased dramatically after the addition of thiol-containing amino acids, but not the other non-sulfhydryl amino acids. UV-vis absorption and fluorescence spectra indicated that compound 1-Cu2+ had good selectivity and sensitivity for thiols such as glutathione in CH3CN:H2O (3:2, v/v) PBS solution. The fluorescence imaging experiments implied that compound 1-Cu2+ has potential application in thiol-containing amino acids detection in living cells.

  6. Synthesis and Optical Properties of Thiol Functionalized CdSe/ZnS (Core/Shell) Quantum Dots by Ligand Exchange

    Zhu, Huaping [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); South China Univ. of Technology (SCUT), Gangzhou (China); Hu, Michael Z. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Shao, Lei [Beijing Univ. of Chemical Technology (China); Yu, Kui [National Research Council of Canada, Ottawa, ON (Canada); Dabestani, Reza T [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Zaman, Md. Badruz [National Research Council of Canada, Ottawa, ON (Canada); Liao, Dr. Shijun [South China University of Technology, Guangzhou, PR China


    The colloidal photoluminescent quantum dots (QDs) of CdSe (core) and CdSe/ZnS (core/shell) were synthesized at different temperatures with different growth periods. The optical properties (i.e., UV/Vis spectra and photoluminescent emission spectra) of the resulting QDs were investigated. The CdSe/ZnS QDs exhibited higher photoluminescent (PL) efficiency and stability than their corresponding CdSe core QDs. Ligand exchange with various thiol molecules was performed to replace the initial surface passivation ligands, that is, trioctylphosphine oxide (TOPO) and trioctylphosphine (TOP), and the optical properties of the surface-modified QDs were studied. The thiol ligand molecules used included 1,4-benzenedimethanethiol, 1,16-hexadecanedithiol, 1,11-undecanedithiol, 11-mercapto-1-undecanol, and 1,8 octanedithiol. After the thiol functionalization, the CdSe/ZnS QDs exhibited significantly enhanced PL efficiency and storage stability. Besides surface passivation effect, such enhanced performance of thiol-functionalized QDs could be due to self-assembly formation of dimer/trimer clusters, in which QDs are linked by dithiol molecules. Effects of ligand concentration, type of ligand, and heating on the thiol stabilization of QDs were also discussed.

  7. Synthesis and Optical Properties of Thiol Functionalized CdSe/ZnS (Core/Shell Quantum Dots by Ligand Exchange

    Huaping Zhu


    Full Text Available The colloidal photoluminescent quantum dots (QDs of CdSe (core and CdSe/ZnS (core/shell were synthesized at different temperatures with different growth periods. Optical properties (i.e., UV/Vis spectra and photoluminescent emission spectra of the resulting QDs were investigated. The shell-protected CdSe/ZnS QDs exhibited higher photoluminescent (PL efficiency and stability than their corresponding CdSe core QDs. Ligand exchange with various thiol molecules was performed to replace the initial surface passivation ligands, that is, trioctylphosphine oxide (TOPO and trioctylphosphine (TOP, and the optical properties of the surface-modified QDs were studied. The thiol ligand molecules in this study included 1,4-benzenedimethanethiol, 1,16-hexadecanedithiol, 1,11-undecanedithiol, biphenyl-4,4′-dithiol, 11-mercapto-1-undecanol, and 1,8-octanedithiol. After the thiol functionalization, the CdSe/ZnS QDs exhibited significantly enhanced PL efficiency and storage stability. Besides surface passivation effect, such enhanced performance of thiol-functionalized QDs could be due to cross-linked assembly formation of dimer/trimer clusters, in which QDs are linked by dithiol molecules. Furthermore, effects of ligand concentration, type of ligand, and heating on the thiol stabilization of QDs were also discussed.

  8. Copper and ceruloplasmin levels in relation to total thiols and GST in type 2 diabetes mellitus patients.

    Sarkar, A; Dash, S; Barik, B K; Muttigi, Manjunatha S; Kedage, V; Shetty, J K; Prakash, M


    Presence of oxidative stress in type 2 diabetes mellitus (DM) is well proved. Current study was undertaken to know the relation between fasting plasma glucose (FPG) and copper along with antioxidants like total thiols and ceruloplasmin, and antioxidant enzyme glutathione S transferase (GST). The study group consisted of a total of 201 subjects which included nondiabetic healthy control subjects (n = 78) and diabetic patients (n = 123). Plasma total thiols, GST, copper and ceruloplasmin levels were measured all the subjects using spectrophotometric methods and FPG levels were determined in clinical chemistry analyzer Hitachi 912. There was significant increase in FPG (P<0.001) and copper (P<0.001) and decrease in ceruloplasmin (P<0.001) and protein thiols (P<0.001) in type 2 DM cases compared to healthy controls. There was no significant change in GST between type 2 DM cases and controls. There was significant negative correlation of FPG with antioxidants like ceruloplasmin (r = -0.420, P<0.001) and total thiols (r = -0.565, P<0.001). Protein thiols correlated positively with ceruloplasmin (r = 0.364, P<0.001). Our study indicates possible increase in copper mediated generation of ROS leading to increased consumption of available antioxidants in the body.

  9. “Oxygen sensing” by Na,K-ATPase: these miraculous thiols

    Anna Bogdanova


    Full Text Available Control over the Na,K-ATPase function plays a central role in adaptation of the organisms to hypoxic and anoxic conditions. As the enzyme itself does not possess O2 binding sites its oxygen-sensitivity is mediated by a variety of redox-sensitive modifications including S-glutathionylation, S-nitrosylation and redox-sensitive phosphorylation. This is an overview of the current knowledge on the plethora of molecular mechanisms tuning the activity of the ATP-consuming Na,K-ATPase to the cellular metabolic activity. Recent findings suggest that oxygen-derived free radicals and H2O2, NO, and oxidised glutathione are the signalling messengers that make the Na,K-ATPase oxygen-sensitive. This very ancient signalling pathway targeting thiols of all three subunits of the Na,K-ATPase as well as redox-sensitive kinases sustains the enzyme activity at the optimal level avoiding terminal ATP depletion and maintaining the transmembrane ion gradients in cells of anoxia-tolerant species. We acknowledge the complexity of the underlying processes as we characterise the sources of reactive oxygen and nitrogen species production in hypoxic cells, and identify their targets, the reactive thiol groups which, upon modification, impact the enzyme activity. Structured accordingly, this review presents a summery on (i the sources of free radical production in hypoxic cells, (ii localisation of regulatory thiols within the Na,K-ATPase and the role reversible thiol modifications play in responses of the enzymes to a variety of stimuli (hypoxia, receptors’ activation control of the enzyme activity (iii redox-sensitive regulatory phosphorylation, and (iv the role of fine modulation of the Na,K-ATPase function in survival success under hypoxic conditions. The co-authors attempted to cover all the contradictions and standing hypotheses in the field and propose the possible future developments in this dynamic area of research, the importance of which is hard to overestimate

  10. Reactivity of vegetable oil macromonomers in thiol-ene, cationic, and emulsion polymerizations

    Black, Micah Stephen

    Vegetable oils were, and continue, to be a mainstay in unsaturated polyester ("alkyd") technology. Our endeavor is to use vegetable oil-based polymers in environmentally-friendly coatings. The role of vegetable oil cis-unsaturation has not been fully explored in polymers. To that end, vegetable oil macromonomers (VOMMs) in three different systems were investigated to determine the involvement of cis-unsaturation in chain transfer, auto-oxidation, and copolymerization reactions. VOMMs were incorporated into UV curable thiol-ene coatings, UV cationic coatings, and acrylic solution copolymers and fundamental studies were conducted to determine how and to what extent cis-unsaturation contributes to film performance properties. In thiol-ene UV curable coatings, cis-unsaturation was involved in the initial curing reaction and to lesser degrees, in postcure crosslinking. Its behavior was determined to be dependent on the ene component. Thiol-ene photopolymerization yielded homogeneous networks but formulations containing VOMMs exhibited greater heterogeneity due to non-uniformity in the VOMM chemical structures, and the concurrent reactions occurring during thiol-ene photopolymerization and "dark cure". Partially epoxidized soybean oil was synthesized to contain varying levels of residual cis-unsaturation. Cationic photopolymerization of partially epoxidized soybean oil yielded lightly crosslinked films, but the influence of free radical decomposition byproducts has not been fully investigated. The low involvement of the cis-unsaturation in photopolymerization was attributed to its low reactivity and/or radical combination with antioxidants and molecular oxygen dissolved in the films. When used in emulsion polymerization, VOMMs lower the minimum filming temperature during coalescence and increase the Tg after application via auto-oxidation. Free radical polymerization of VOMMs is accompanied by chain transfer reactions between polymer radicals and VOMMs that reduces

  11. Characterization of thiol-functionalised silica films deposited on electrode surfaces

    Ivana Cesarino


    Full Text Available Thiol-functionalised silica films were deposited on various electrode surfaces (gold, platinum, glassy carbon by spin-coating sol-gel mixtures in the presence of a surfactant template. Film formation occurred by evaporation induced self-assembly (EISA involving the hydrolysis and (cocondensation of silane and organosilane precursors on the electrode surface. The characterization of such material was performed by IR spectroscopy, thermogravimetry (TG, elemental analysis (EA, atomic force microscopy (AFM, scanning electron microscopy (SEM and cyclic voltammetry (CV.

  12. 巯基-烯点击化学%Thiol-Ene Click Chemistry

    徐源鸿; 熊兴泉; 蔡雷; 唐忠科; 叶章基


    "Click chemistry", introduced by Sharpless in 2001, becomes a new synthetic method used in areas such as drugs, polymers and materials rapidly because of its high efficiency, high selectivity and reliable characteristics. With the further research on click chemistry, the types of it are increasing continuously and the scope of application is also expanding. Radical-mediated or nucleophile-initiated thiol-ene reaction is a novel kind of click reactions, which shows the characteristics of click chemistry. Starting from the concept, characteristic and types of click chemistry, the mechanism and the wide applications of thiol-ene reaction in preparation of functional polymers and topologic macromolecules, polymeric materials surface modification and biomaterials are emphasized. Furthermore, the latest research based on thiol-ene chemistry is summarized. The prospects of thiol-ene reaction are also discussed.%点击化学自2001年由Sharpless提出后,由于其高效、可靠、高选择性的特点迅速成为药物和高分子材料合成的新方法。随着对点击化学研究的深入,其反应类型在不断增多,应用范围也在不断扩大。自由基或亲核试剂引发的巯基.烯反应作为其中一种新型的点击反应具有点击化学的所有特性。本文从点击化学的概念、特征和类型出发,重点介绍了巯基-烯反应的机理和在合成功能聚合物、制备拓扑结构高分子、表面修饰以及生物药物等方面的应用,并对巯基.烯反应的最新研究成果进行综述,最后展望了巯基-烯的点击化学的发展前景。

  13. Thiol-functionalized MCM-48: an Effective Absorbent of Mercury Ions

    Shu Guo WANG; Jin Lin LI


    Mercaptopropyl groups were grafted onto the pore walls of mesoporous molecular sieves MCM-48. The pore structures were characterized by powder X-ray diffraction and N2 adsorption analysis. Elemental analysis confirmed that the material with high organic moiety (2.2 mmol/g) had been obtained. 13C MAS NMR verified the tethered organic groups. The thiol-functionalized MCM-48 showed effective capture of mercury ions, and all of the organic ligands were accessible for the binding of mercury ions.

  14. Self-consistent GW calculations of electronic transport in thiol- and amine-linked molecular junctions

    Strange, M.; Rostgaard, Carsten; Hakkinen, H.


    The electronic conductance of a benzene molecule connected to gold electrodes via thiol, thiolate, or amino anchoring groups is calculated using nonequilibrium Green functions in combination with the fully self-consistent GW approximation for exchange and correlation. The calculated conductance...... suggest that more complex gold-thiolate structures where the thiolate anchors are chemically passivated by Au adatoms are responsible for the measured conductance. Analysis of the energy level alignment obtained with DFT, Hartree-Fock, and GW reveals the importance of self-interaction corrections...

  15. Redox Characteristics of Thiol Compounds Using Radicals Produced by Water Vapor Radio Frequency Discharge

    Hayashi, Nobuya; Nakahigashi, Akari; Goto, Masaaki; Kitazaki, Satoshi; Koga, Kazunori; Shiratani, Masaharu


    The redox reaction between cystein and cystine is observed using radicals produced in water vapor plasma for the control of plant growth. Cystein is oxidized to cystine using the OH radical in the higher-pressure regime and cystine is reduced to cystein by the H radical generated in the lower-pressure regime. Also, the oxidative stress reaction of plants is observed when water vapor plasma is irradiated onto seeds of plants such as radish sprouts. The mechanism of the control of plant growth is explained by the change in thiol compound quantity of the plant cells induced by the radical reaction.

  16. Thiol-disulfide balance: from the concept of oxidative stress to that of redox regulation.

    Ghezzi, Pietro; Bonetto, Valentina; Fratelli, Maddalena


    Originally, small thiols, including glutathione, were viewed as protective antioxidants, acting as free radical scavengers in the context of oxidative damage. Recently, there is a growing literature showing that protein glutathionylation (formation of protein-glutathione mixed disulfides) and other forms of cysteine oxidation may be a means of redox regulation under physiological conditions. This review discusses the importance of protein oxidation in redox regulation in view of the recent data originating from the application of redox proteomics to identify redox-sensitive targets.

  17. Tailoring the physical properties of thiol-capped PbS quantum dots by thermal annealing.

    Turyanska, L; Elfurawi, U; Li, M; Fay, M W; Thomas, N R; Mann, S; Blokland, J H; Christianen, P C M; Patanè, A


    We show that the thermal annealing of thiol-capped PbS colloidal quantum dots provides a means of narrowing the nanoparticle size distribution, increasing the size of the quantum dots and facilitating their coalescence preferentially along the 100 crystallographic axes. We exploit these phenomena to tune the photoluminescence emission of an ensemble of dots and to narrow the optical linewidth to values that compare with those reported at room temperature for single PbS quantum dots. We probe the influence of annealing on the electronic properties of the quantum dots by temperature dependent studies of the photoluminescence and magneto-photoluminescence.

  18. Considering Hydrophobicity via Contact Angle Stability of Organic Thiols Measured with a Homemade Goniometer

    Seraly, Mark; Ollander, Brooke; Statman, Ariel; Poynor, Adele


    When water meets an extended hydrophobic surface, an ultra-thin, low-density depletion layer is expected at the interface. Exactly how the depletion layer changes with change in hydrophobicity is still an open question. An accurate measure of contact angle is essential in determining how water meets a hydrophobic surface. Utilizing a homemade goniometer with ImageJ software we investigate the stability of self-assembled organic thiol monolayers, 1-octadecanethiol (ODT) and 11-mercaptoundecanoic acid (MUA). We report the changes in contact angle due to exposure to air, water, and ethanol. Other factors that affect contact angles were also considered in our investigation.

  19. Functions of Flavin Reductase and Quinone Reductase in 2,4,6-Trichlorophenol Degradation by Cupriavidus necator JMP134▿

    Belchik, Sara Mae; Xun, Luying


    The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), a toxic pollutant. TcpA is a reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase that converts 2,4,6-TCP to 6-chlorohydroxyquinone. It has been implied via genetic analysis that TcpX acts as an FAD reductase to supply TcpA with FADH2, whereas the function of TcpB in 2,4,6-TCP degradation is still unclear. In order to provide direct biochemical evidence for t...

  20. Properties of the arsenate reductase of plasmid R773.

    Gladysheva, T B; Oden, K L; Rosen, B P


    Resistance to toxic oxyanions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells of E. coli, thus providing resistance to those toxic oxyanions. In addition, resistance to arsenate is conferred by the product of the arsC gene. In this report, purified ArsC protein was shown to catalyze reduction of arsenate to arsenite. The enzymatic activity of the ArsC protein required glutaredoxin as a source of reducing equivalents. Other reductants, including glutathione and thioredoxin, were not effective electron donors. A spectrophotometric assay was devised in which arsenate reduction was coupled to NADPH oxidation. The results obtained with the coupled assay corresponded to those found by direct reduction of radioactive arsenate to arsenite. The only substrate of the reaction was arsenate (Km = 8 mM); other oxyanions including phosphate, sulfate, and antimonate were not reduced. Phosphate and sulfate were weak inhibitors, while the product, arsenite, was a stronger inhibitor (Ki = 0.1 mM). Arsenate reductase activity exhibited a pH optimum of 6.3-6.8. These results indicate that the ArsC protein is a novel reductase, and elucidation of its enzymatic mechanism should be of interest.

  1. Dimethyl Fumarate Induces Glutathione Recycling by Upregulation of Glutathione Reductase

    Hoffmann, Christina; Dietrich, Michael; Herrmann, Ann-Kathrin; Schacht, Teresa


    Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate (DMF) is an effective oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. DMF activates the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) leading to increased synthesis of the major cellular antioxidant glutathione (GSH) and prominent neuroprotection in vitro. We previously demonstrated that DMF is capable of raising GSH levels even when glutathione synthesis is inhibited, suggesting enhanced GSH recycling. Here, we found that DMF indeed induces glutathione reductase (GSR), a homodimeric flavoprotein that catalyzes GSSG reduction to GSH by using NADPH as a reducing cofactor. Knockdown of GSR using a pool of E. coli RNase III-digested siRNAs or pharmacological inhibition of GSR, however, also induced the antioxidant response rendering it impossible to verify the suspected attenuation of DMF-mediated neuroprotection. However, in cystine-free medium, where GSH synthesis is abolished, pharmacological inhibition of GSR drastically reduced the effect of DMF on glutathione recycling. We conclude that DMF increases glutathione recycling through induction of glutathione reductase. PMID:28116039

  2. Stereospecificity of (+)-pinoresinol and (+)-lariciresinol reductases from Forsythia intermedia.

    Chu, A; Dinkova, A; Davin, L B; Bedgar, D L; Lewis, N G


    Pinoresinol/lariciresinol reductase catalyzes the first known example of a highly unusual benzylic ether reduction in plants; its mechanism of hydride transfer is described. The enzyme was found in Forsythia intermedia and catalyzes the presumed regulatory branch-points in the pathway leading to benzylaryltetrahydrofuran, dibenzylbutane, dibenzylbutyrolactone, and aryltetrahydronaphthalene lignans. Using [7,7'-2H2]-pinoresinol and [7,7'-2H3]lariciresinol as substrates, the hydride transfers of the highly unusual reductase were demonstrated to be completely stereospecific (> 99%). The incoming hydrides were found to take up the pro-R position at C-7' (and/or C-7) in lariciresinol and secoisolariciresinol, thereby eliminating the possibility of random hydride delivery to a planar quinone methide intermediate. As might be expected, the mode of hydride abstraction from NADPH was also stereospecific: using [4R-3H] and [4S-3H]NADPH, it was found that only the 4 pro-R hydrogen was abstracted for enzymatic hydride transfer.

  3. Dimethyl Fumarate Induces Glutathione Recycling by Upregulation of Glutathione Reductase

    Christina Hoffmann


    Full Text Available Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate (DMF is an effective oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. DMF activates the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2 leading to increased synthesis of the major cellular antioxidant glutathione (GSH and prominent neuroprotection in vitro. We previously demonstrated that DMF is capable of raising GSH levels even when glutathione synthesis is inhibited, suggesting enhanced GSH recycling. Here, we found that DMF indeed induces glutathione reductase (GSR, a homodimeric flavoprotein that catalyzes GSSG reduction to GSH by using NADPH as a reducing cofactor. Knockdown of GSR using a pool of E. coli RNase III-digested siRNAs or pharmacological inhibition of GSR, however, also induced the antioxidant response rendering it impossible to verify the suspected attenuation of DMF-mediated neuroprotection. However, in cystine-free medium, where GSH synthesis is abolished, pharmacological inhibition of GSR drastically reduced the effect of DMF on glutathione recycling. We conclude that DMF increases glutathione recycling through induction of glutathione reductase.

  4. Determination of potential N2O-reductase activity in soil

    Qin, S.P.; Yuan, H.J.; Hu, C.S.; Oenema, O.; Zhang, Y.M.; Li, X.X.


    Determination of N2O-reductase activity in soil is important for understanding the microbial regulation of nitrous oxide (N2O) concentrations in soil. Unfortunately, there are no easily applicable and accurate methods for determining N2O-reductase activity, which frustrates the understanding of the




    The polyol pathway has long been associated with diabetic retinopathy. Glucose is converted to sorbitol with the aid of the enzyme aldose reductase. Aldose reductase inhibitors can prevent changes induced by diabetes. A total of 30 patients with minimal background retinopathy were randomly divided i

  6. Separation and distribution of thiosulfate-oxidizing enzyme, tetrathionate reductase, and thiosulfate reductase in extracts of marine heterotroph strain 16B.

    Whited, G M; Tuttle, J.H.


    Thiosulfate-oxidizing enzyme (TSO), tetrathionate reductase (TTR), and thiosulfate reductase (TSR) were demonstrated in cell-free extracts of the marine heterotrophic thiosulfate-oxidizing bacterium strain 16B. Extracts prepared from cells cultured aerobically in the absence of thiosulfate or tetrathionate exhibited constitutive TSO and TTR activity which resided in the soluble fraction of ultracentrifuged crude extracts. Constitutive TSO and TTR cochromatographed on DEAE-Sephadex A-50, Celle...

  7. The effect of ionic and non-ionic surfactants on the growth, nitrate reductase and nitrite reductase activities of Spirodela polyrrhiza (L. Schleiden

    Józef Buczek


    Full Text Available Inclusion into the medium of 5 mg•dm-3 of non-ionic (ENF or ionic (DBST surfactant caused 50-60% inhibition of nitrite reductase MR activity in S. polyrrhiza. At the same time, increased accumulation of NO2- in the plant tissues and lowering of the total and soluble protein contents were found. DBST also lowered the nitrate reductase (NR activity and the dry mass of the plants.

  8. Antioxidative Mechanisms of Sulfite and Protein-Derived Thiols during Early Stages of Metal Induced Oxidative Reactions in Beer.

    Lund, Marianne N; Krämer, Anna C; Andersen, Mogens L


    The radical-mediated reactions occurring during the early stages of beer storage were studied by following the rate of oxygen consumption, radical formation as detected by electron spin resonance spectroscopy, and concentrations of the antioxidant compounds sulfite and thiols. Addition of either Fe(III) or Fe(II) had similar effects, indicating that a fast redox equilibrium is obtained between the two species in beer. Addition of iron in combination with hydrogen peroxide gave the most pronounced levels of oxidation due to a direct initiation of ethanol oxidation through generation of hydroxyl radicals by the Fenton reaction. The concentration of sulfite decreased more than the thiol concentration, suggesting that thiols play a secondary role as antioxidants by mainly quenching 1-hydroxyethyl radicals that are intermediates in the oxidation of ethanol. Increasing the temperature had a minor effect on the rate of oxygen consumption.

  9. Quantifying changes in the cellular thiol-disulfide status during differentiation of B cells into antibody-secreting plasma cells

    Hansen, Rosa Rebecca Erritzøe; Otsu, Mieko; Braakman, Ineke


    Plasma cells produce and secrete massive amounts of disulfide-containing antibodies. To accommodate this load on the secretory machinery, the differentiation of resting B cells into antibody-secreting plasma cells is accompanied by a preferential expansion of the secretory compartments of the cells...... and by an up-regulation of enzymes involved in redox regulation and protein folding. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated proteins in whole cells. The results show that while the global thiol-disulfide state is affected to some extent...... by the differentiation, steady-state levels of glutathionylated protein thiols are less than 0.3% of the total protein cysteines, even in fully differentiated cells, and the overall protein redox state is not affected until late in differentiation, when large-scale IgM production is ongoing. A general expansion...

  10. Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 2: Iron- and Copper-Catalyzed Oxidation.

    Kreitman, Gal Y; Danilewicz, John C; Jeffery, David W; Elias, Ryan J


    Sulfidic off-odors arising during wine production are frequently removed by Cu(II) fining. In part 1 of this study ( 10.1021/acs.jafc.6b00641 ), the reaction of H2S and thiols with Cu(II) was examined; however, the interaction of iron and copper is also known to play an important synergistic role in mediating non-enzymatic wine oxidation. The interaction of these two metals in the oxidation of H2S and thiols (cysteine, 3-sulfanylhexan-1-ol, and 6-sulfanylhexan-1-ol) was therefore examined under wine-like conditions. H2S and thiols (300 μM) were reacted with Fe(III) (100 or 200 μM) alone and in combination with Cu(II) (25 or 50 μM), and concentrations of H2S and thiols, oxygen, and acetaldehyde were monitored over time. H2S and thiols were shown to be slowly oxidized in the presence of Fe(III) alone and were not bound to Fe(III) under model wine conditions. However, Cu(II) added to model wine containing Fe(III) was quickly reduced by H2S and thiols to form Cu(I) complexes, which then rapidly reduced Fe(III) to Fe(II). Oxidation of Fe(II) in the presence of oxygen regenerated Fe(III) and completed the iron redox cycle. In addition, sulfur-derived oxidation products were observed, and the formation of organic polysulfanes was demonstrated.

  11. Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases.

    Gutscher, Marcus; Sobotta, Mirko C; Wabnitz, Guido H; Ballikaya, Seda; Meyer, Andreas J; Samstag, Yvonne; Dick, Tobias P


    H(2)O(2) acts as a signaling molecule by oxidizing critical thiol groups on redox-regulated target proteins. To explain the efficiency and selectivity of H(2)O(2)-based signaling, it has been proposed that oxidation of target proteins may be facilitated by H(2)O(2)-scavenging peroxidases. Recently, a peroxidase-based protein oxidation relay has been identified in yeast, namely the oxidation of the transcription factor Yap1 by the peroxidase Orp1. It has remained unclear whether the protein oxidase function of Orp1 is a singular adaptation or whether it may represent a more general principle. Here we show that Orp1 is in fact not restricted to oxidizing Yap1 but can also form a highly efficient redox relay with the oxidant target protein roGFP (redox-sensitive green fluorescent protein) in mammalian cells. Orp1 mediates near quantitative oxidation of roGFP2 by H(2)O(2), and the Orp1-roGFP2 redox relay effectively converts physiological H(2)O(2) signals into measurable fluorescent signals in living cells. Furthermore, the oxidant relay phenomenon is not restricted to Orp1 as the mammalian peroxidase Gpx4 also mediates oxidation of proximal roGFP2 in living cells. Together, these findings support the concept that certain peroxidases harbor an intrinsic and powerful capacity to act as H(2)O(2)-dependent protein thiol oxidases when they are recruited into proximity of oxidizable target proteins.

  12. Selective Targeting of Extracellular Insulin-Degrading Enzyme by Quasi-Irreversible Thiol-Modifying Inhibitors.

    Abdul-Hay, Samer O; Bannister, Thomas D; Wang, Hui; Cameron, Michael D; Caulfield, Thomas R; Masson, Amandine; Bertrand, Juliette; Howard, Erin A; McGuire, Michael P; Crisafulli, Umberto; Rosenberry, Terrone R; Topper, Caitlyn L; Thompson, Caroline R; Schürer, Stephan C; Madoux, Franck; Hodder, Peter; Leissring, Malcolm A


    Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC50(app) = 63 nM), nontoxic at concentrations up to 100 μM, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets.

  13. The isolation of nuclear envelopes. Effects of thiol-group oxidation and of calcium ions.

    Comerford, S A; McLuckie, I F; Gorman, M; Scott, K A; Agutter, P S


    The effects of (a) oxidative cross-linking of protein thiol groups and (b) the presence or absence of Ca2+ ions on rat liver nuclear-envelope isolation were studied. Two envelope-isolation procedures were compared: a well characterized low-ionic-strength method and a recently developed high-ionic-strength method. The latter method seems preferable to the former in respect of lower intranuclear contamination of the envelopes, suppression of endogenous serine proteinase, and maintenance of high specific activities of envelope-associated enzymes. In both procedures, however, the presence of Ca2+ gave rise to a rapid, apparently irreversible, contamination of the envelopes by intranuclear material. This effect was half-maximal at 20 microM-Ca2+. In addition, the envelopes became contaminated with intranuclear material by a Ca2+-independent mechanism, apparently resulting from N-ethylmaleimide-sensitive intermolecular disulphide-bond formation. This oxidative process seemed to have two major kinetic components (half-life, t1/2, approx. 2 min and 10 min). In view of these findings, it is recommended that (i) for most purposes, nuclear envelopes be isolated by the newly developed high-ionic-strength procedure, (ii) irrespective of the method used, Ca2+-chelators be included in all the buffers, (iii) thiol-group oxidation be prevented or reversed during the procedure.

  14. Mixing thiols on the surface of silver nanoparticles: preserving antibacterial properties while introducing SERS activity

    Taglietti, Angelo, E-mail: [Università di Pavia, Dipartimento di Chimica (Italy); Diaz Fernandez, Yuri A. [Chalmers University of Technology, Department of Chemical and Biological Engineering (Sweden); Galinetto, Pietro [Università di Pavia, Dipartimento di Fisica (Italy); Grisoli, Pietro [Università di Pavia, Dipartimento di Scienze del Farmaco (Italy); Milanese, Chiara; Pallavicini, Piersandro [Università di Pavia, Dipartimento di Chimica (Italy)


    Controlling the surface composition of self-assembled monolayers is one of the major experimental challenges in nanotechnology. Despite the significant interest of the scientific community and the considerable number of publications related to this topic, the potential in this field is still far from being fully exploited.We present in this study a versatile method to coat silver nanoparticles (AgNPs) having average diameter of 7 nm with mixed monolayers of two thiols, achieving a precise control of surface composition. Different combinations of thiols have been investigated, and the nanomaterials obtained have been characterized by complementary experimental techniques, addressing the composition of the mixed monolayer. The surface-enhanced Raman spectroscopy (SERS) effect on a Raman reporter (7-mercapto-4-methylcoumarine) introduced into the mixed monolayers has also been investigated. The antibacterial activity of the coated AgNPs was investigated, showing that the colloids were active against Escherichia coli and Staphilococcus aureus irrespective of the nature of the mixed monolayer. These materials are good candidates as SERS-tags for biological applications.

  15. Thiol peroxidase-like activity of some intramolecularly coordinated diorganyl diselenides

    Sangit Kumar; Harkesh B Singh


    Several new diaryl diselenides having intramolecular coordinating groups have been synthesized by ortho-lithiation/Na2Se2 routes in good yield. Bis[2-(N-phenylferrocenecarboxamide)] diselenide (10), bis[2-(N-tert-butylferrocenecarboxamide)] diselenide (11), ()()-bis[2(--phenethylferrocenecarboxamide)] diselenide (12) were synthesized by the ortho-lithiation route. Bis[2-(N,N-dimethylaminomethylnaphthyl)] diselenide (13) was synthesized by lithium/bromide exchange reaction whereas bis(2,4-dinitrophenyl) diselenide (14) was prepared by the reaction of disodium diselenide with 2,4- dinitro-1-chlorobenzene. Thiol peroxidase-like activities of the diorganodiselenides have been evaluated by using H2O2 as substrate and PhSH as cosubstrate. Diselenides (13) and (14) with dimethylaminomethyl- or nitro-donor groups in close proximity to selenium, show much better thiol peroxidase-like activities compared to diselenides 10-12 with amide donor groups. Cyclic voltammetry study of diselenides 10-12 derived from redox-active ferrocenamide has been carried out.

  16. Calcium transport, thiol status, and hepatotoxicity following N-nitrosodimethylamine exposure in mice

    Reitman, F.A.; Berger, M.L.; Minnema, D.J.; Shertzer, H.G.


    The hepatotoxicant N-nitrosodimethylamine (NDMA) is presumed to exert toxicity through reactive metabolites. NDMA is similar in this respect to numerous other hepatotoxicants, for which hepatotoxicity is also associated with a rapid depletion of soluble and/or protein thiols, and an inhibition of calcium transport systems. The authors examined the hypothesis that hepatotoxicity for NDMA is preceded by thiol depletion and/or inhibition of calcium transport in isolated liver subcellular fractions. Centrizonal liver necrosis in mice was evident at 24 but not at 12 h subsequent to intraperitoneal administration of 40 mg NDMA/kg. Hepatotoxicity was not preceded by depletion of liver protein-free sulfhydryls, nor by protein sulfhydryl depletion in liver whole homogenate, microsomal, or plasma membrane fractions. NDMA-mediated toxicity was also not preceded by inhibition of calcium uptake capability by microsomal, mitochondrial, or plasma membrane fractions. In contrast, carbon tetrachloride produced the expected rapid decrease in microsomal calcium uptake capability, followed by a centrizonal necrosis that was maximal at about 24 h. These studies suggest that the mechanism of NDMA hepatotoxicity may differ from that of a number of other hepatotoxicants (e.g., carbon tetrachloride, acetaminophen, bromobenzene) for which toxicity is also mediated through reactive metabolites.

  17. Thiol-based redox regulation in sexual plant reproduction: new insights and perspectives.

    Traverso, Jose A; Pulido, Amada; Rodríguez-García, María I; Alché, Juan D


    The success of sexual reproduction in plants involves (i) the proper formation of the plant gametophytes (pollen and embryo sac) containing the gametes, (ii) the accomplishment of specific interactions between pollen grains and the stigma, which subsequently lead to (iii) the fusion of the gametes and eventually to (iv) the seed setting. Owing to the lack of mobility, plants have developed specific regulatory mechanisms to control all developmental events underlying the sexual plant reproduction according to environmental challenges. Over the last decade, redox regulation and signaling have come into sight as crucial mechanisms able to manage critical stages during sexual plant reproduction. This regulation involves a complex redox network which includes reactive oxygen species (ROS), reactive nitrogen species (RNS), glutathione and other classic buffer molecules or antioxidant proteins, and some thiol/disulphide-containing proteins belonging to the thioredoxin superfamily, like glutaredoxins (GRXs) or thioredoxins (TRXs). These proteins participate as critical elements not only in the switch between the mitotic to the meiotic cycle but also at further developmental stages of microsporogenesis. They are also implicated in the regulation of pollen rejection as the result of self-incompatibility. In addition, they display precise space-temporal patterns of expression and are present in specific localizations like the stigmatic papillae or the mature pollen, although their functions and subcellular localizations are not clear yet. In this review we summarize insights and perspectives about the presence of thiol/disulphide-containing proteins in plant reproduction, taking into account the general context of the cell redox network.

  18. Irreversible bonding of polyimide and polydimethylsiloxane (PDMS) based on a thiol-epoxy click reaction

    Hoang, Michelle V.; Chung, Hyun-Joong; Elias, Anastasia L.


    Polyimide is one of the most popular substrate materials for the microfabrication of flexible electronics, while polydimethylsiloxane (PDMS) is the most widely used stretchable substrate/encapsulant material. These two polymers are essential in fabricating devices for microfluidics, bioelectronics, and the internet of things; bonding these materials together is a crucial challenge. In this work, we employ click chemistry at room temperature to irreversibly bond polyimide and PDMS through thiol-epoxy bonds using two different methods. In the first method, we functionalize the surfaces of the PDMS and polyimide substrates with mercaptosilanes and epoxysilanes, respectively, for the formation of a thiol-epoxy bond in the click reaction. In the second method, we functionalize one or both surfaces with mercaptosilane and introduce an epoxy adhesive layer between the two surfaces. When the surfaces are bonded using the epoxy adhesive without any surface functionalization, an extremely small peel strength (0.3 N mm-1 (method 2) are observed, and failure occurs by tearing of the PDMS layer. We envision that the novel processing route employing click chemistry can be utilized in various cases of stretchable and flexible device fabrication.

  19. Effect of silver nanomaterials on the activity of thiol-containing antioxidants.

    Zhou, Yu-Ting; He, Weiwei; Lo, Y Martin; Hu, Xiaona; Wu, Xiaochun; Yin, Jun-Jie


    The use of nanomaterials in consumer products is rapidly expanding. In most studies, nanomaterials are examined as isolated ingredients. However, consumer products such as foods, cosmetics, and dietary supplements are complex chemical matrixes. Therefore, interactions between nanomaterials and other components of the product must be investigated to ensure the product's performance and safety. Silver nanomaterials are increasingly being used in food packaging as antimicrobial agents. Thiol-containing compounds, such as reduced glutathione (GSH), cysteine, and dihydrolipoic acid, are used as antioxidants in many consumer products. In the current study, we have investigated the interaction between silver nanomaterials and thiol-containing antioxidants. The selected Ag nanomaterials were Ag coated with citrate, Ag coated with poly(vinylpyrrolidone), and Au nanorods coated with Ag in a core/shell structure. We observed direct quenching of the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) by all three Ag nanomaterials to varying degrees. The Ag nanomaterials also reduced the quenching of DPPH by GSH to varying degrees. In addition, we determined that the mixture of GSH and Au@Ag nanorods held at 37 °C was less effective at quenching azo radical than at ambient temperature. Furthermore, we determined that Au@Ag nanorods significantly reduced the ability of GSH and cysteine to quench hydroxyl and superoxide radicals. The work presented here demonstrates the importance of examining the chemical interactions between nanomaterials used in products and physiologically important antioxidants.

  20. Preparation and properties of cyclic acetal based biodegradable gel by thiol-ene photopolymerization.

    Wang, Kemin; Lu, Jian; Yin, Ruixue; Chen, Lu; Du, Shuang; Jiang, Yan; Yu, Qiang


    Synthetic, hydrolytically degradable biomaterials have been widely developed for biomedical use; however, most of them will form acidic products upon degradation of polymer backbone. In order to address this concern, we proposed to fabricate a biodegradable gel based on the crosslinking of a cyclic acetal monomer with reactable diallyl group and multifunctional thiols by thiol-ene photopolymerization. This gel produces diols and carbonyl end groups upon hydrolytic degradation and could be entirely devoid of acidic by-products. Real time infrared spectroscopy was employed to investigate the effect of different light intensities and concentrations of photoinitiator on the polymerization kinetics. With the increase of the concentration of photoinitiator and light intensity, both the rate of polymerization and final double bond conversion increased. Degradation of cyclic acetal based networks was investigated in PBS medium so as to simulate physiological conditions. The remaining mass of the materials after 25 days incubation was 84%. TGA analysis showed that the gels exhibited a typical weight loss (97.2%) at around 378 °C. In vitro cytotoxicity showed that the cyclic acetal based gels had non-toxicity to cell L-929 and had good biocompatibility.

  1. Peach MYB7 activates transcription of the proanthocyanidin pathway gene encoding leucoanthocyanidin reductase, but not anthocyanidin reductase

    Hui eZhou


    Full Text Available Proanthocyanidins (PAs are a group of natural phenolic compounds that have a great effect on both flavour and nutritious value of fruit. It has been shown that PA synthesis is regulated by R2R3-MYB transcription factors (TFs via activation of PA-specific pathway genes encoding leucoanthocyanidin reductase (LAR and anthocyanidin reductase (ANR. Here, we report the isolation and characterization of a MYB gene designated PpMYB7 in peach. The peach PpMYB7 represents a new group of R2R3-MYB genes regulating PA synthesis in plants. It is able to activate transcription of PpLAR1 but not PpANR, and has a broader selection of potential bHLH partners compared with PpMYBPA1. Transcription of PpMYB7 can be activated by the peach basic leucine-zipper 5 TF (PpbZIP5 via response to ABA. Our study suggests a transcriptional network regulating PA synthesis in peach, with the results aiding the understanding of the functional divergence between R2R3-MYB TFs in plants.

  2. Proximal FAD histidine residue influences interflavin electron transfer in cytochrome P450 reductase and methionine synthase reductase.

    Meints, Carla E; Parke, Sarah M; Wolthers, Kirsten R


    Cytochrome P450 reductase (CPR) and methionine synthase reductase (MSR) transfer reducing equivalents from NADPH to FAD to FMN. In CPR, hydride transfer and interflavin electron transfer are kinetically coupled steps, but in MSR the two catalytic steps are represented by two distinct kinetic phases leading to transient formation of the FAD hydroquinone. In human CPR, His(322) forms a hydrogen-bond with the highly conserved Asp(677), a member of the catalytic triad. The catalytic triad is present in MSR, but Ala(312) replaces the histidine residue. To examine if this structural variation accounts for differences in their kinetic behavior, reciprocal substitutions were created. Substitution of His(322) for Ala in CPR does not affect the rate of NADPH hydride transfer or the FAD redox potentials, but does impede interflavin electron transfer. For MSR, swapping Ala(312) for a histidine residue resulted in the kinetic coupling of hydride and interflavin electron transfer, and eliminated the formation of the FAD hydroquinone intermediate. For both enzymes, placement of the His residue in the active site weakens coenzyme binding affinity. The data suggest that the proximal FAD histidine residue accelerates proton-coupled electron transfer from FADH2 to the higher potential FMN; a mechanism for this catalytic role is discussed.

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

    Ali, Mohammed; Ghosh, Sujit Kumar


    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.

  4. Formation of Monolayers by the Coadsorption of Thiols on Gold: Variation in the Length of the Alkyl Chain


    Methyl- terminated thiols generate surfaces that are composed of densely packed methyl groups and are both hydrophobic (Oa(H20) = 1120) and oleophobic ...together with monolayers of the two pure thiols. Both pure monolayers were autophobic and oleophobic : Oa(HD) = 470 for HS(CH2)2 1CH 3, 0a(HD) = 460 for...would be oleophobic , and we would expect Oa(HD) to be independent of the composition of the monolayer. The contact angles in Fig. 3 were measured

  5. Dynamic Changes of Nitrate Reductase Activity within 24 Hours


    [Objective] The research aimed to study the circadian rhythm of nitrate re- ductase activity (NRA) in plant. [Method] The wheat plants at heading stage were used as the materials for the measurement of dynamic changes of nitrate reductase activity (NRA) within 24 h under the conditions of constant high temperature. [Resulti The fluctuation of NRA in wheat changed greatly from 20:00 pm to 11:00 am. The enzyme activity remained constant, but at 14:00 the enzyme activity was the high- est, higher than all the other time points except the enzyme activity measured at11:00. The enzyme activity was the lowest of 17:00, which was lower than all the other time points except the enzyme activity measured at 2:00. [Conclusion] There were autonomous rhythm changes of NRA in wheat in a certain degree.

  6. Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).

    Wang, Xiaoqiang; He, Xianzhi; Lin, Jianqiao; Shao, Hui; Chang, Zhenzhan; Dixon, Richard A


    Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.

  7. Two methylenetetrahydrofolate reductase gene (MTHFR) polymorphisms, schizophrenia and bipolar disorder

    Jönsson, Erik G; Larsson, Kristina; Vares, Maria


    disorder. In a replication attempt the MTHFR C677T and A1298C SNPs were analyzed in three Scandinavian schizophrenia case-control samples. In addition, Norwegian patients with bipolar disorder were investigated. There were no statistically significant allele or genotype case-control differences....... The present Scandinavian results do not verify previous associations between the putative functional MTHFR gene polymorphisms and schizophrenia or bipolar disorder. However, when combined with previous studies in meta-analyses there is still evidence for association between the MTHFR C677T polymorphism......Recent meta-analyses of the methylenetetrahydrofolate reductase gene (MTHFR) have suggested association between two of its functional single gene polymorphisms (SNPs; C677T and A1298C) and schizophrenia. Studies have also suggested association between MTHFR C677T and A1298C variation and bipolar...

  8. Two methylenetetrahydrofolate reductase gene (MTHFR) polymorphisms, schizophrenia and bipolar disorder

    Jönsson, Erik G; Larsson, Kristina; Vares, Maria;


    Recent meta-analyses of the methylenetetrahydrofolate reductase gene (MTHFR) have suggested association between two of its functional single gene polymorphisms (SNPs; C677T and A1298C) and schizophrenia. Studies have also suggested association between MTHFR C677T and A1298C variation and bipolar....... The present Scandinavian results do not verify previous associations between the putative functional MTHFR gene polymorphisms and schizophrenia or bipolar disorder. However, when combined with previous studies in meta-analyses there is still evidence for association between the MTHFR C677T polymorphism...... disorder. In a replication attempt the MTHFR C677T and A1298C SNPs were analyzed in three Scandinavian schizophrenia case-control samples. In addition, Norwegian patients with bipolar disorder were investigated. There were no statistically significant allele or genotype case-control differences...

  9. Go Green: The Antiinflammatory Effects of Biliverdin Reductase

    Barbara eWegiel


    Full Text Available Biliverdin (BV has emerged as a cytoprotective and important anti-inflammatory molecule. Conversion of BV to bilirubin (BR is catalyzed by biliverdin reductase (BVR and is required for the downstream signaling and nuclear localization of BVR. Recent data by others and us make clear that BVR is a critical regulator of innate immune responses resulting from acute insult and injury and moreover, that a lack of BVR results in an enhanced pro-inflammatory phenotype. In macrophages, BVR is regulated by its substrate BV which leads to activation of the PI3K-Akt-IL10 axis and inhibition of TLR4 expression via direct binding of BVR to the TLR4 promoter. In this review, we will summarize recent findings on the role of BVR and the bile pigments in inflammation in context with its activity as an enzyme, receptor and transcriptional regulator.

  10. Pulse radiolysis studies on superoxide reductase from Treponema pallidum

    Nivière, V; Fontecave, M; Houée-Levin, C


    Superoxide reductases (SORs) are small metalloenzymes, which catalyze reduction of O2*- to H2O2. The reaction of the enzyme from Treponema pallidum with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bi-molecular reaction of the ferrous center with O2, with a rate constant of 6 x 10 (8) M(-1) s(-1). A first intermediate is formed which is converted to a second one with a slower rate constant of 4800 s(-1). This latter value is 10 times higher than the corresponding one previously reported in the case of SOR from Desulfoarculus baarsii. The reconstituted spectra for the two intermediates are consistent with formation of transient iron-peroxide species.

  11. Fatty acyl-CoA reductases of birds

    Hellenbrand Janine


    Full Text Available Abstract Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba, domestic chicken (Gallus gallus domesticus and domestic goose (Anser anser domesticus. Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis.

  12. Comparison of hydrazone heterobifunctional cross-linking agents for reversible conjugation of thiol-containing chemistry.

    Christie, R James; Anderson, Diana J; Grainger, David W


    Reversible covalent conjugation chemistries that allow site- and condition-specific coupling and uncoupling reactions are attractive components in nanotechnologies, bioconjugation methods, imaging, and drug delivery systems. Here, we compare three heterobifunctional cross-linkers, containing both thiol- and amine-reactive chemistries, to form pH-labile hydrazones with hydrazide derivatives of the known and often published water-soluble polymer, poly[N-(2-hydroxypropyl methacrylamide)] (pHPMA), while subsequently coupling thiol-containing molecules to the cross-linker via maleimide addition. Two novel cross-linkers were prepared from the popular heterobifunctional cross-linking agent, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), modified to contain either terminal aldehyde groups (i.e., 1-(N-3-propanal)-4-(N-maleimidomethyl) cyclohexane carboxamide, PMCA) or methylketone groups (i.e., 1-(N-3-butanone)-4-(N-maleimidomethyl) cyclohexane carboxamide, BMCA). A third cross-linking agent was the commercially available N-4-acetylphenyl maleimide (APM). PMCA and BMCA exhibited excellent reactivity toward hydrazide-derivatized pHPMA with essentially complete hydrazone conjugation to polymer reactive sites, while APM coupled only ∼60% of available reactive sites on the polymer despite a 3-fold molar excess relative to polymer hydrazide groups. All polymer hydrazone conjugates bearing these bifunctional agents were then further reacted with thiol-modified tetramethylrhodamine dye, confirming cross-linker maleimide reactivity after initial hydrazone polymer conjugation. Incubation of dye-labeled polymer conjugates in phosphate buffered saline at 37 °C showed that hydrazone coupling resulting from APM exhibited the greatest difference in stability between pH 7.4 and 5.0, with hydrolysis and dye release increased at pH 5.0 over a 24 h incubation period. Polymer conjugates bearing hydrazones formed from cross-linker BMCA exhibited intermediate stability

  13. Steroid 5β-Reductase from Leaves of Vitis vinifera: Molecular Cloning, Expression, and Modeling.

    Ernst, Mona; Munkert, Jennifer; Campa, Manuela; Malnoy, Mickael; Martens, Stefan; Müller-Uri, Frieder


    A steroid 5β-reductase gene corresponding to the hypothetical protein LOC100247199 from leaves of Vitis vinifera (var. 'Chardonnay') was cloned and overexpressed in Escherichia coli. The recombinant protein showed 5β-reductase activity when progesterone was used as a substrate. The reaction was stereoselective, producing only 5β-products such as 5β-pregnane-3,20-dione. Other small substrates (terpenoids and enones) were also accepted as substrates, indicating the highly promiscuous character of the enzyme class. Our results show that the steroid 5β-reductase gene, encoding an orthologous enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in leaves of the cardenolide-free plant V. vinifera. We emphasize the fact that, on some occasions, different reductases (e.g., progesterone 5β-reductase and monoterpenoid reductase) can also use molecules that are similar to the final products as a substrate. Therefore, in planta, the different reductases may contribute to the immense number of diverse small natural products finally leading to the flavor of wine.

  14. Glyphosate inhibition of ferric reductase activity in iron deficient sunflower roots.

    Ozturk, Levent; Yazici, Atilla; Eker, Selim; Gokmen, Ozgur; Römheld, Volker; Cakmak, Ismail


    Iron (Fe) deficiency is increasingly being observed in cropping systems with frequent glyphosate applications. A likely reason for this is that glyphosate interferes with root uptake of Fe by inhibiting ferric reductase in roots required for Fe acquisition by dicot and nongrass species. This study investigated the role of drift rates of glyphosate (0.32, 0.95 or 1.89 mm glyphosate corresponding to 1, 3 and 6% of the recommended herbicidal dose, respectively) on ferric reductase activity of sunflower (Helianthus annuus) roots grown under Fe deficiency conditions. Application of 1.89 mm glyphosate resulted in almost 50% inhibition of ferric reductase within 6 h and complete inhibition 24 h after the treatment. Even at lower rates of glyphosate (e.g. 0.32 mm and 0.95 mm), ferric reductase was inhibited. Soluble sugar concentration and the NAD(P)H oxidizing capacity of apical roots were not decreased by the glyphosate applications. To our knowledge, this is the first study reporting the effects of glyphosate on ferric reductase activity. The nature of the inhibitory effect of glyphosate on ferric reductase could not be identified. Impaired ferric reductase could be a major reason for the increasingly observed Fe deficiency in cropping systems associated with widespread glyphosate usage.

  15. Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

    Benedito Barraviera


    Full Text Available The authors have standardized methods for evaluation of the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The general principle of the first method was based on methemoglobin formation by sodium nitrite followed by stimulation of the glucose-6-phosphate dehydrogenase with methylene blue. Forty six adults (23 males and 23 females were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. The results showed that methemoglobin reduction by methylene blue was 154.40 and 139.90 mg/min (p<0.05 for males and females, respectively, in whole blood, and 221.10 and 207.85 mg/min (n.s., respectively, in washed red cells. These data showed that using washed red cells and 0.7g% sodium nitrite concentration produced no differences between sexes and also shortened reading time for the residual amount of methemoglobin to 90 minutes. Glutathione reductase activity was evaluated on the basis of the fact that cystamine (a thiol agent binds to the SH groups of hemoglobin, forming complexes. These complexes are reversed by the action of glutathione reductase, with methemoglobin reduction occurring simultaneously with this reaction. Thirty two adults (16 males and 16 females were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. Methemoglobin reduction by cystamine was 81.27 and 91.13 mg/min (p<0.01 for males and females, respectively. These data showed that using washed red cells and 0.1 M cystamine concentration permits a reading of the residual amount of methemoglobin at 180 minutes of incubation. Glutathione reductase activity was evaluated by methemoglobin reduction by cystamine in 14 females before and after treatment with 10 mg riboflavin per day for 8 days. The results were 73.69 and 94.26 jug/min (p<0.01 before and after treatment, showing that riboflavin treatment increase glutathione reductase activity even in normal individuals. Three Black G6PD-deficient individuals (2 males and 1

  16. The Rearrangement of 2-Benzothiazolylthioacetyl Hydrazide in Synthesis of s-Triazolo[3,4-b]benzothiazole-3-thiol


    The rearrangement reaction of 2-benzothiazolylthioacetyl hydrazide 1 with potassium hydroxide and carbon disulfide in ethanol to produce s-triazolo[3,4-b]benzothiazole-3-thiol 3 was described.3 also can be obtained from 2-benzothiazolylhydazine 2 and the two methods for getting 3 were compared.Mannich reaction of compounds 3 was reported too.

  17. Hydrotalcite as an Efficient and Reusable Catalyst for Acylation of Phenols, Amines and Thiols Under Solvent-free Conditions

    A. R. Massah


    Full Text Available A wide variety of alcohols, phenols, amines and thiols were efficiently acylated with carboxylic acid anhydrides and chlorides in the presence of Hydrotalcite under solvent-free conditions at room temperature in good to high yields. Eco-friendly conditions and reusability of the catalyst are the most important advantages of this protocol.

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

    Weber, Arthur L.


    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.

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

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


    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.

  20. Self-assembled monolayers of terminal acetylenes as replacements for thiols in bottom-up tunneling junctions

    Fracasso, Davide; Kumar, Sumit; Rudolf, Petra; Chiechi, Ryan C.


    Why use thiols in Molecular Electronics? They stink, oxidize readily, poison catalysts, and often require nontrivial protection/deprotection chemistry. In this communication we demonstrate the fabrication of tunneling junctions formed by contact of self-assembled monolayers (SAMs) of terminal alkyne

  1. Thiol-disulfide Oxidoreductases TRX1 and TMX3 Decrease Neuronal Atrophy in a Lentiviral Mouse Model of Huntington's Disease.

    Fox, Jonathan; Lu, Zhen; Barrows, Lorraine


    Huntington's disease (HD) is caused by a trinucleotide CAG repeat in the huntingtin gene (HTT) that results in expression of a polyglutamine-expanded mutant huntingtin protein (mHTT). N-terminal fragments of mHTT accumulate in brain neurons and glia as soluble monomeric and oligomeric species as well as insoluble protein aggregates and drive the disease process. Decreasing mHTT levels in brain provides protection and reversal of disease signs in HD mice making mHTT a prime target for disease modification. There is evidence for aberrant thiol oxidation within mHTT and other proteins in HD models. Based on this, we hypothesized that a specific thiol-disulfide oxidoreductase exists that decreases mHTT levels in cells and provides protection in HD mice. We undertook an in-vitro genetic screen of key thiol-disulfide oxidoreductases then completed secondary screens to identify those with mHTT decreasing properties. Our in-vitro experiments identified thioredoxin 1 and thioredoxin-related transmembrane protein 3 as proteins that decrease soluble mHTT levels in cultured cells. Using a lentiviral mouse model of HD we tested the effect of these proteins in striatum. Both proteins decreased mHTT-induced striatal neuronal atrophy. Findings provide evidence for a role of dysregulated protein-thiol homeostasis in the pathogenesis of HD.

  2. Thiol accumulation and cysteine desulfhydrase activity in H2S-fumigated leaves and leaf homogenates of cucurbit plants

    Schütz, Bärbel; De Kok, Luit J.; Rennenberg, Heinz


    Fumigation of both, cucurbit plants and cucurbit leaf homogenates with hydrogen sulfide (H2S) resulted in an increase in soluble thiol, mainly glutathione and cysteine. In leaf homogenates this increase was counteracted or prevented by the addition at 1 mM of inhibitors of pyridoxalphosphate depende

  3. New non-hydroxamic ADAMTS-5 inhibitors based on the 1,2,4-triazole-3-thiol scaffold.

    Maingot, Lucie; Leroux, Florence; Landry, Valérie; Dumont, Julie; Nagase, Hideaki; Villoutreix, Bruno; Sperandio, Olivier; Deprez-Poulain, Rebecca; Deprez, Benoit


    In this Letter we describe the design, synthesis, screening, and optimization of a new family of ADAMTS-5 inhibitors. These inhibitors display an original 1,2,4-triazole-3-thiol scaffold as a putative zinc binding-group. In vitro results are rationalized by in silico docking of the compounds in ADAMTS-5's crystal structure.

  4. Adsorption kinetics and mechanical properties of thiol-modified DNA-oligos on gold investigated by microcantilever sensors

    Marie, Rodolphe Charly Willy; Jensenius, Henriette; Thaysen, Jacob;


    -mer thiol-modified DNA-oligo layer. The self-assembly induces a surface-stress change, which closely follows Langmuir adsorption model. The adsorption results in compressive surface-stress formation, which might be due to intermolecular repulsive forces in the oligo layer. The rate constant...

  5. Effects of simultaneous expression of heterologous genes involved in phytochelatin biosynthesis on thiol content and cadmium accumulation in tobacco plants.

    Wawrzynski, Adam; Kopera, Edyta; Wawrzynska, Anna; Kaminska, Jolanta; Bal, Wojciech; Sirko, Agnieszka


    Transgenic tobacco (Nicotiana tabacum cv. LA Burley 21) lines expressing three genes encoding enzymes thought to be critical for the efficient production of phytochelatins, (i) serine acetyltransferase (EC involved in the production of O-acetylserine, the cysteine precursor, (ii) gamma-glutamylcysteine synthetase (EC involved in the production of gamma-glutamylcysteine, the precursor of glutathione, and (iii) phytochelatin synthase (EC, were obtained and analysed for non-protein thiol content and cadmium accumulation. After a 3 week exposure to 15 microM CdCl2, plants expressing transgenes (either separately or in combination) had increased cadmium concentration in roots but not in shoots compared with the wild type. Nearly all transgenic lines analysed had more non-protein thiols than the wild type. The greatest effects (about 8-fold elevation of thiols) were found in one of the lines simultaneously expressing the three transgenes. Despite the fact that a multi-transgene strategy described in this work resulted in a strong increase in the levels of several classes of non-protein thiols in transgenic plants, other factors appeared to restrict cadmium accumulation in shoots.

  6. In situ generated hypoiodous acid in an efficient and heterogeneous catalytic system for the homo-oxidative coupling of thiols

    Ghorbani-Choghamarani Arash


    Full Text Available Supported hydrogen peroxide on polyvinylpolypyrrolidone (PVPH2O2, silica sulfuric acid (SiO2-OSO3H and catalytic amounts of potassium iodide (KI has been developed as a heterogeneous medium for the rapid oxidative coupling of thiols into symmetrical homodisulfides. This oxidizing system proceeds under extremely mild conditions and gives no other oxidized side products.

  7. Glutathione S-Transferase activity and total thiol status in chronic alcohol abusers before and 30 days after alcohol abstinence

    Manjunatha S Muttigi


    Full Text Available Background: Glutathione S Transferase (GST has been involved in detoxification process in the liver and its activity has been shown to be increased in alcohol abusers. In the current work we measured the GST activity, total thiol status, AST, ALT, and direct bilirubin in chronic alcohol abusers before and 30 days after alcohol abstinence and lifestyle modification. Methods: Serum and urine GST activity and total thiol status were determined using spectrophotometric methods and serum transaminases were determined using clinical chemistry analyzer. Results: We found,significant increase in serum and urine GST (p<0.001, AST (p<0.001, ALT (p<0.001, and decrease in total thiol status (p<0.001 in chronic alcohol abusers. GST activity significantly decreased (p<0.001 and total thiol status were improved significantly (p<0.001 30 days after alcohol abstinence and lifestyle modification. Conclusion: This study provides preliminary data to suggest the role of GST as prognostic indicator of alcohol abstinence with possible trend towards an improvement in liver function.

  8. Biomimetic surface modification of polypropylene by surface chain transfer reaction based on mussel-inspired adhesion technology and thiol chemistry

    Niu, Zhijun; Zhao, Yang; Sun, Wei; Shi, Suqing; Gong, Yongkuan


    Biomimetic surface modification of polypropylene (PP) is conducted by surface chain transfer reaction based on the mussel-inspired versatile adhesion technology and thiol chemistry, using 2-methacryloyloxyethylphosphorylcholine (MPC) as a hydrophilic monomer mimicking the cell outer membrane structure and 2,2-azobisisobutyronitrile (AIBN) as initiator in ethanol. A layer of polydopamine (PDA) is firstly deposited onto PP surface, which not only offers good interfacial adhesion with PP, but also supplies secondary reaction sites (-NH2) to covalently anchor thiol groups onto PP surface. Then the radical chain transfer to surface-bonded thiol groups and surface re-initiated polymerization of MPC lead to the formation of a thin layer of polymer brush (PMPC) with cell outer membrane mimetic structure on PP surface. X-ray photoelectron spectrophotometer (XPS), atomic force microscopy (AFM) and water contact angle measurements are used to characterize the PP surfaces before and after modification. The protein adsorption and platelet adhesion experiments are also employed to evaluate the interactions of PP surface with biomolecules. The results show that PMPC is successfully grafted onto PP surface. In comparison with bare PP, the resultant PP-PMPC surface exhibits greatly improved protein and platelet resistance performance, which is the contribution of both increased surface hydrophilicity and zwitterionic structure. More importantly, the residue thiol groups on PP-PMPC surface create a new pathway to further functionalize such zwitterion modified PP surface.

  9. Interfacial thiol-isocyanate reactions for functional nanocarriers: a facile route towards tunable morphologies and hydrophilic payload encapsulation.

    Kuypers, Sören; Pramanik, Sumit Kumar; D'Olieslaeger, Lien; Reekmans, Gunter; Peters, Martijn; D'Haen, Jan; Vanderzande, Dirk; Junkers, Thomas; Adriaensens, Peter; Ethirajan, Anitha


    Functional nanocarriers were synthesized using an in situ inverse miniemulsion polymerization employing thiol-isocyanate reactions at the droplet interface to encapsulate hydrophilic payloads. The morphology of the nanocarriers is conveniently tunable by varying the reaction conditions and the dispersions are easily transferable to the aqueous phase.

  10. Quinone-induced inhibition of urease: elucidation of its mechanisms by probing thiol groups of the enzyme.

    Zaborska, Wiesława; Krajewska, Barbara; Kot, Mirosława; Karcz, Waldemar


    In this work we studied the reaction of four quinones, 1,4-benzoquinone (1,4-BQ), 2,5-dimethyl-1,4-benzoquinone (2,5-DM-1,4-BQ), tetrachloro-1,4-benzoquinone (TC-1,4-BQ) and 1,4-naphthoquinone (1,4-NQ) with jack bean urease in phosphate buffer, pH 7.8. The enzyme was allowed to react with different concentrations of the quinones during different incubation times in aerobic conditions. Upon incubation the samples had their residual activities assayed and their thiol content titrated. The titration carried out with use of 5,5'-di-thiobis(2-nitrobenzoic) acid was done to examine the involvement of urease thiol groups in the quinone-induced inhibition. The quinones under investigation showed two distinct patterns of behaviour, one by 1,4-BQ, 2,5-DM-1,4-BQ and TC-1,4-BQ, and the other by 1,4-NQ. The former consisted of a concentration-dependent inactivation of urease where the enzyme-inhibitor equilibrium was achieved in no longer than 10min, and of the residual activity of the enzyme being linearly correlated with the number of modified thiols in urease. We concluded that arylation of the thiols in urease by these quinones resulting in conformational changes in the enzyme molecule is responsible for the inhibition. The other pattern of behaviour observed for 1,4-NQ consisted of time- and concentration-dependent inactivation of urease with a nonlinear residual activity-modified thiols dependence. This suggests that in 1,4-NQ inhibition, in addition to the arylation of thiols, operative are other reactions, most likely oxidations of thiols provoked by 1,4-NQ-catalyzed redox cycling. In terms of the inhibitory strength, the quinones studied formed a series: 1,4-NQ approximately 2,5-DM-1,4-BQ<1,4-BQ

  11. Kinetic and Thermodynamic Aspects of Cellular Thiol-Disulfide Redox Regulation

    Jensen, Kristine Steen; Hansen, Rosa Erritzøe; Winther, Jakob R


    Regulation of intracellular thiol-disulfide redox status is an essential part of cellular homeostasis. This involves the regulation of both oxidative and reductive pathways, production of oxidant scavengers and, importantly, the ability of cells to respond to changes in the redox environment....... In the cytosol regulatory disulfide bonds are typically formed in spite of the prevailing reducing conditions and may thereby function as redox switches. Such disulfide bonds are protected from enzymatic reduction by kinetic barriers and are thus allowed to exist long enough to elicit the signal. Factors......-disulfide exchange reaction is thermodynamically favourable it will only take place if the activation energy to form the transition state complex can be overcome. This is accomplished by enzymes, such as the oxidoreductases, that direct reactions in thermodynamically favourable directions by decreasing...

  12. Surface analysis of gold nanoparticles functionalized with thiol-modified glucose SAMs for biosensor applications.

    Valentina eSpampinato


    Full Text Available In this work, Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS, Principal Component Analysis (PCA and X-ray Photoelectron Spectroscopy (XPS have been used to characterize the surface chemistry of gold substrates before and after functionalization with thiol-modified glucose self-assembled monolayers and subsequent biochemical specific recognition of maltose binding protein (MBP.The results indicate that the surface functionalization is achieved both on flat and nanoparticles gold substrates thus showing the potential of the developed system as biodetection platform. Moreover, the method presented here has been found to be a sound and valid approach to characterize the surface chemistry of nanoparticles functionalized with large molecules.Both techniques were proved to be very useful tools for monitoring all the functionalization steps, including the investigation of the biological behaviour of the glucose-modified particles in presence of the maltose binding protein.

  13. Inhibition of enzymatic browning and protection of sulfhydryl enzymes by thiol compounds.

    Negishi, O; Ozawa, T


    In a reaction between (-)-epicatechin (EC) and 2-mercaptoethanol (2ME), catalyzed by partially purified polyphenol oxidase (PPO) extracted from the style of Rhododendron mucronatum, 2'-(2-hydroxyethylthio)-(-)-epicatechin (2'-HETEC), 5'-(2-hydroxyethylthio)-(-)-epicatechin (5'-HETEC), and 2',5'-bis(2-hydroxyethylthio)-(-)-epicatechin (2',5'-HETEC) were formed. The rate of formation of 2',5'-HETEC from 5'-HETEC was faster than that from 2'-HETEC. In the absence of 2ME, the concentration of EC decreased rapidly and the reaction mixture turned brown; 2'-, 5'-, and 2',5'-HETEC, especially 2'-substituted HETECs. reacted more slowly. These data indicate that 2ME acts both as an inhibitor of the polymerization of O-quinone, presumably by binding to it and as a reductant involved in the conversion of O-quinone to O-dihydroxyphenol, Inhibition of enzymatic browning by other thiol compounds such as cysteine and dithiothreitol was also investigated.

  14. Polysiloxane-based luminescent elastomers prepared by thiol-ene "click" chemistry.

    Zuo, Yujing; Lu, Haifeng; Xue, Lei; Wang, Xianming; Wu, Lianfeng; Feng, Shengyu


    Side-chain vinyl poly(dimethylsiloxane) has been modified with mercaptopropionic acid, methyl 3-mercaptopropionate, and mercaptosuccinic acid. Coordinative bonding of Eu(III) to the functionalized polysiloxanes was then carried out and crosslinked silicone elastomers were prepared by thiol-ene curing reactions of these composites. All these europium complexes could be cast to form transparent, uniform, thin elastomers with good flexibility and thermal stability. The networks were characterized by FTIR, NMR, UV/Vis, and luminescence spectroscopy as well as by scanning electron microscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy. The europium elastomer luminophores exhibited intense red light at 617 nm under UV excitation at room temperature due to the (5)D0 →(7)F2 transition in Eu(III) ions. The newly synthesized luminescent materials offer many advantages, including the desired mechanical flexibility. They cannot be dissolved or fused, and so they have potential for use in optical and electronic applications.

  15. A Fluorescence Turn-On Probe for Thiols with a Tunable Dynamic Range.

    Li, Qian; Guo, Rui; Lin, Weiying


    We designed and synthesized a novel probe Cou-Rho-SA-Cu (II) for detection of thiols based on a coumarin-rhodamine FRET system. The compound Cou-Rho-SA exhibited strong blue fluorescence as the rhodamine moiety of the compound Cou-Rho-SA was in a ring-closing form and no FRET process occurred. The probe Cou-Rho-SA-Cu (II) was prepared by addition of Cu(2+) to the solution of Cou-Rho-SA. In the absence of GSH, the complex Cou-Rho-SA-Cu (II) showed almost no fluorescence owing to the FRET process and the paramagnetic nature of Cu(2+) . However, upon addition of GSH, the rhodamine moiety of the probe Cou-Rho-SA-Cu (II) recovered to its ring-closing form, and the FRET process was prohibited to show the blue fluorescence again. Moreover, via tuning the Cu(2+) concentrations, the probe could detect the different levels of GSH.

  16. Modeling of the Bioactivation of an Organic Nitrate by a Thiol to Form a Thionitrate Intermediate.

    Sano, Tsukasa; Shimada, Keiichi; Aoki, Yohei; Kawashima, Takayuki; Sase, Shohei; Goto, Kei


    Thionitrates (R-SNO₂) have been proposed as key intermediates in the biotransformation of organic nitrates that have been used for the clinical treatment of angina pectoris for over 100 years. It has been proposed and widely accepted that a thiol would react with an organic nitrate to afford a thionitrate intermediate. However, there has been no example of an experimental demonstration of this elementary chemical process in organic systems. Herein, we report that aryl- and primary-alkyl-substituted thionitrates were successfully synthesized by the reaction of the corresponding lithium thiolates with organic nitrates by taking advantage of cavity-shaped substituents. The structure of a primary-alkyl-substituted thionitrate was unambiguously established by X-ray crystallographic analysis.

  17. Photolithographic fabrication of solid–liquid core waveguides by thiol-ene chemistry

    Sagar, Kaushal Shashikant; Gopalakrishnan, Nimi; Christiansen, Mads Brøkner;


    are comparable with literature values for state-of-the-art liquid-core waveguide devices. The demonstrated waveguide function added to the nanoporous polymer with a very high internal surface area makes the system interesting for many applications in different areas, such as diagnostics and bio-chemical sensing.......In this work we demonstrate an efficient and cleanroom compatible method for the fabrication of solid–liquid core waveguides based on nanoporous polymers. We have used thiol-ene photo-grafting to tune and pattern the hydrophilicity of an originally hydrophobic nanoporous 1, 2-polybutadiene....... The generated refractive index contrast between the patterned water-filled volume and the surrounding empty hydrophobic porous polymer allows for light confinement within the water-filled volume—the solid–liquid core. The presented fabrication process is simple and fast. It allows a high degree of flexibility...

  18. Thiol-yne Click Adamantane Monolithic Stationary Phase for Capillary Electrochromatography

    Dao, Thi Thu Hien; Guerrouache, Mohamed; Carbonnier, Benjamin


    A porous crosslinked organic polymer based on N-acryloxysuccinimide (NAS) and ethylene dimethacrylate (EDMA) was prepared inside 75 μm i.d. fused silica capillary as functionalizable monolithic stationary phase for electrochromatographic applications. Succinimide groups on the monolith surface provide reactive sites able to re- act readily through standard electrophile-nucleophile chemistry. Propargylamine was used to prepare alkyne func- tionalized poly(NAS-co-EDMA). Onto this thiol-reactive polymer surface was grafted adamantane units via a photochemically-driven addition reaction. Chemical characterization was performed in situ after each synthetic step by means of Raman spectroscopy and grafting kinetics was investigated to ensure quantitative grafting of 1-adamantanethiol. The as-designed monolithic stationary phase exhibited typical reversed-phase separation mechanism as evidenced by the linear increase of the logarithm of retention factor of neutral aromatic solutes with the increase of the aqueous buffer content in the mobile phase.

  19. Deposition of gold nanoparticles onto thiol-functionalized multiwalled carbon nanotubes.

    Zanella, Rodolfo; Basiuk, Elena V; Santiago, Patricia; Basiuk, Vladimir A; Mireles, Edgar; Puente-Lee, Iván; Saniger, José M


    Gold nanoparticles were deposited on the surface of multiwalled carbon nanotubes (MWNTs) functionalized with aliphatic bifunctional thiols (1,4-butanedithiol, 1,6-hexanedithiol, 1,8-octanedithiol, and 2-aminoethanethiol) through a direct solvent-free procedure. Small gold particles, with a narrow particle size distribution around 1.7 nm, were obtained on 1,6-hexanedithiol-functionalized MWNTs. For MWNTs functionalized with the aminothiol, the average Au particle size was larger, 5.5 nm, apparently due to a coalescence phenomenon. Gatan image filter (GIF) observations show that sulfur is at the nanotube surface with a non-homogeneous distribution. A higher sulfur concentration was observed around the gold nanoparticles' location.

  20. Conserved water-mediated H-bonding dynamics of catalytic Asn 175 in plant thiol protease

    Tapas K Nandi; Hridoy R Bairagya; Bishnu P Mukhopadhyay; K Sekar; Dipankar Sukul; Asim K Bera


    The role of invariant water molecules in the activity of plant cysteine protease is ubiquitous in nature. On analysing the 11 different Protein DataBank (PDB) structures of plant thiol proteases, the two invariant water molecules W1 and W2 (W220 and W222 in the template 1PPN structure) were observed to form H-bonds with the Ob atom of Asn 175. Extensive energy minimization and molecular dynamics simulation studies up to 2 ns on all the PDB and solvated structures clearly revealed the involvement of the H-bonding association of the two water molecules in fixing the orientation of the asparagine residue of the catalytic triad. From this study, it is suggested that H-bonding of the water molecule at the W1 invariant site better stabilizes the Asn residue at the active site of the catalytic triad.

  1. Atomistic simulations of thiol-terminated modifiers for hybrid photovoltaic interfaces

    Malloci, G. [Istituto Officina dei Materiali (CNR-IOM), Unità di Cagliari, Cittadella Universitaria, I-09042 Monserrato (Italy); Petrozza, A. [Center for Nano Science and Technology @Polimi, Istituto Italiano di Tecnologia, Via Pascoli 70/3, I-20133 Milano (Italy); Mattoni, A., E-mail: [Istituto Officina dei Materiali (CNR-IOM), Unità di Cagliari, Cittadella Universitaria, I-09042 Monserrato (Italy)


    Small aromatic molecules such as benzene or pyridine derivatives are often used as interface modifiers (IMs) at polymer/metal oxide hybrid interfaces. We performed a theoretical investigation on prototypical thiol-terminated IMs aimed at improving the photovoltaic performances of poly(3-hexylthiophene)/TiO{sub 2} devices. By means of first-principles calculations in the framework of the density functional theory we investigate 3-furanthiol (3FT), 4-mercaptobenzoicacid (4MB), and 6-isoquinolinethiol (6QT) molecules. We discuss the role of these molecules as modifiers alternative to 4-mercaptopyridine (4MP) which has recently shown to induce a large improvement in the overall power conversion efficiency of mesoporous films of TiO{sub 2} infiltrated by poly(3-hexylthiophene). The IMs investigated are expected to keep the beneficial features of 4MP giving at the same time the possibility to further tune the interlayer properties (e.g., its thickness, stability, and density). Dense interlayers of 6QT turn out to be slightly unstable since the titania substrate induces a compressive strain in the molecular film. On the contrary, we predict very stable films for both 3FT and 4MB molecules, which makes them interesting candidates for future experimental investigations. - Highlights: • We performed a theoretical investigation on thiol-terminated interface modifiers. • We investigate 3-furanthiol (3FT), 4-mercaptobenzoicacid (4MB), and 6-isoquinolinethiol molecules. • We discuss the role of these molecules as modifiers alternative to 4-mercaptopyridine. • Dense interlayers of 6-isoquinolinethiol turn out to be slightly unstable. • We predict very stable self-assembled thin-films for both 3FT and 4MB molecules.

  2. Modification of nanoelectrode ensembles by thiols and disulfides to prevent non specific adsorption of proteins

    Silvestrini, M. [Department of Molecular Sciences and Nanosystems, University Ca' Foscari of Venice, Santa Marta 2137, 30123 Venice (Italy); Schiavuta, P.; Scopece, P. [Associazione CIVEN, via delle Industrie 5, 30175 Marghera - Venice (Italy); Pecchielan, G.; Moretto, L.M. [Department of Molecular Sciences and Nanosystems, University Ca' Foscari of Venice, Santa Marta 2137, 30123 Venice (Italy); Ugo, P., E-mail: [Department of Molecular Sciences and Nanosystems, University Ca' Foscari of Venice, Santa Marta 2137, 30123 Venice (Italy)


    Highlights: > Complex nanostructures are built on the gold surface of ensembles of nanoelectrodes. > Gold surface of nanoelectrodes was functionalized with SAM of organic sulphurs. > The polycarbonate surrounding nanoelectrodes was functionalized with proteins. > SAMs protect the nanoelectrodes from undesired proteins adsorption. - Abstract: The possibility to functionalize selectively with thiols or disulfides the surface of the gold nanoelectrodes of polycarbonate templated nanoelectrode ensembles (NEEs) is studied. It is shown that the Au nanoelectrodes can be coated by a self assembled monolayer (SAM) of thioctic acid (TA) or 2-mercaptoethanesulfonic (MES) acid. The study of the electrochemical behavior of SAM-modified NEEs by cyclic voltammetry (CV) at different solution pH, using ferrocenecarboxylate as an anionic redox probe (FcCOO{sup -}) and (ferrocenylmethyl)trimethylammonium (FA{sup +}) as a cationic redox probe, demonstrate that the SAM-modified nanoelectrodes are permselective, in that only cationic or neutral probes can access the SAM-coated nanoelectrode surface. CV, AFM and FTIR-ATR data indicate that proteins such as casein or bovine serum albumin, which are polyanionic at pH 7, adsorb on the surface of NEEs untreated with thiols, tending to block the electron transfer of the ferrocenyl redox probes. On the contrary, the pre-treatment of the NEE with an anionic SAM protects the nanoelectrodes from protein fouling, allowing the detection of well shaped voltammetric patterns for the redox probe. Experimental results indicate that, in the case of MES treated NEEs, the protein is bound only onto the polycarbonate surface which surrounds the nanoelectrodes, while the tips of the gold nanoelectrodes remain protein free.

  3. Thiol-based redox regulation in sexual plant reproduction: new insights and perspectives

    Traverso, Jose A.; Pulido, Amada; Rodríguez-García, María I.; Alché, Juan D.


    The success of sexual reproduction in plants involves (i) the proper formation of the plant gametophytes (pollen and embryo sac) containing the gametes, (ii) the accomplishment of specific interactions between pollen grains and the stigma, which subsequently lead to (iii) the fusion of the gametes and eventually to (iv) the seed setting. Owing to the lack of mobility, plants have developed specific regulatory mechanisms to control all developmental events underlying the sexual plant reproduction according to environmental challenges. Over the last decade, redox regulation and signaling have come into sight as crucial mechanisms able to manage critical stages during sexual plant reproduction. This regulation involves a complex redox network which includes reactive oxygen species (ROS), reactive nitrogen species (RNS), glutathione and other classic buffer molecules or antioxidant proteins, and some thiol/disulphide-containing proteins belonging to the thioredoxin superfamily, like glutaredoxins (GRXs) or thioredoxins (TRXs). These proteins participate as critical elements not only in the switch between the mitotic to the meiotic cycle but also at further developmental stages of microsporogenesis. They are also implicated in the regulation of pollen rejection as the result of self-incompatibility. In addition, they display precise space-temporal patterns of expression and are present in specific localizations like the stigmatic papillae or the mature pollen, although their functions and subcellular localizations are not clear yet. In this review we summarize insights and perspectives about the presence of thiol/disulphide-containing proteins in plant reproduction, taking into account the general context of the cell redox network. PMID:24294217

  4. Biomimetic surface modification of polypropylene by surface chain transfer reaction based on mussel-inspired adhesion technology and thiol chemistry

    Niu, Zhijun; Zhao, Yang; Sun, Wei; Shi, Suqing, E-mail:; Gong, Yongkuan


    Highlights: • Biomimetic surface modification of PP was successfully conducted by integrating mussel-inspired technology, thiol chemistry and cell outer membranes-like structures. • The resultant biomimetic surface exhibits good interface and surface stability. • The obvious suppression of protein adsorption and platelet adhesion is also achieved. • The residue thoil groups on the surface could be further functionalized. - Abstract: Biomimetic surface modification of polypropylene (PP) is conducted by surface chain transfer reaction based on the mussel-inspired versatile adhesion technology and thiol chemistry, using 2-methacryloyloxyethylphosphorylcholine (MPC) as a hydrophilic monomer mimicking the cell outer membrane structure and 2,2-azobisisobutyronitrile (AIBN) as initiator in ethanol. A layer of polydopamine (PDA) is firstly deposited onto PP surface, which not only offers good interfacial adhesion with PP, but also supplies secondary reaction sites (-NH{sub 2}) to covalently anchor thiol groups onto PP surface. Then the radical chain transfer to surface-bonded thiol groups and surface re-initiated polymerization of MPC lead to the formation of a thin layer of polymer brush (PMPC) with cell outer membrane mimetic structure on PP surface. X-ray photoelectron spectrophotometer (XPS), atomic force microscopy (AFM) and water contact angle measurements are used to characterize the PP surfaces before and after modification. The protein adsorption and platelet adhesion experiments are also employed to evaluate the interactions of PP surface with biomolecules. The results show that PMPC is successfully grafted onto PP surface. In comparison with bare PP, the resultant PP-PMPC surface exhibits greatly improved protein and platelet resistance performance, which is the contribution of both increased surface hydrophilicity and zwitterionic structure. More importantly, the residue thiol groups on PP-PMPC surface create a new pathway to further functionalize such

  5. Toward the Facile and Ecofriendly Fabrication of Quantum Dot-Sensitized Solar Cells via Thiol Coadsorbent Assistance.

    Chang, Jia-Yaw; Li, Chen-Hei; Chiang, Ya-Han; Chen, Chia-Hung; Li, Pei-Ni


    This paper reports a facile and environmentally friendly approach to the preparation of highly efficient quantum dot-sensitized solar cells (QDSSCs) based on a combination of aqueous CuInS2 quantum dots (QDs) and thiol coadsorbents. The photovoltaic properties of the QDSSCs were found to be dependent on the type and concentration of the thiol coadsorbent. The incorporation of thiol coadsorbents results in improved JSC and VOC because (1) they provide disulfide reductants during the QD sensitization process and (2) the coadsorbent molecules are anchored on the TiO2 surface, thus affecting the movement of the conduction band of TiO2. To the best of the our knowledge, this is the first demonstrated use of various thiol coadsorbents as reducing agents in the fabrication of high-efficiency QDSSCs. CuInS2 QDSSCs fabricated with the assistance of thioglycolic acid coadsorbents exhibited efficiencies as high as 5.90%, which is 20 times higher than that of the control device without thiol coadsorbents (0.29%). In addition, the photovoltaic properties of a device fabricated using the colloidal CuInS2 QDs coated with different bifunctional linkers were investigated for comparison. The versatility of this facile fabrication process was demonstrated in the preparation of solar cells sensitized with aqueous AgInS2 or CdSeTe QDs. The AgInS2 QDSSC showed a conversion efficiency of 2.72%, which is the highest reported for Ag-based metal sulfides QDSSCs thus far.

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

    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.


    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

  7. Nitro-fatty acid reaction with glutathione and cysteine. Kinetic analysis of thiol alkylation by a Michael addition reaction.

    Baker, Laura M S; Baker, Paul R S; Golin-Bisello, Franca; Schopfer, Francisco J; Fink, Mitchell; Woodcock, Steven R; Branchaud, Bruce P; Radi, Rafael; Freeman, Bruce A


    Fatty acid nitration by nitric oxide-derived species yields electrophilic products that adduct protein thiols, inducing changes in protein function and distribution. Nitro-fatty acid adducts of protein and reduced glutathione (GSH) are detected in healthy human blood. Kinetic and mass spectrometric analyses reveal that nitroalkene derivatives of oleic acid (OA-NO2) and linoleic acid (LNO2) rapidly react with GSH and Cys via Michael addition reaction. Rates of OA-NO2 and LNO2 reaction with GSH, determined via stopped flow spectrophotometry, displayed second-order rate constants of 183 M(-1)S(-1) and 355 M(-1)S(-1), respectively, at pH 7.4 and 37 degrees C. These reaction rates are significantly greater than those for GSH reaction with hydrogen peroxide and non-nitrated electrophilic fatty acids including 8-iso-prostaglandin A2 and 15-deoxy-Delta(12,14)-prostaglandin J2. Increasing reaction pH from 7.4 to 8.9 enhanced apparent second-order rate constants for the thiol reaction with OA-NO2 and LNO2, showing dependence on the thiolate anion of GSH for reactivity. Rates of nitroalkene reaction with thiols decreased as the pKa of target thiols increased. Increasing concentrations of the detergent octyl-beta-d-glucopyranoside decreased rates of nitroalkene reaction with GSH, indicating that the organization of nitro-fatty acids into micellar or membrane structures can limit Michael reactivity with more polar nucleophilic targets. In aggregate, these results reveal that the reversible adduction of thiols by nitro-fatty acids is a mechanism for reversible post-translational regulation of protein function by nitro-fatty acids.

  8. One-step interfacial thiol-ene photopolymerization for metal nanoparticle-decorated microcapsules (MNP@MCs).

    Liu, Dandan; Jiang, Xuesong; Yin, Jie


    We herein reported a one-step strategy to prepare the noble metal nanoparticle-decorated microcapsules (MNP@MCs) through the interfacial thiol-ene photopolymerization. In the presence of amphiphlic polyhedral oligomeric silsesquioxane (POSS) containing thiol groups (PTPS) as a reactive surfactant and trimethylolpropane triacrylate (TMPTA) as a cross-linker, the oil phase of toluene dissolved with a photoinitiator was emulsified into a water phase containing a metal precursor to form an oil-in-water (O/W) emulsion. Upon irradiation of ultraviolet (UV) light, the thiol-ene photoploymerization and photoreduction at the interface of toluene/water lead to the formation of the cross-linked wall and metal nanoparticles, respectively. A series of gold, silver, and platinum nanoparticle-decorated microcapsules (AuNP@MC, AgNP@MC, and PtNP@MC) were prepared through this one-step interfacial thiol-ene photopolymerization and were characterized carefully by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM). The results revealed that the obtained MNP@MCs were 2.2-2.7 μm in diameter with a wall of 40-70 nm in thickness, which was covered with the metal nanoparticles. The size and amount of metal nanoparticles increased with the increasing concentration of the metal precursor in water. Furthermore, the catalyst performance of AuNP@MC was studied by reduction of aromatic nitro compounds and exhibited the enhanced catalytic activity and good stability in the reduction of hydrophobic nitrophenol. It is believed that this robust, convenient, simple strategy based on the one-step interfacial thiol-ene photopolymerization will provide an important alternative to fabricate the functional metal nanoparticle-modified microcapsules.

  9. Production of low-molecular weight thiols as a response to cadmium uptake by tumbleweed (Salsola kali).

    De la Rosa, Guadalupe; Martínez-Martínez, Alejandro; Pelayo, Helvia; Peralta-Videa, José R; Sanchez-Salcido, Blanca; Gardea-Torresdey, Jorge L


    Tumbleweed (Salsola kali) is a desert plant species that has shown to be a potential Cd hyperaccumulator. In this study, the production of low-molecular weight thiols (LMWT) as a response to cadmium stress was determined in hydroponically grown seedlings exposed to 0, 45, 89, and 178 microM Cd(2+). The treatment of 89 microM Cd(2+) was tested alone and supplemented with an equimolar concentration of ethylenediaminetetraacetic acid (EDTA) to determine the effect of this chelating agent on Cd uptake and thiols production. After 6 days of growth, the Cd concentration in plant tissues was determined by using inductively coupled plasma/optical emission spectroscopy (ICP/OES). Results indicated that Cd uptake by plants was concentration-dependent. Plants treated with 178 microM Cd(2+), had 10+/-0.62, 9.7+/-1.4, and 4.3+/-0.83 mmol Cd kg(-1) dry tissue in roots, stems, and leaves, respectively. The production of thiols was dependent on Cd concentration in tissues. According to the stoichiometry performed, plants treated with Cd concentrations up to 178 muM produced 0.131+/-0.02, and 0.087+/-0.012 mmol SH per mmol Cd present in roots and stems. In leaves, the production of thiols decreased at the highest Cd concentration tested. Thus, up to 89 microM Cd in the media, 0.528+/-0.004 mmol SH per mmol Cd in leaf tissues were produced. EDTA equimolar to Cd reduced both Cd uptake and thiols production. Catalase activity (CAT) (EC was significantly depressed at the lowest Cd concentration. None of the conditions tested affected biomass or plant elongation.

  10. Inhibition of glutathione synthesis eliminates the adaptive response of ascitic hepatoma 22 cells to nedaplatin that targets thioredoxin reductase

    Wang, Yijun [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China); Lu, Hongjuan [Productivity Center of Jiangsu Province, Nanjing 210042, Jiangsu (China); Wang, Dongxu; Li, Shengrong; Sun, Kang; Wan, Xiaochun [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China); Taylor, Ethan Will [Department of Nanoscience, Joint School of Nanoscience and Nanoengineering, University of North Carolina at Greensboro, Greensboro, NC 27402 (United States); Zhang, Jinsong, E-mail: [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China)


    Thioredoxin reductase (TrxR) is a target for cancer therapy and the anticancer mechanism of cisplatin involves TrxR inhibition. We hypothesize that the anticancer drug nedaplatin (NDP), an analogue of cisplatin and a second-generation platinum complex, also targets TrxR. Furthermore, we investigate whether the therapeutic efficacy of NDP can be enhanced by simultaneous modulation of 1) TrxR, via NDP, and 2) glutathione (GSH), via the GSH synthesis inhibitor buthionine sulfoximine (BSO). Mice bearing ascitic hepatoma 22 (H22) cells were treated with NDP alone or NDP plus BSO. TrxR activity of H22 cells was inhibited by NDP in a dose-dependent manner. A high correlation between the inhibition of TrxR activity at 6 h and the inhibition of ascitic fluid volume at 72 h was established (r = 0.978, p < 0.01). As an adaptive response, the viable ascitic cancer cells after NDP treatment displayed an enlarged cell phenotype, assembled with several-fold more antioxidant enzymes and GSH-predominant non-protein free thiols. This adaptive response was largely eliminated when BSO was co-administered with NDP, leading to the decimation of the H22 cell population without enhancing renal toxicity, since at this dose, NDP did not inhibit renal TrxR activity. In conclusion, the pharmacological effect of NDP involves TrxR inhibition, and the adaptive response of NDP-treated ascitic H22 cells can be efficiently counteracted by BSO. Simultaneous modulation of TrxR and GSH on ascitic H22 cells using NDP plus BSO greatly enhances therapeutic efficacy as compared with the single modulation of TrxR using NDP alone. -- Highlights: ► Nedaplatin at a pharmacological dose inhibits TrxR in cancer cells but not in kidney. ► The nedaplatin-treated cancer cells exhibit adaptive response. ► Buthionine sulfoximine inhibits glutathione in both cancer cells and kidney. ► Buthionine sulfoximine counteracts the adaptive response to the nedaplatin treatment. ► Buthionine sulfoximine does not

  11. Changes in protein and nonprotein thiol contents in bladder, kidney and liver of mice by the pesticide sodium-o-phenylphenol and their possible role in cellular toxicity.

    Narayan, S; Roy, D


    Acute treatment of mice with Na-o-phenylphenol or phenylbenzoquinone, an electrophilic metabolite of o-phenylphenol, resulted in differential depletion of contents of protein and nonprotein thiols in bladder, kidney and liver. Maximum decrease in the levels of protein and nonprotein reduced thiols was observed in bladder (by both agents) and was followed by kidney (by both agents) and liver (phenylbenzoquinone only). The reason for this differential changes in reduced thiol contents remains to be understood. The content of protein and nonprotein disulfides was higher in bladder of mice treated with Na-o-phenylphenol compared to that observed in untreated mice bladder. Phenyl 2,5'-p-benzoquinone mediated in vivo depletion of nonprotein and protein thiols suggests that Na-o-phenylphenol treatment may decrease in vivo thiols via the formation of phenylbenzoquinone. Increased disulfide formation is considered to represent an index of oxidative stress produced by chemical. Increases in the level of protein and nonprotein disulfides in bladder suggest as observed in this study that administration of Na-o-phenylphenol to mice produced oxidative stress in bladder. Products of redox cycling of xenobiotics are known to cause cellular toxicity via altering the homeostasis of thiol status. Therefore, it is concluded that decreases in protein thiol contents either via alkylation and/or oxidation of sulfhydryl groups of proteins and increases in disulfide contents presumably by products of redox cycling of Na-o-phenylphenol may play a role in Na-o-phenylphenol-induced cellular toxicity.

  12. Survival and psychomotor development with early betaine treatment in patients with severe methylenetetrahydrofolate reductase deficiency

    Diekman, E.F.; Koning, T.J. de; Verhoeven-Duif, N.M.; Rovers, M.M.; Hasselt, P.M. van


    IMPORTANCE The impact of betaine treatment on outcome in patients with severe methylenetetrahydrofolate reductase (MTHFR) deficiency is presently unclear. OBJECTIVE To investigate the effect of betaine treatment on development and survival in patients with severe MTHFR deficiency. DATA SOURCES MEDLI

  13. Survival and Psychomotor Development With Early Betaine Treatment in Patients With Severe Methylenetetrahydrofolate Reductase Deficiency

    Diekman, Eugene F.; de Koning, Tom J.; Verhoeven-Duif, Nanda M.; Rovers, Maroeska M.; van Hasselt, Peter M.


    IMPORTANCE The impact of betaine treatment on outcome in patients with severe methylenetetrahydrofolate reductase (MTHFR) deficiency is presently unclear. OBJECTIVE To investigate the effect of betaine treatment on development and survival in patients with severe MTHFR deficiency. DATA SOURCES MEDLI

  14. Positive pleiotropic effects of HMG-CoA reductase inhibitor on vitiligo


    Abstract Background HMG-CoA reductase inhibitors (statins) are commonly used in medicine to control blood lipid disorder. Large clinical trials have demonstrated that statins greatly reduces cardiovascular-related morbidity and mortality in patients with and without coronary artery disease. Also, the use of HMG-CoA reductase inhibitors has been reported to have immunosuppressive effects. Case presentation We describe an unusual case of regression of vitiligo in a patient treated with high dos...

  15. Measurement of nitrite reductase in leaf tissue of Vigna mungo : A new method.

    Srivastava, R C; Bose, B; Mukerji, D; Mathur, S N; Srivastava, H S


    The enzyme nitrite reductase (EC is generally assayed in terms of disappearance of nitrite from the assay medium. We describe a technique which allowed estimation of the enzyme level in leaf tissues of Vigna mungo (L). Hepper in terms of the release of the product (NH3) of the enzyme reaction. The technique is offered as an alternative, possibly more convenient method for assay of nitrite reductase in plant tissue in vivo.

  16. Localization and Solubilization of the Iron(III) Reductase of Geobacter sulfurreducens


    The iron(III) reductase activity of Geobacter sulfurreducens was determined with the electron donor NADH and the artificial electron donor horse heart cytochrome c. The highest reduction rates were obtained with Fe(III) complexed by nitrilotriacetic acid as an electron acceptor. Fractionation experiments indicated that no iron(III) reductase activity was present in the cytoplasm, that approximately one-third was found in the periplasmic fraction, and that two-thirds were associated with the m...

  17. A neutral polyacrylate copolymer coating for surface modification of thiol-ene microchannels for improved performance of protein separation by microchip electrophoresis

    Mesbah, Kiarach; Mai, T.D.; Jensen, Thomas Glasdam


    We have investigated the behavior of thiol-ene substrates that is a class of promising materials for lab-on-a-chip electrophoresis applications. Two polymeric materials were prepared by copolymerization of N, N-dimethylacrylamide (DMA), (3-(methacryloyl-oxy)propyl)trimethoxysilane (PMA) and 3......-(DMA-PMAMAPS) copolymer were evaluated in terms of surface hydrophilicity, suppression and stability of electro-osmotic flow and prevention of protein adsorption. Surface modification of thiol-ene containing a 20 % excess of thiols with the terpolymer p-(DMA-PMA-MAPS) was found to offer the most stable coating and most...

  18. Elaboration de revêtements à base d'huile végétale par chimie thiol-X photoamorcée

    Zhao, Yu Hui


    Two types of coatings constituted about 80 wt% of vegetable oil were prepared by photoinitiated thiol-X chemistry. The first one has been prepared by crosslinking of native and stand linseed oil triglycerides in the presence of 0.5 equivalent of various thiols (SH/ene) under air and UV radiation, without photoinitiator or solvent. The oxidation of fatty acids has shown a beneficial effect on the thiol-ene addition as well as on the final properties of coatings. This process is thus an interes...

  19. Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis

    Mee-Kyung; Cha; Yoo-Jeen; Bae; Kyu-Jeong; Kim; Byung-Joon; Park; Il-Han; Kim


    AIM: To identify alkyl hydroperoxide reductase subunit C(AhpC) homologs in Bacillus subtilis(B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria.METHODS: Two AhpC homologs(AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues(C37S, C47 S, C166 S, C37/47 S, C37/166 S, C47/166 S, and C37/47/166 S for AhpC_H1; C52 S, C169 S, and C52/169 S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahp C genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined.RESULTS: Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis

  20. X-ray structure of trypanothione reductase from Crithidia fasciculata at 2. 4- angstrom resolution

    Kuriyan, J.; Xiangpeng Kong; Krishna, T.S.R.; Murgolo, N.J.; Field, H.; Cerami, A.; Henderson, G.B. (Rockefeller Univ., New York, NY (United States)); Sweet, R.M. (Brookhaven National Lab., Upton, NY (United States))


    Trypanosomes and related protozoan parasites lack glutathione reductase and possess instead a closely related enzyme that serves as the reductant of a bis(glutathione)-spermidien conjugate, trypanothione. The human and parasite enzymes have mutually exclusive substrate specificities, providing a route for the design of therapeutic agents by specific inhibition of the parasite enzyme. The authors report here the three-dimensional structure of trypanothione reductase from Crithidia fasciculata and show that it closely resembles the structure of human glutathione reductase. In particular, the core structure surrounding the catalytic machinery is almost identical in the two enzymes. However, significant differences are found at the substrate binding sites. A cluster of basic residues in glutathione reductase is replaced by neutral, hydrophobic, or acidic residues in trypanothione reductase, consistent with the nature of the spermidine linkage and the change in overall charge of the substrate from {minus}2 to +1, respectively. The binding site is more open in trypanothione reductase due to rotations of about 4{degree} in the domains that form in site, with relative shifts of as much as 2-3 {angstrom} in residues that can interact with potential inhibitors and complement previous modeling and mutagenesis studies on the two enzymes.

  1. Structural Basis for the Thermostability of Sulfur Oxygenase Reductases

    尤晓颜; 孟珍; 陈栋炜; 郭旭; Josef Zeyer; 刘双江; 姜成英


    The thermostability of three sulfur oxygenase reductases (SORs) was investigated from thermoacidophilic achaea Acidianus tengchongensis (SORAT) and Sulfolobus tokodaii (SORsT) as well as the moderately thermophilic bacterium Acidithiobacillus sp. SM-1 (SORsB). The optimal temperatures for catalyzing sulfur oxidation were 80 ℃ (SORAT), 85 ℃ (SORsT), and 70 ℃ (SORsB), respectively. The half-lives of the three SORs at their optimal catalytic conditions were 100 min (SORAT), 58 min (SORsT), and 37 min (SORsB). In order to reveal the structural basis of the thermostability of these SORs, three-dimensional structural models of them were generated by homology modeling using the previously reported high-resolution X-ray structure of SORAA (from Acidianus ambivalens) as a template. The results suggest that thermostability was dependent on: (a) high number of the charged amino acid glutamic acid and the flexible amino acid proline, (b) low number of the therraolabile amino acid glutamine, (c) increased number of ion pairs, (d) decreased ratio of hydrophobie accessible solvent surface area (ASA) to charged ASA, and (e) increased volumes of the cavity. The number of cavities and the number of hydrogen bonds did not signifieantly affect the thermostability of SORs, whereas the cavity volumes increased as the thermal stability increased.

  2. Binding of Natural and Synthetic Polyphenols to Human Dihydrofolate Reductase

    José Neptuno Rodríguez-López


    Full Text Available Dihydrofolate reductase (DHFR is the subject of intensive investigation since it appears to be the primary target enzyme for antifolate drugs. Fluorescence quenching experiments show that the ester bond-containing tea polyphenols (--epigallocatechin gallate (EGCG and (--epicatechin gallate (ECG are potent inhibitors of DHFR with dissociation constants (KD of 0.9 and 1.8 μM, respectively, while polyphenols lacking the ester bound gallate moiety [e.g., (--epigallocatechin (EGC and (--epicatechin (EC] did not bind to this enzyme. To avoid stability and bioavailability problems associated with tea catechins we synthesized a methylated derivative of ECG (3-O-(3,4,5-trimethoxybenzoyl-(--epicatechin; TMECG, which effectively binds to DHFR (KD = 2.1 μM. In alkaline solution, TMECG generates a stable quinone methide product that strongly binds to the enzyme with a KD of 8.2 nM. Quercetin glucuronides also bind to DHFR but its effective binding was highly dependent of the sugar residue, with quercetin-3-xyloside being the stronger inhibitor of the enzyme with a KD of 0.6 μM. The finding that natural polyphenols are good inhibitors of human DHFR could explain the epidemiological data on their prophylactic effects for certain forms of cancer and open a possibility for the use of natural and synthetic polyphenols in cancer chemotherapy.

  3. Molecular Characterization of a Dehydroascorbate Reductase from Pinus bungeana

    Hai-Ling Yang; Ying-Ru Zhao; Cai-Ling Wang; Zhi-Ling Yang; Qing-Yin Zeng; Hai Lu


    Dehydroascorbate reductase (DHAR) plays a critical role in the ascorbate-glutathione recycling reaction for most higher plants. To date, studies on DHAR in higher plants have focused largely on Arabidopsis and agricultural plants, and there is virtually no information on the molecular characteristics of DHAR in gymnosperms. The present study reports the cloning and characteristics of a DHAR (PbDHAR) from a pine, Pinus bungeana Zucc. ex Endl. The PbDHAR gene encodes a protein of 215 amino acid residues with a calculated molecular mass of 24.26 kDa. The predicted 3-D structure of PbDHAR showed a typical glutathione S-transferase fold. Reverse transcription-polymerase chain reaction revealed that the PbDHAR was a constitutive expression gene in P. bungeana. The expression level of PbDHAR mRNA in P. bungeana seedlings did not show significant change under high temperature stress. The recombinant PbDHAR was overexpressed in Escherichia coll following purification with affinity chromatography. The recombinant PbDHAR exhibited enzymatic activity (19.84μmol/min per mg) and high affinity (a K_m of 0.08 mM) towards the substrates dehydroascorbate (DHA). Moreover, the recombinant PbDHAR was a thermostable enzyme, and retained 77% of its initial activity at 55 ℃. The present study is the first to provide a detailed molecular characterization of the DHAR in P. bungeana.

  4. Methylenetetrahydrofolate Reductase Genotypes, Dietary Habits and Susceptibility to Stomach Cancer

    ChangmingGao; TakezakiToshiro; JianzhongWu; JianhuoDing; YantingLiu; SupingLi; PingSu; XuHu; TianliongXu; HamajimaNobuyuki; TajimaKazuo


    OBJECTIVE To study the relation among methylenetetrahydrofolate reductase (MTHFR) C677T genotypes, dietary habits and the risk of stomach cancer (SC).METHODS A case-control study was conducted with 107 cases of SC and 200 population-based controls in Chuzhou district, Huaian, Jiangsu province, China. The epidemiological data were collected, and DNA of peripheral blood leukocytes was obtained from all of the subjects..MTHFR genotypes were detected by PCR-RFLP. RESULTS (1) The prevalence of the MTHFR C/T or T/T genotypes was found to be significantly different between controls (68.5%) and SC cases (79.4%,P=0.0416), the increased risk had an adjusted OR of 1.79 (95%C1:1.01-3.19). (2) Among subjects who had a low intake of garlic or Chinese onion, MTHFR C/T or T/T genotypes significantly increased the risk of developing SC. Among non-tea drinkers or among subjects who had a frequent intakeof meat, the carriers of the MTHFR C/T or T/T genotypes had a higher risk of SC than individuals with the C/C type MTHFR. CONCLUSION The polymorphism of MTHFR C677T was associated with increased risk of developing SC, and that individuals with differing genotypes may have different susceptibilities to SC, based on their exposure level to environmental factors.

  5. Solvent effects on catalysis by Escherichia coli dihydrofolate reductase.

    Loveridge, E Joel; Tey, Lai-Hock; Allemann, Rudolf K


    Hydride transfer catalyzed by dihydrofolate reductase (DHFR) has been described previously within an environmentally coupled model of hydrogen tunneling, where protein motions control binding of substrate and cofactor to generate a tunneling ready conformation and modulate the width of the activation barrier and hence the reaction rate. Changes to the composition of the reaction medium are known to perturb protein motions. We have measured kinetic parameters of the reaction catalyzed by DHFR from Escherichia coli in the presence of various cosolvents and cosolutes and show that the dielectric constant, but not the viscosity, of the reaction medium affects the rate of reaction. Neither the primary kinetic isotope effect on the reaction nor its temperature dependence were affected by changes to the bulk solvent properties. These results are in agreement with our previous report on the effect of solvent composition on catalysis by DHFR from the hyperthermophile Thermotoga maritima. However, the effect of solvent on the temperature dependence of the kinetic isotope effect on hydride transfer catalyzed by E. coli DHFR is difficult to explain within a model, in which long-range motions couple to the chemical step of the reaction, but may indicate the existence of a short-range promoting vibration or the presence of multiple nearly isoenergetic conformational substates of enzymes with similar but distinct catalytic properties.

  6. A second target of benzamide riboside: dihydrofolate reductase.

    Roussel, Breton; Johnson-Farley, Nadine; Kerrigan, John E; Scotto, Kathleen W; Banerjee, Debabrata; Felczak, Krzysztof; Pankiewicz, Krzysztof W; Gounder, Murugesan; Lin, HongXia; Abali, Emine Ercikan; Bertino, Joseph R


    Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth.

  7. Inhibition of Aldose Reductase by Gentiana lutea Extracts

    Chandrasekhar Akileshwari


    Full Text Available Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2 activity has been implicated in the development of various secondary complications of diabetes. Thus, ALR2 inhibition could be an effective strategy in the prevention or delay of certain diabetic complications. Gentiana lutea grows naturally in the central and southern areas of Europe. Its roots are commonly consumed as a beverage in some European countries and are also known to have medicinal properties. The water, ethanol, methanol, and ether extracts of the roots of G. lutea were subjected to in vitro bioassay to evaluate their inhibitory activity on the ALR2. While the ether and methanol extracts showed greater inhibitory activities against both rat lens and human ALR2, the water and ethanol extracts showed moderate inhibitory activities. Moreover, the ether and methanol extracts of G. lutea roots significantly and dose-dependently inhibited sorbitol accumulation in human erythrocytes under high glucose conditions. Molecular docking studies with the constituents commonly present in the roots of G. lutea indicate that a secoiridoid glycoside, amarogentin, may be a potential inhibitor of ALR2. This is the first paper that shows G. lutea extracts exhibit inhibitory activity towards ALR2 and these results suggest that Gentiana or its constituents might be useful to prevent or treat diabetic complications.

  8. Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis

    Bodra, Nandita; Young, David; Astolfi Rosado, Leonardo; Pallo, Anna; Wahni, Khadija; De Proft, Frank; Huang, Jingjing; Van Breusegem, Frank; Messens, Joris


    Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release. PMID:28195196

  9. Functional characterization of methionine sulfoxide reductase A from Trypanosoma spp.

    Arias, Diego G; Cabeza, Matías S; Erben, Esteban D; Carranza, Pedro G; Lujan, Hugo D; Téllez Iñón, María T; Iglesias, Alberto A; Guerrero, Sergio A


    Methionine is an amino acid susceptible to being oxidized to methionine sulfoxide (MetSO). The reduction of MetSO to methionine is catalyzed by methionine sulfoxide reductase (MSR), an enzyme present in almost all organisms. In trypanosomatids, the study of antioxidant systems has been mainly focused on the involvement of trypanothione, a specific redox component in these organisms. However, no information is available concerning their mechanisms for repairing oxidized proteins, which would be relevant for the survival of these pathogens in the various stages of their life cycle. We report the molecular cloning of three genes encoding a putative A-type MSR in trypanosomatids. The genes were expressed in Escherichia coli, and the corresponding recombinant proteins were purified and functionally characterized. The enzymes were specific for L-Met(S)SO reduction, using Trypanosoma cruzi tryparedoxin I as the reducing substrate. Each enzyme migrated in electrophoresis with a particular profile reflecting the differences they exhibit in superficial charge. The in vivo presence of the enzymes was evidenced by immunological detection in replicative stages of T. cruzi and Trypanosoma brucei. The results support the occurrence of a metabolic pathway in Trypanosoma spp. involved in the critical function of repairing oxidized macromolecules.

  10. Thiol-thione tautomeric analysis, spectroscopic (FT-IR, Laser-Raman, NMR and UV-vis) properties and DFT computations of 5-(3-pyridyl)-4H-1,2,4-triazole-3-thiol molecule

    Gökce, Halil; Öztürk, Nuri; Ceylan, Ümit; Alpaslan, Yelda Bingöl; Alpaslan, Gökhan


    In this study, the 5-(3-pyridyl)-4H-1,2,4-triazole-3-thiol molecule (C7H6N4S) molecule has been characterized by using FT-IR, Laser-Raman, NMR and UV-vis spectroscopies. Quantum chemical calculations have been performed to investigate the molecular structure (thione-thiol tautomerism), vibrational wavenumbers, electronic transition absorption wavelengths in DMSO solvent and vacuum, proton and carbon-13 NMR chemical shifts and HOMOs-LUMOs energies at DFT/B3LYP/6-311 ++G(d,p) level for all five tautomers of the title molecule. The obtained results show that the calculated vibrational wavenumbers, NMR chemical shifts and UV-vis wavelengths are in a good agreement with experimental data.

  11. Electronic Transport through Self Assembled Thiol Molecules: Effect of Monolayer Order, Dynamics and Temperature

    Dholakia, Geetha; Fan, Wendy; Meyyappan, M.


    We present the charge transport and tunneling conductance of self assembled organic thiol molecules and discuss the influence of order and dynamics in the monolayer on the transport behavior and the effect of temperature. Conjugated thiol molecular wires and organometals such as terpyridine metal complexes provide a new platform for molecular electronic devices and we study their self assembly on Au(111) substrates by the scanning tunneling microscope. Determining the organization of the molecule and the ability to control the nature of its interface with the substrate is important for reliable performance of the molecular electronic devices. By concurrent scanning tunneling microscopy and spectroscopy studies on SAMs formed from oligo (phenelyne ethynelyne) monolayers with and without molecular order, we show that packing and order determine the response of a self assembled monolayer (SAM) to competing interactions. Molecular resolution STM imaging in vacuum shows that the OPES adopt an imcommensurate SAM structure on Au(111) with a rectangular unit cell. Tunneling spectroscopic measurements were performed on the SAM as a function of junction resistance. STS results show that the I-Vs are non linear and asymmetric due to the inherent asymmetry in the molecular structure, with larger currents at negative sample biases. The asymmetry increases with increasing junction resistance due to the asymmetry in the coupling to the leads. This is brought out clearly in the differential conductance, which also shows a gap at the Fermi level. We also studied the effect of order and dynamics in the monolayer on the charge transport and found that competing forces between the electric field, intermolecular interactions, tip-molecule physisorption and substrate-molecule chemisorption impact the transport measurements and its reliability and that the presence of molecular order is very important for reproducible transport measurements. Thus while developing new electronic platforms

  12. Decaborane thiols as building blocks for self-assembled monolayers on metal surfaces.

    Bould, Jonathan; Macháček, Jan; Londesborough, Michael G S; Macías, Ramón; Kennedy, John D; Bastl, Zdeněk; Rupper, Patrick; Baše, Tomáš


    Three nido-decaborane thiol cluster compounds, [1-(HS)-nido-B(10)H(13)] 1, [2-(HS)-nido-B(10)H(13)] 2, and [1,2-(HS)(2)-nido-B(10)H(12)] 3 have been characterized using NMR spectroscopy, single-crystal X-ray diffraction analysis, and quantum-chemical calculations. In the solid state, 1, 2, and 3 feature weak intermolecular hydrogen bonding between the sulfur atom and the relatively positive bridging hydrogen atoms on the open face of an adjacent cluster. Density functional theory (DFT) calculations show that the value of the interaction energy is approximately proportional to the number of hydrogen atoms involved in the interaction and that these values are consistent with a related bridging-hydrogen atom interaction calculated for a B(18)H(22)·C(6)H(6) solvate. Self-assembled monolayers (SAMs) of 1, 2, and 3 on gold and silver surfaces have been prepared and characterized using X-ray photoelectron spectroscopy. The variations in the measured sulfur binding energies, as thiolates on the surface, correlate with the (CC2) calculated atomic charge for the relevant boron vertices and for the associated sulfur substituents for the parent B(10)H(13)(SH) compounds. The calculated charges also correlate with the measured and DFT-calculated thiol (1)H chemical shifts. Wetting-angle measurements indicate that the hydrophilic open face of the cluster is directed upward from the substrate surface, allowing the bridging hydrogen atoms to exhibit a similar reactivity to that of the bulk compound. Thus, [PtMe(2)(PMe(2)Ph)(2)] reacts with the exposed and acidic B-H-B bridging hydrogen atoms of a SAM of 1 on a gold substrate, affording the addition of the metal moiety to the cluster. The XPS-derived stoichiometry is very similar to that for a SAM produced directly from the adsorption of [1-(HS)-7,7-(PMe(2)Ph)(2)-nido-7-PtB(10)H(11)] 4. The use of reactive boron hydride SAMs as templates on which further chemistry may be carried out is unprecedented, and the principle may be

  13. Pseudomonas aeruginosa IscR-Regulated Ferredoxin NADP(+ Reductase Gene (fprB Functions in Iron-Sulfur Cluster Biogenesis and Multiple Stress Response.

    Adisak Romsang

    Full Text Available P. aeruginosa (PAO1 has two putative genes encoding ferredoxin NADP(+ reductases, denoted fprA and fprB. Here, the regulation of fprB expression and the protein's physiological roles in [4Fe-4S] cluster biogenesis and stress protection are characterized. The fprB mutant has defects in [4Fe-4S] cluster biogenesis, as shown by reduced activities of [4Fe-4S] cluster-containing enzymes. Inactivation of the gene resulted in increased sensitivity to oxidative, thiol, osmotic and metal stresses compared with the PAO1 wild type. The increased sensitivity could be partially or completely suppressed by high expression of genes from the isc operon, which are involved in [Fe-S] cluster biogenesis, indicating that stress sensitivity in the fprB mutant is partially caused by a reduction in levels of [4Fe-4S] clusters. The pattern and regulation of fprB expression are in agreement with the gene physiological roles; fprB expression was highly induced by redox cycling drugs and diamide and was moderately induced by peroxides, an iron chelator and salt stress. The stress-induced expression of fprB was abolished by a deletion of the iscR gene. An IscR DNA-binding site close to fprB promoter elements was identified and confirmed by specific binding of purified IscR. Analysis of the regulation of fprB expression supports the role of IscR in directly regulating fprB transcription as a transcription activator. The combination of IscR-regulated expression of fprB and the fprB roles in response to multiple stressors emphasizes the importance of [Fe-S] cluster homeostasis in both gene regulation and stress protection.

  14. Mitochondrial respiratory chain and thioredoxin reductase regulate intermembrane Cu,Zn-superoxide dismutase activity: implications for mitochondrial energy metabolism and apoptosis.

    Iñarrea, Pedro; Moini, Hadi; Han, Derick; Rettori, Daniel; Aguiló, Ignacio; Alava, Maria Angeles; Iturralde, María; Cadenas, Enrique


    IMS (intermembrane space) SOD1 (Cu/Zn-superoxide dismutase) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS SOD1 activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H2O2 (5 microM) activated SOD1 in intact mitochondria. However, neither H2O2 alone nor H2O2 in the presence of mitochondrial peroxiredoxin III activated SOD1, which was purified from mitochondria and subsequently reduced by dithiothreitol to an inactive state. The reduced enzyme was activated following incubation with the superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of SOD1 activation was inversely correlated with mitochondrial superoxide production. The presence of TxrR-1 (thioredoxin reductase-1) was demonstrated in the mitochondrial IMS by Western blotting. Inhibitors of TxrR-1, CDNB (1-chloro-2,4-dinitrobenzene) or auranofin, prolonged the duration of H2O2-induced SOD1 activity in intact mitochondria. TxrR-1 inactivated SOD1 purified from mitochondria in an active oxidized state. Activation of IMS SOD1 by exogenous H2O2 delayed CaCl2-induced loss of transmembrane potential, decreased cytochrome c release and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that H2O2, superoxide and TxrR-1 regulate IMS SOD1 activity reversibly, and that the active enzyme is implicated in protecting vital mitochondrial functions.

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

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


    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.

  16. Spectroscopic Characterization of Extracellular Polymeric Substances from Escherichia coli and Serratia marcescens: Suppression using Sub-Inhibitory Concentrations of Bismuth Thiols

    Badireddy, Appala R.; Korpol, Bhoom Reddy; Chellam, Shankararaman; Gassman, Paul L.; Engelhard, Mark H.; Lea, Alan S.; Rosso, Kevin M.


    Free and capsular EPS produced by Escherichia coli and Serratia marcescens were characterized in detail using Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and Auger electron spectroscopy (AES). Total EPS production decreased upon treatment with sub-inhibitory concentrations of lipophilic bismuth thiols (bismuth dimercaptopropanol, BisBAL; bismuth ethanedithiol, BisEDT; and bismuth pyrithione, BisPYR), BisBAL being most effective. Bismuth thiols also influenced acetylation and carboxylation of polysaccharides in EPS from S. marcescens. Extensive homology between EPS samples in the presence and absence of bismuth was observed with proteins, polysaccharides, and nucleic acids varying predominantly only in the total amount expressed. Second derivative analysis of the amide I region of FTIR spectra revealed decreases in protein secondary structures in the presence of bismuth thiols. Hence, anti-fouling properties of bismuth thiols appear to originate in their ability to suppress O-acetylation and protein secondary structures in addition to total EPS secretion.

  17. 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).

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


    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.

  18. Photostability and Performance of Polystyrene Films Containing 1,2,4-Triazole-3-thiol Ring System Schiff Bases.

    Ali, Gassan Q; El-Hiti, Gamal A; Tomi, Ivan Hameed R; Haddad, Raghad; Al-Qaisi, Alaa J; Yousif, Emad


    Series of 4-(4-substituted benzylideneamino)-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-3-thiols were synthesized and their structures were confirmed. The synthesized Schiff bases were used as photostabilizers for polystyrene against photodegradation. Polystyrene polymeric films containing synthesized Schiff bases (0.5% by weight) were irradiated (λmax = 365 nm and light intensity = 6.43 × 10(-9) ein·dm(-3)·s(-1)) at room temperature. The photostabilization effect of 1,2,4-triazole-3-thiols Schiff bases was determined using various methods. All the additives used enhanced the photostability of polystyrene films against irradiation compared with the result obtained in the absence of Schiff base. The Schiff bases can act as photostabilizers for polystyrene through the direct absorption of UV radiation and/or radical scavengers.

  19. Glutamine synthetase isoforms in nitrogen-fixing soybean nodules: distinct oligomeric structures and thiol-based regulation.

    Masalkar, Pintu D; Roberts, Daniel M


    Legume root nodule glutamine synthetase (GS) catalyzes the assimilation of ammonia produced by nitrogen fixation. Two GS isoform subtypes (GS1β and GS1γ) are present in soybean nodules. GS1γ isoforms differ from GS1β isoforms in terms of their susceptibility to reversible inhibition by intersubunit disulfide bond formation between C159 and C92 at the shared active site at subunit interfaces. Although nodule GS enzymes share 86% amino acid sequence identity, analytical ultracentrifugation experiments showed that GS1γ is a dodecamer, whereas the GS1β is a decamer. It is proposed that this difference contributes to the differential thiol sensitivity of each isoform, and that GS1γ1 may be a target of thiol-based regulation.

  20. Crystal Structure of the ERp44-Peroxiredoxin 4 Complex Reveals the Molecular Mechanisms of Thiol-Mediated Protein Retention.

    Yang, Kai; Li, De-Feng; Wang, Xi'e; Liang, Jinzhao; Sitia, Roberto; Wang, Chih-Chen; Wang, Xi


    ERp44 controls the localization and transport of diverse proteins in the early secretory pathway. The mechanisms that allow client recognition and the source of the oxidative power for forming intermolecular disulfides are as yet unknown. Here we present the structure of ERp44 bound to a client, peroxiredoxin 4. Our data reveal that ERp44 binds the oxidized form of peroxiredoxin 4 via thiol-disulfide interchange reactions. The structure explains the redox-dependent recognition and characterizes the essential non-covalent interactions at the interface. The ERp44-Prx4 covalent complexes can be reduced by glutathione and protein disulfide isomerase family members in the ER, allowing the two components to recycle. This work provides insights into the mechanisms of thiol-mediated protein retention and indicates the key roles of ERp44 in this biochemical cycle to optimize oxidative folding and redox homeostasis.

  1. Thiol click chemistry on gold-decorated MoS2: elastomer composites and structural phase transitions

    Topolovsek, Peter; Cmok, Luka; Gadermaier, Christoph; Borovsak, Milos; Kovac, J.; Mrzel, Ales


    We show that gold decorated MoS2 flakes are amenable to thiol chemistry by blending them with a cross-linkable thiolated polysiloxane (PMMS). PMMS prevents restacking of dispersed MoS2 when transforming the metallic to the semiconducting phase. Cross-linking PMMS yields an elastomer of good optical quality, containing individual, mostly single-layer MoS2 flakes.We show that gold decorated MoS2 flakes are amenable to thiol chemistry by blending them with a cross-linkable thiolated polysiloxane (PMMS). PMMS prevents restacking of dispersed MoS2 when transforming the metallic to the semiconducting phase. Cross-linking PMMS yields an elastomer of good optical quality, containing individual, mostly single-layer MoS2 flakes. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01490a

  2. Monocatenary, branched, double-headed, and bolaform surface active carbohydrate esters via photochemical thiol-ene/-yne reactions.

    Boyère, Cédric; Broze, Guy; Blecker, Christophe; Jérôme, Christine; Debuigne, Antoine


    An original and versatile method for the synthesis of a range of novel mannose-based surfactants was developed via metal-free photo-induced thiol-ene/-yne 'click' reactions. This light-mediated hydrothiolation reaction involving a thiolated mannose was successfully applied to terminal and internal alkenes, dienes, and alkynes, leading to monocatenary, branched, double-headed, and bolaform amphiphilic carbohydrate esters, respectively. A surface activity study showed that these new compounds possess valuable properties and display specific behavior at the air-water interface. It also demonstrated the greater flexibility of the thioether moiety in the spacer of the surfactants produced via a thiol-ene reaction in comparison with the triazole heterocyclic rings in similar glucose-based surfactants synthesized elsewhere by the alkyne-azide 1,3-dipolar addition.

  3. Synthesis of antibacterial amphiphilic elastomer based on polystyrene-block-polyisoprene-block-polystyrene via thiol-ene addition.

    Keleş, Elif; Hazer, Baki; Cömert, Füsun B


    A new type of amphiphilic antibacterial elastomer has been described. Thermoplastic elastomer, polystyrene-block-polyisoprene-block-polystyrene (PS-b-PI-b-PS) triblock copolymer was functionalized in toluene solution by free radical mercaptan addition in order to obtain an amphiphilic antibacterial elastomer. Thiol terminated PEG was grafted through the double bonds of PS-b-PI-b-PS via free radical thiol-ene coupling reaction. The antibacterial properties of the amphiphilic graft copolymers were observed. The original and the modified polymers were used to create microfibers in an electro-spinning process. Topology of the electrospun micro/nanofibers were studied by using scanning electron microscopy (SEM). The chemical structures of the amphiphilic comb type graft copolymers were elucidated by the combination of elemental analysis, (1)H NMR, (13)C NMR, GPC and FTIR.

  4. X-ray magnetic circular dichroism and small angle neutron scattering study of thiol capped gold nanoparticles.

    de la Venta, J.; Bouzas, V.; Pucci, A.; Laguna-Marco, M. A.; Haskel, D.; Pinel, E. F.; te Velthuis, S. G. E.; Hoffmann, A.; Lal, J.; Bleuel, M.; Ruggeri, G.; de Julian, C.; Garcia, M. A.; Univ. Complutense de Madrid; Inst. de Magnetismo Aplicado UCM; Univ. Pisa; Univ. di Padova


    X-ray magnetic circular dichroism (XMCD) and Small Angle Neutron Scattering (SANS) measurements were performed on thiol capped Au nanoparticles (NPs) embedded into polyethylene. An XMCD signal of 0.8 {center_dot} 10{sup -4} was found at the Au L{sub 3} edge of thiol capped Au NPs embedded in a polyethylene matrix for which Superconducting Quantum Interference Device (SQUID) magnetometry yielded a saturation magnetization, M{sub s}, of 0.06 emu/g{sub Au}. SANS measurements showed that the 3.2 nm average-diameter nanoparticles are 28% polydispersed, but no detectable SANS magnetic signal was found with the resolution and sensitivity accessible with the neutron experiment. A comparison with previous experiments carried out on Au NPs and multilayers, yield to different values between XMCD signals and magnetization measured by SQUID magnetometer. We discuss the origin of those differences.

  5. X-ray magnetic circular dichroism and small angle neutron scattering studies of thiol capped gold nanoparticles.

    de la Venta, J.; Bouzas, V.; Pucci, A.; Laguna-Marco, M. A.; Haskel, D.; te Velthuis, S. G. E; Hoffmann, A.; Lal, J.; Bleuel, M.; Ruggeri, G.; de Julian Fernandez, C.; Garcia, M. A.; Univ.Complutense de Madrid; Inst. de Magnetismo Aplicado; Univ. of Pisa; Lab. di Magnetismo Molecolare


    X-ray magnetic circular dichroism (XMCD) and Small Angle Neutron Scattering (SANS) measurements were performed on thiol capped Au nanoparticles (NPs) embedded into polyethylene. An XMCD signal of 0.8 {center_dot} 10{sup -4} was found at the Au L{sub 3} edge of thiol capped Au NPs embedded in a polyethylene matrix for which Superconducting Quantum Interference Device (SQUID) magnetometry yielded a saturation magnetization, M{sub s}, of 0.06 emu/g{sub Au}. SANS measurements showed that the 3.2 nm average-diameter nanoparticles are 28% polydispersed, but no detectable SANS magnetic signal was found with the resolution and sensitivity accessible with the neutron experiment. A comparison with previous experiments carried out on Au NPs and multilayers, yield to different values between XMCD signals and magnetization measured by SQUID magnetometer. We discuss the origin of those differences.

  6. Photostability and Performance of Polystyrene Films Containing 1,2,4-Triazole-3-thiol Ring System Schiff Bases

    Gassan Q. Ali


    Full Text Available Series of 4-(4-substituted benzylideneamino-5-(3,4,5-trimethoxyphenyl-4H-1,2,4-triazole-3-thiols were synthesized and their structures were confirmed. The synthesized Schiff bases were used as photostabilizers for polystyrene against photodegradation. Polystyrene polymeric films containing synthesized Schiff bases (0.5% by weight were irradiated (λmax = 365 nm and light intensity = 6.43 × 10−9 ein·dm−3·s−1 at room temperature. The photostabilization effect of 1,2,4-triazole-3-thiols Schiff bases was determined using various methods. All the additives used enhanced the photostability of polystyrene films against irradiation compared with the result obtained in the absence of Schiff base. The Schiff bases can act as photostabilizers for polystyrene through the direct absorption of UV radiation and/or radical scavengers.

  7. Potassium carbonate: a highly efficient catalyst for the acylation of alco-hols, phenols and thiols under mild conditions

    Mosstafa Kazemi


    Full Text Available A general, mild and efficient protocol has been developed for the synthesis of esters and thioesters. The process has been taking place using tetra n-butylammonium iodide (TBAI as a phase-transfer catalyst and in the presence of potassium carbonate (K2CO3. A wide range of esters and thioesters was prepared in high yields and suitable times by the treatment of alcohols, phenols and thiols with acetic anhydride. Acylation reactions of a number of alcohols, phenols and thiols with acetic anhydride demonstrated that potassium carbonate is a convenient and efficient catalyst for the synthesis of esters and thioesters. This is a mild, general and practical procedure for the synthesis of esters and thioesters in high yields and suitable times.

  8. Synthesis of thiol-functionalized TiO_2 nanocomposite and photocatalytic degradation for PAH under visible light irradiation

    Long Liu; Jia Hui Kou; Da Meng Guo; Jing Yang; Hong Lin Liu; Hong Xia Yu; Sheng Chu; Ke Ren Jiang; Ying Wang; Zhi Gang Zou


    In this paper, a thiol-functionalized nanophotocatalyst MPTES/TiO_2 was first synthesized by one-pot method using P123 as a template. X-ray diffraction confirms the complete anatasc crystalline of thiol-funetionalized TiO_2, N_2 adsorption-desorption isotherm demonstrated that these materials possess high surface area and mesoporous structure. The results of XPS show that MPTES has been successfully polymerized in mesoporous structured TiO_2. The photodegradation of phenanthrene (PHE) was investigated under visible light irradiation (λ>420 nm) to evaluate the photocatalytic activity of these materials. Based the experiment results of GC-Mass analysis, a possible mechanism was proposed.

  9. Drastic difference in luminescence stability between amine- and thiol-capped quantum dots treated with CO2

    Vokhmintcev, Kirill V.; Nabiev, Igor R.; Samokhvalov, Pavel S.


    Research on the surface chemistry of quantum dots (QDs) has been rapidly developing in recent years, since the understanding of the processes that occur on their surface is prerequisite for successful exploration of the outstanding fluorescence properties and superior stability of these nanomaterials in numerous applications. The lack of stability during long-term storage under atmospheric conditions restricts QD applications. Here, we have investigated the interaction of QDs with carbon dioxide as a model system for studying their long-term storage or operation in atmospheric environment. Quenching of the photoluminescence of CdSe/ZnS semiconductor QDs continuously treated with CO2 has shown that this process depends on the type of the QD surface ligands. The luminescence of QDs capped with amine ligands is quenched to a higher degree, the quenching being caused by the formation of carbamic acid precipitate. The luminescence of QDs capped with thiols remain absolutely stable upon CO2 treatment due to the chemical resistance of thiol functional groups to CO2, which makes this type of QDs suitable for long-term storage and operation under atmospheric conditions. However, further functionalization of such QDs may be difficult, because the strong bond between thiol ligands and QD surface may limit the efficiency of ligand-exchange procedures. A new ligand system of alkylamine salts of fatty acids has been proposed as an alternative to thiols. It has been shown to be inert to CO2, and also can be easily replaced with functional surface ligands. The results are important for development of nextgeneration QDs with superior stability suitable for various applications requiring efficient ligand exchange and operation in the atmospheric environment.

  10. Improving gelation efficiency and cytocompatibility of visible light polymerized thiol-norbornene hydrogels via addition of soluble tyrosine.

    Shih, Han; Liu, Hung-Yi; Lin, Chien-Chi


    Hydrogels immobilized with biomimetic peptides have been used widely for tissue engineering and drug delivery applications. Photopolymerization has been among the most commonly used techniques to fabricate peptide-immobilized hydrogels as it offers rapid and robust peptide immobilization within a crosslinked hydrogel network. Both chain-growth and step-growth photopolymerizations can be used to immobilize peptides within covalently crosslinked hydrogels. A previously developed visible light mediated step-growth thiol-norbornene gelation scheme has demonstrated efficient crosslinking of hydrogels composed of an inert poly(ethylene glycol)-norbornene (PEGNB) macromer and a small molecular weight bis-thiol linker, such as dithiothreitol (DTT). Compared with conventional visible light mediated chain-polymerizations where multiple initiator components are required, step-growth photopolymerized thiol-norbornene hydrogels are more cytocompatible for the in situ encapsulation of radical sensitive cells (e.g., pancreatic β-cells). This contribution explored visible light based crosslinking of various bis-cysteine containing peptides with macromer 8-arm PEGNB to form biomimetic hydrogels suitable for in situ cell encapsulation. It was found that the addition of soluble tyrosine during polymerization not only significantly accelerated gelation, but also improved the crosslinking efficiency of PEG-peptide hydrogels as evidenced by a decreased gel point and enhanced gel modulus. In addition, soluble tyrosine drastically enhanced the cytocompatibility of the resulting PEG-peptide hydrogels, as demonstrated by in situ encapsulation and culture of pancreatic MIN6 β-cells. This visible light based thiol-norbornene crosslinking mechanism provides an attractive gelation method for preparing cytocompatible PEG-peptide hydrogels for tissue engineering applications.

  11. In Situ forming poly(ethylene glycol)-based hydrogels via thiol-maleimide Michael-type addition

    Fu, Yao; Kao, Weiyuan John


    The incorporation of cells and sensitive compounds can be better facilitated without the presence of UV or other energy sources that are common in the formation of biomedical hydrogels such as poly(ethylene glycol) hydrogels. The formation of hydrogels by the step-growth polymerization of maleimide- and thiol-terminated poly(ethylene glycol) macromers via Michael-type addition is described. The effects of macromer concentration, pH, temperature, and the presence of biomolecule gelatin on gel ...

  12. Dissolved low-molecular weight thiol concentrations from the U.S. GEOTRACES North Atlantic Ocean zonal transect

    Swarr, Gretchen J.; Kading, Tristan; Lamborg, Carl H.; Hammerschmidt, Chad R.; Bowman, Katlin L.


    Low-molecular weight thiols, including cysteine and glutathione, are biomolecules involved in a variety of metabolic pathways and act as important antioxidant and metal buffering agents. In this last capacity, they represent a potential mechanism for modulating the bioavailability and biogeochemistry of many trace elements in the ocean, particularly for chalcophilic elements (e.g., Cu, Zn, Cd, Ag and Hg). For this reason, and in the context of the international GEOTRACES program that seeks to understand the biogeochemistry of trace elements in the ocean, we measured the concentration of individual dissolved low-molecular weight thiols during the U.S. GEOTRACES North Atlantic Zonal Transect (USGNAZT). Only two thiols were identified, cysteine and glutathione, in contrast to results from the northeast subarctic Pacific Ocean, where the dipeptides glycine-cysteine and arginine-cysteine were also present and γ-glutamylcysteine was dominant. Concentrations of cysteine and glutathione in the North Atlantic Ocean were lower than in the Pacific and ranged from below detection ( 0.01 nM) to 0.61 nM of cysteine and up to 1.0 nM of glutathione, with cysteine generally more abundant than glutathione. Vertical profiles of cysteine and glutathione were broadly consistent with their biological production, being more abundant in surface water and usually below detection at depths greater than about 200 m. Subsurface concentration maxima, often co-incident with the deep chlorophyll maximum, were frequently observed but not universal. We conclude that cysteine and glutathione do not make up significant portions of complexation capacity for Cu and Zn in the upper open ocean but could be important for Cd, Hg, and potentially other chalcophiles. Extremely low concentrations of cysteine and glutathione in deep water suggest that higher molecular-weight thiols are a more important ligand class for chalcophiles in that portion of the ocean.

  13. Influence of liposome forms of the rhenium compounds and cis-platin on thiol-disulfide coefficient in the rats’ blood

    I. V. Klenina


    Full Text Available Thiol-disulfide coefficient (TDC and its different modifications in model in vivo were studied. Introduction of the liposome forms of cluster rhenium compounds with organic ligands (CROL leads to both TDC increasing and to the constancy of the TDC. Thus, CROLs aren’t toxic agents and some compounds could mobilize organisms’ thiol defence system. Liposome form of cis-platin leads to the TDC decreasing. Important CROL capacities for its future medical treatment practice were shown.

  14. Solvent-free Thia-Michael Addition Reactions Using 3-[Bis(alkylthio)methylene]pentane-2,4-diones as Efficient and Odorless Thiol Equivalents

    LIN Chun; ZHAO Xiao-Liang; OUYANG Yan; YU Hai-Feng; DONG De-Wen


    3-[Bis(alkylthio)methylene]pentane-2,4-diones (1a and 1b) have been investigated as nonthiolic and odorless thiol equivalents for thia-Michael addition reactions under solvent-free conditions. Promoted by HCl (aq.), the cleavage of compounds 1 took place, and the in-situ generated thiols underwent facile conjugate addition to α,β-unsaturated carbonyl compounds 2 affording the corresponding β-keto sulfides 3 in high yields.

  15. Phenolics from Garcinia mangostana Inhibit Advanced Glycation Endproducts Formation: Effect on Amadori Products, Cross-Linked Structures and Protein Thiols

    Hossam M. Abdallah


    Full Text Available Accumulation of Advanced Glycation Endproducts (AGEs in body tissues plays a major role in the development of diabetic complications. Here, the inhibitory effect of bioactive metabolites isolated from fruit hulls of Garcinia mangostana on AGE formation was investigated through bio-guided approach using aminoguanidine (AG as a positive control. Including G. mangostana total methanol extract (GMT in the reaction mixture of bovine serum albumin (BSA and glucose or ribose inhibited the fluorescent and non-fluorescent AGEs formation in a dose dependent manner. The bioassay guided fractionation of GMT revealed isolation of four bioactive constituents from the bioactive fraction; which were identified as: garcimangosone D (1, aromadendrin-8-C-glucopyranoside (2, epicatechin (3, and 2,3′,4,5′,6-pentahydroxybenzophenone (4. All the tested compounds significantly inhibited fluorescent and non-fluorescent AGEs formation in a dose dependent manner whereas compound 3 (epicatechin was found to be the most potent. In search for the level of action, addition of GMT, and compounds 2–4 inhibited fructosamine (Amadori product and protein aggregation formation in both glucose and ribose. To explore the mechanism of action, it was found that addition of GMT and only compound (3 to reaction mixture increased protein thiol in both glucose and ribose while compounds 1, 2 and 4 only increased thiol in case of ribose. In conclusion, phenolic compounds 1–4 inhibited AGEs formation at the levels of Amadori product and protein aggregation formation through saving protein thiol.

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

    Krajewska, Barbara; Zaborska, Wiesława


    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.

  17. Cysteine analogs with a free thiol group promote fertilization by reducing disulfide bonds in the zona pellucida of mice.

    Takeo, Toru; Horikoshi, Yuka; Nakao, Satohiro; Sakoh, Kazuhito; Ishizuka, Yuta; Tsutsumi, Aki; Fukumoto, Kiyoko; Kondo, Tomoko; Haruguchi, Yukie; Takeshita, Yumi; Nakamuta, Yuko; Tsuchiyama, Shuuji; Nakagata, Naomi


    Archives of cryopreserved sperm harvested from genetically engineered mice, in mouse resource centers, are a readily accessible genetic resource for the scientific community. We previously reported that exposure of oocytes to reduced glutathione (GSH) greatly improves the fertilization rate of frozen-thawed mouse sperm. Application of GSH to in vitro fertilization techniques is widely accepted as a standard protocol to produce sufficient numbers of mice from cryopreserved sperm. However, the detailed mechanism of the enhancement of fertilization mediated by GSH in vitro is not fully understood. Here we focused on the chemical by determining the effects of its amino acid constituents and cysteine analogs on the fertilization of oocytes by frozen-thawed sperm. Furthermore, we determined the stability of these compounds in aqueous solution. We show here that l-cysteine (l-Cys), d-cysteine (d-Cys), or N-acetyl-l-cysteine (NAC) increased the rate of fertilization when added to the medium but did not adversely affect embryo development in vitro or in vivo. The levels of thiol groups of proteins in the zona pellucida (ZP) and the expansion of the ZP were increased by l-Cys, d-Cys, and NAC. These effects were abrogated by the methylation of the thiol group of l-Cys. NAC was the most stable of these compounds in the fertilization medium at 4°C. These results suggest that the thiol groups of cysteine analogs markedly enhance the fertilization rate of mouse oocytes.

  18. Tip-enhanced Raman spectroscopic imaging shows segregation within binary self-assembled thiol monolayers at ambient conditions.

    Lin, Wan-Ing; Shao, Feng; Stephanidis, Bruno; Zenobi, Renato


    Phase segregation of coadsorbed thiol molecules on a gold surface was investigated with nanoscale chemical imaging using tip-enhanced Raman spectroscopy (TERS). Samples were prepared using mixed solutions containing thiophenol (PhS) and an oligomeric phenylene-ethynylene (OPE) thiol, with 10:1, 2:1, and 1:1 molar ratios. Phase segregation into domains with sizes from ≈30 to 240 nm is observed with these molar ratios. A comparison of TERS images with different pixel sizes indicates that a pixel size bigger than 15 nm is not reliable in defining nanodomains, because of undersampling. In this study, the formation of nanodomains was clearly evident based on the molecular fingerprints provided by TERS, while ambient scanning tunneling microscopy (STM) was not capable of discerning individual domains via their apparent height difference. TERS therefore allows to image nanodomains in binary self-assembled monolayers, which are invisible to methods solely relying on topographic or electron density characteristics of self-assembled monolayers. Moreover, TERS mapping provides statistical data to describe the distribution of molecules on the sample surface in a well-defined manner. Peak ratio histograms of selected TERS signals from samples prepared with different mixing ratios give a better understanding of the adsorption preference of the thiols studied, and the relationship of their mixing ratio in solution and adsorbed on the surface.

  19. Synthesis of antibacterial amphiphilic elastomer based on polystyrene-block-polyisoprene-block-polystyrene via thiol-ene addition

    Keleş, Elif, E-mail: [Department of Chemistry, Bülent Ecevit University, Zonguldak 67100 (Turkey); Hazer, Baki, E-mail: [Department of Chemistry, Bülent Ecevit University, Zonguldak 67100 (Turkey); Cömert, Füsun B. [Department of Microbiology, Faculty of Medicine, Bülent Ecevit University, 67600 Zonguldak (Turkey)


    A new type of amphiphilic antibacterial elastomer has been described. Thermoplastic elastomer, polystyrene–block-polyisoprene–block-polystyrene (PS–b-PI–b-PS) triblock copolymer was functionalized in toluene solution by free radical mercaptan addition in order to obtain an amphiphilic antibacterial elastomer. Thiol terminated PEG was grafted through the double bonds of PS–b-PI–b-PS via free radical thiol-ene coupling reaction. The antibacterial properties of the amphiphilic graft copolymers were observed. The original and the modified polymers were used to create microfibers in an electro-spinning process. Topology of the electrospun micro/nanofibers were studied by using scanning electron microscopy (SEM). The chemical structures of the amphiphilic comb type graft copolymers were elucidated by the combination of elemental analysis, {sup 1}H NMR, {sup 13}C NMR, GPC and FTIR. - Graphical abstract: Double bonds of polyisoprene units in polystyrene–block-polyisoprene–block-polystyrene triblock copolymer were partially capped with PEG containing mercapto end group via thiol-ene addition in order to obtain antibacterial amphiphilic elastomer. Nano fibers from amphiphilic graft polymers solution were produced by electrospinning. The PEG grafted copolymer inhibits very effectively bacterial growth. Highlights: ► A commercial synthetic elastomer was grafted with PEG to obtain amphiphilic elastomer. ► Amphiphilic elastomer shows antibacterial properties. ► Electrospun micro fibers of the amphiphilic elastomer tend to globular formation.

  20. Species-Specific Standard Redox Potential of Thiol-Disulfide Systems: A Key Parameter to Develop Agents against Oxidative Stress

    Mirzahosseini, Arash; Noszál, Béla


    Microscopic standard redox potential, a new physico-chemical parameter was introduced and determined to quantify thiol-disulfide equilibria of biological significance. The highly composite, codependent acid-base and redox equilibria of thiols could so far be converted into pH-dependent, apparent redox potentials (E’°) only. Since the formation of stable metal-thiolate complexes precludes the direct thiol-disulfide redox potential measurements by usual electrochemical techniques, an indirect method had to be elaborated. In this work, the species-specific, pH-independent standard redox potentials of glutathione were determined primarily by comparing it to 1-methylnicotinamide, the simplest NAD+ analogue. Secondarily, the species-specific standard redox potentials of the two-electron redox transitions of cysteamine, cysteine, homocysteine, penicillamine, and ovothiol were determined using their microscopic redox equilibrium constants with glutathione. The 30 different, microscopic standard redox potential values show close correlation with the respective thiolate basicities and provide sound means for the development of potent agents against oxidative stress.

  1. Aptamer-based organic-silica hybrid affinity monolith prepared via "thiol-ene" click reaction for extraction of thrombin.

    Wang, Zheng; Zhao, Jin-cheng; Lian, Hong-zhen; Chen, Hong-yuan


    A novel strategy for preparing aptamer-based organic-silica hybrid monolithic column was developed via "thiol-ene" click chemistry. Due to the large specific surface area of the hybrid matrix and the simplicity, rapidness and high efficiency of "thiol-ene" click reaction, the average coverage density of aptamer on the organic-silica hybrid monolith reached 420 pmol μL(-1). Human α-thrombin can be captured on the prepared affinity monolithic column with high specificity and eluted by NaClO4 solution. N-p-tosyl-Gly-Pro-Arg p-nitroanilide acetate was used as the sensitive chromogenic substrate of thrombin. The thrombin enriched by this affinity column was detected with a detection of limit of 0.01 μM by spectrophotometry. Furthermore, the extraction recovery of thrombin at 0.15 μM in human serum was 91.8% with a relative standard deviation of 4.0%. These results indicated that "thiol-ene" click chemistry provided a promising technique to immobilize aptamer on organic-inorganic hybrid monolith and the easily-assembled affinity monolithic material could be used to realize highly selective recognition of trace proteins.

  2. Synthesis of the Hemoglobin-Conjugated Polymer Micelles by Thiol Michael Addition Reactions.

    Qi, Yanxin; Li, Taihang; Wang, Yupeng; Wei, Xing; Li, Bin; Chen, Xuesi; Xie, Zhigang; Jing, Xiabin; Huang, Yubin


    Amphiphilic triblock copolymers mPEG-b-PMAC-b-PCL are synthesized using methoxyl poly(ethylene glycol), cyclic carbonic ester monomer including acryloyl group, and ε-caprolactone. Copolymers are self-assembled into core-shell micelles in aqueous solution. Thiolated hemoglobin (Hb) is conjugated with micelles sufficiently through thiol Michael addition reaction to form hemoglobin nanoparticles (HbNs) with 200 nm in diameter. The conjugation of Hb onto the micelle surface is further confirmed by X-ray photoelectron spectroscopy. Feeding ratio of copolymer micelles to Hb at 1:3 would lead to the highest hemoglobin loading efficiency 36.7 wt%. The UV results demonstrate that the gas transporting capacity of HbNs is well remained after Hb is conjugated with polymeric micelles. Furthermore, the obtained HbNs have no obvious detrimental effects on blood components in vitro. This system may thus have great potential as one of the candidates to be developed as oxygen carriers and provide a reference for the modification of protein drugs.

  3. Effect of thiol derivatives on mixed mucus and blood clots in vitro.

    Risack, L E; Vandevelde, M E; Gobert, J G


    The disintegrating effect of three reducing thiol derivatives: [sodium mercaptoethane sulphonate (Mesna), N-acetyl-L-cysteine (NAC) and dithio-1,4-threitol (DTT)] was investigated in vitro upon blood clots formed in the absence or in the presence of tracheobronchial secretions and compared with the effect of iso-osmotic saline solution. The amounts of haemoglobin released from the clots after 30 min incubation and the initial rates of haemoglobin release were compared for the different products at different concentrations. All three reducing agents showed some ability to disintegrate mixed clots to an extent depending on their concentration. After 30 min incubation, statistical analysis showed a highly significant difference in favour of Mesna at the three concentrations used, i.e. 0.1, 1.0 and 10 mmol/1. The initial rate of haemoglobin release in presence of Mesna was at all concentrations significantly higher than that of NAC or DTT. The effects on normal blood clots were much less pronounced. The effectiveness of Mesna in splitting up mixed blood and mucus clots in the management of patients who had inhaled blood is discussed.

  4. The thiol compounds glutathione and homoglutathione differentially affect cell development in alfalfa (Medicago sativa L.).

    Pasternak, Taras; Asard, Han; Potters, Geert; Jansen, Marcel A K


    Glutathione (GSH) is an important scavenger of Reactive Oxygen Species (ROS), precursor of metal chelating phytochelatins, xenobiotic defence compound and regulator of cell proliferation. Homoglutathione (hGSH) is a GSH homologue that is present in several taxa in the family of Fabaceae. It is thought that hGSH performs many of the stress-defence roles typically ascribed to GSH, yet little is known about the potential involvement of hGSH in controlling cell proliferation. Here we show that hGSH/GSH ratios vary across organs and cells and that these changes in hGSH/GSH ratio occur during dedifferentiation and/or cell cycle activation events. The use of a GSH/hGSH biosynthesis inhibitor resulted in impaired cytokinesis in isolated protoplasts, showing the critical importance of these thiol-compounds for cell division. However, exposure of isolated protoplasts to exogenous GSH accelerated cytokinesis, while exogenous hGSH was found to inhibit the same process. We conclude that GSH and hGSH have distinct functional roles in cell cycle regulation in Medicago sativa L. GSH is associated with meristemic cells, and promotes cell cycle activation and induction of somatic embryogenesis, while hGSH is associated with differentiated cells and embryo proliferation.

  5. Modulation of electrochemical hydrogen evolution rate by araliphatic thiol monolayers on gold

    Muglali, Mutlu I.; Erbe, Andreas; Chen, Ying; Barth, Christoph; Koelsch, Patrick; Rohwerder, Michael


    Electroreductive desorption of a highly ordered self-assembled monolayer (SAM) formed by the araliphatic thiol (4-(4-(4-pyridyl)phenyl)phenyl)methanethiol leads to a concurrent rapid hydrogen evolution reaction (HER). The desorption process and resulting interfacial structure were investigated by voltammetric techniques, in situ spectroscopic ellipsometry, and in situ vibrational sum–frequency–generation (SFG) spectroscopy. Voltammetric experiments on SAM-modified electrodes exhibit extraordinarily high peak currents, which di er between Au(111) and polycrystalline Au substrates. Association of reductive desorption with HER is shown to be the origin of the observed excess cathodic charges. The studied SAM preserves its two–dimensional order near Au surface throughout a fast voltammetric scan even when the vertex potential is set several hundred millivolt beyond the desorption potential. A model is developed for the explanation of the observed rapid HER involving ordering and pre–orientation of water present in the nanometer–sized reaction volume between desorbed SAM and the Au electrode, by the structurally extremely stable monolayer, leading to the observed catalysis of the HER. PMID:24235778

  6. Modulation of electrochemical hydrogen evolution rate by araliphatic thiol monolayers on gold.

    Muglali, Mutlu I; Erbe, Andreas; Chen, Ying; Barth, Christoph; Koelsch, Patrick; Rohwerder, Michael


    Electroreductive desorption of a highly ordered self-assembled monolayer (SAM) formed by the araliphatic thiol (4-(4-(4-pyridyl)phenyl)phenyl)methanethiol leads to a concurrent rapid hydrogen evolution reaction (HER). The desorption process and resulting interfacial structure were investigated by voltammetric techniques, in situ spectroscopic ellipsometry, and in situ vibrational sum-frequency-generation (SFG) spectroscopy. Voltammetric experiments on SAM-modified electrodes exhibit extraordinarily high peak currents, which di er between Au(111) and polycrystalline Au substrates. Association of reductive desorption with HER is shown to be the origin of the observed excess cathodic charges. The studied SAM preserves its two-dimensional order near Au surface throughout a fast voltammetric scan even when the vertex potential is set several hundred millivolt beyond the desorption potential. A model is developed for the explanation of the observed rapid HER involving ordering and pre-orientation of water present in the nanometer-sized reaction volume between desorbed SAM and the Au electrode, by the structurally extremely stable monolayer, leading to the observed catalysis of the HER.

  7. Simple thiol-ene click chemistry modification of SBA-15 silica pores with carboxylic acids.

    Bordoni, Andrea V; Lombardo, M Verónica; Regazzoni, Alberto E; Soler-Illia, Galo J A A; Wolosiuk, Alejandro


    A straightforward approach for anchoring tailored carboxylic groups in mesoporous SiO2 colloidal materials is presented. The thiol-ene photochemical reaction between vinyltrimethoxysilane precursors and various thiocarboxylic acids which has, click chemistry features (i.e. high conversion yields, insensitivity to oxygen, mild reaction conditions), results in carboxylated silane precursors that can be readily used as surface modifiers. The carboxylic groups of acetic, undecanoic and succinic acid were immobilized on the silica mesopore walls of SBA-15 powders employing the synthesized silane precursors. Post-grafting has been confirmed through infrared spectrometry (FTIR), energy dispersive X-ray spectroscopy (EDS), elemental analysis (EA) and zeta potential measurements. Detailed field-emission gun scanning electron microscopy (FESEM) images and small angle X-ray scattering (SAXS) data revealed parallel mesopores and ordered mesostructures. It is shown that the immobilized COOH groups are chemically accessible for acid-base reactions as well as copper adsorption. Immobilization of easily synthesized tailored carboxylic modified alkoxide precursors within mesoporous systems provides a unique chemical nanoenvironment within these ordered frameworks.

  8. Cystatin like thiol proteinase inhibitor from pancreas of Capra hircus: purification and detailed biochemical characterization.

    Priyadarshini, Medha; Bano, Bilqees


    A thiol proteinase inhibitor from Capra hircus (goat) pancreas (PTPI) isolated by ammonium sulphate precipitation (20-80%) and gel filtration chromatography on Sephacryl S-100HR, with 20.4% yield and 500-fold purification, gave molecular mass of 44 kDa determined by its electrophoretic and gel filtration behavior, respectively. The stokes radius, diffusion and sedimentation coefficients of PTPI were 27.3 A, 7.87 x 10(-7) cm(2) s(-1) and 3.83 s, respectively. It was stable in pH range 3-10 and up to 70 degrees C (critical temperature, E (a) = 21 kJ mol(-1)). Kinetic analysis revealed reversible and competitive mode of inhibition with PTPI showing the highest inhibitory efficiency against papain (K ( i ) = 5.88 nM). The partial amino acid sequence analysis showed that it shared good homology with bovine parotid and skin cystatin C. PTPI possessed 17.18% alpha helical content assessed by CD spectroscopy. The hydropathy plot of first 24 residues suggested that most amino acids of this stretch might be in the hydrophobic core of the protein.

  9. Site-dependent atomic and molecular affinities of hydrocarbons, amines and thiols on diamond nanoparticles

    Lai, Lin; Barnard, Amanda S.


    Like many of the useful nanomaterials being produced on the industrial scale, the surface of diamond nanoparticles includes a complicated mixture of various atomic and molecular adsorbates, attaching to the facets following synthesis. Some of these adsorbates may be functional, and adsorption is encouraged to promote applications in biotechnology and nanomedicine, but others are purely adventurous and must be removed prior to use. In order to devise more effective treatments it is advantageous to know the relative strength of the interactions of the adsorbates with the surface, and ideally how abundant they are likely to be under different conditions. In this paper we use a series of explicit electronic structure simulations to map the distribution of small hydrocarbons, amines and thiols on a 2.9 nm diamond nanoparticle, with atomic level resolution, in 3-D. We find a clear relationship between surface reconstructions, facet orientation, and the distribution of the different adsorbates; with a greater concentration expected on the (100) and (110) facets, particularly when the supersaturation in the reservoir is high. Adsorption on the (111) facets is highly unlikely, suggesting that controlled graphitization may be a useful stage in the cleaning and treatment of nanodiamonds, prior to the deliberate coating with functional adsorbates needed for drug delivery applications.

  10. Structural modifications of gold thin films produced by thiol-derivatized single-stranded DNA immobilization.

    Arroyo-Hernández, María; Svec, Martin; Rogero, Celia; Briones, Carlos; Martín-Gago, José Angel; Costa-Krämer, José Luis


    Recent experiments have reported an opposite sign of the differential surface stress produced on gold-coated cantilevers by a thiol-derivatized single-stranded DNA (SH-DNA) immobilization process. The sign of the surface stress depends on the method used to evaporate the gold thin film, being compressive (negative) or tensile (positive) for e-beam or resistively deposited gold, respectively. This study investigates the origin of this effect by means of a combination of x-ray diffraction and x-ray photoelectron spectroscopy. Both e-beam and resistively grown gold thin films are characterized to find the subtle differences responsible for this intriguing stress behaviour. Somewhat remarkably, these studies show a tight relation between the surface structure of the gold overlayer and the SH-DNA immobilization efficiency. The average grain size variation seems to correlate well with the differential surface stress triggered by the SH-DNA immobilization previously reported. These results suggest that the relation of the probe molecules with the surface structure must be considered to understand surface stress changes.

  11. Effects of hydrogen peroxide treatment on thiol contents in fresh-cut asparagus (Asparagus officinalis) spears.

    Demrkol, Omca


    In this work, the impact of hydrogen peroxide (H2O2) was investigated on the thiol content of asparagus. Fresh-cut asparagus was treated with H2O2 at varied oxidant concentrations and contact times. A significant decrease (alpha=0.05) was observed in N-acetylcysteine levels treated with 2.5% H2O2 for 10 min and with 5% H2O2 for 3, 5 and 10 min. Captopril and cysteine levels significantly decreased (alpha=0.05) in all and most treatment conditions, respectively. Glutathione levels only significantly decreased with 2.5% and 5% H2O2 for 10 min treatment. In order to determine whether asparagus undergoes oxidative stress, a well-known oxidative stress indicator-the glutathione/oxidized glutathione ratio-was calculated. This study showed that the common use of H2O2 as a disinfectant/sterilizer by the food industry could markedly diminish the important biothiols and develop oxidative stress in asparagus, and potentially in other vegetables as well.

  12. Immunological role of thiol-dependent peroxiredoxin gene in Macrobrachium rosenbergii.

    Arockiaraj, Jesu; Easwvaran, Sarasvathi; Vanaraja, Puganeshwaran; Singh, Arun; Othman, Rofina Yasmin; Bhassu, Subha


    In this study, we have reported a full length of peroxiredoxin (designated MrPrdx) gene, identified from the transcriptome of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrPrdx is 940 base pairs in length, and encodes 186 amino acids. MrPrdx contains a long thioredoxin domain in the amino acid sequence between 34 and 186. The gene expressions of MrPrdx in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction. MrPrdx is highly expressed in all the other tissues of M. rosenbergii considered for analysis and the highest in gills. The expression is strongly up-regulated in gills after IHHNV infection. To understand MrPrdx functional properties, the recombinant MrPrdx protein was expressed in Escherichia coli BL21 (DE3) and purified. A peroxidise activity assay was conducted using recombinant MrPrdx protein at different concentrations. This peroxidises activity showed that the recombinant MrPrdx is a thiol-dependant protein. Additionally, this result showed that recombinant MrPrdx protein, as a secretory protein can remove H₂O₂ and protect DNA damage. This finding leads a possible way to propose the recombinant MrPrdx protein as an effective medicine for reactive oxygen species (ROS) related diseases.

  13. Analysis of Structural Flexibility of Damaged DNA Using Thiol-Tethered Oligonucleotide Duplexes.

    Masashi Fujita

    Full Text Available Bent structures are formed in DNA by the binding of small molecules or proteins. We developed a chemical method to detect bent DNA structures. Oligonucleotide duplexes in which two mercaptoalkyl groups were attached to the positions facing each other across the major groove were prepared. When the duplex contained the cisplatin adduct, which was proved to induce static helix bending, interstrand disulfide bond formation under an oxygen atmosphere was detected by HPLC analyses, but not in the non-adducted duplex, when the two thiol-tethered nucleosides were separated by six base pairs. When the insert was five and seven base pairs, the disulfide bond was formed and was not formed, respectively, regardless of the cisplatin adduct formation. The same reaction was observed in the duplexes containing an abasic site analog and the (6–4 photoproduct. Compared with the cisplatin case, the disulfide bond formation was slower in these duplexes, but the reaction rate was nearly independent of the linker length. These results indicate that dynamic structural changes of the abasic site- and (6–4 photoproduct-containing duplexes could be detected by our method. It is strongly suggested that the UV-damaged DNA-binding protein, which specifically binds these duplexes and functions at the first step of global-genome nucleotide excision repair, recognizes the easily bendable nature of damaged DNA.

  14. Enhanced adsorption of mercury ions on thiol derivatized single wall carbon nanotubes.

    Bandaru, Narasimha Murthy; Reta, Nekane; Dalal, Habibullah; Ellis, Amanda V; Shapter, Joseph; Voelcker, Nicolas H


    Thiol-derivatized single walled carbon nanotube (SWCNT-SH) powders were synthesized by reacting acid-cut SWCNTs with cysteamine hydrochloride using carbodiimide coupling. Infrared (IR) spectroscopy, Raman spectroscopy and thermogravimetric analysis confirmed the successful functionalization of the SWCNTs. SWCNT-SH powders exhibited a threefold higher adsorption capacity for Hg(II) ions compared to pristine SWCNTs, and a fourfold higher adsorption capacity compared to activated carbon. The influence of adsorption time, pH, initial metal concentration and adsorbent dose on Hg(II) ion removal was investigated. The maximum adsorption capacity of the SWCNT-SH powders was estimated by using equilibrium isotherms, such as Freundlich and Langmuir, and the maximum adsorption capacity of the SWCNT-SH powder was found to be 131 mg/g. A first-order rate model was employed to describe the kinetic adsorption process of Hg(II) ions onto the SWCNT-SH powders. Desorption studies revealed that Hg(II) ions could be easily removed from the SWCNT-SH powders by altering the pH. Further, the adsorption efficiency of recovered SWCNT-SH powders was retained up to 91%, even after 5 adsorption/desorption cycles.

  15. From cysteine to longer chain thiols: thermodynamic analysis of cadmium binding by phytochelatins and their fragments.

    Chekmeneva, Elena; Gusmão, Rui; Díaz-Cruz, José Manuel; Ariño, Cristina; Esteban, Miquel


    Isothermal Titration Calorimetry (ITC) was used to study the binding of Cd(2+) by phytochelatins ((γGlu-Cys)(n)-Gly, PC(n); n = 1-5) and their selected fragments (Cys, Cys-Gly and γGlu-Cys) in order to understand the influence of the chain length on the complex stabilities and the origin of the enhanced affinities in Tris buffer at pH 7.5 and 8.5 and at 25 °C. Different complexes are formed with glutathione (GSH) and its fragments, Cys, Cys-Gly and γGlu-Cys, and their stabilities depend on the corresponding pK(a) value of the thiol group in the ligands. The stability of Cd-PC(n) complexes increases moving towards higher PC(2-5), as well as the complexing capacity expressed as the number of metal ions that can be bound by one ligand molecule. The affinity of Cd(2+) for the PC(n) can be described by the following GSH < PC(2) < PC(3)≤ PC(4)≤ PC(5) sequence. On the basis of these thermodynamic data it is possible to explain the abundance of certain Cd-PC(n) complexes found in nature. The comprehension of the thermodynamic rules that govern the interactions of Cd(2+) with PC(n) and their constituents is of great service in the research with real plant samples subjected to metal stress and in the development of new strategies of bio/phytoremediation.

  16. Contribution of cell walls, nonprotein thiols, and organic acids to cadmium resistance in two cabbage varieties.

    Sun, Jianyun; Cui, Jin; Luo, Chunling; Gao, Lu; Chen, Yahua; Shen, Zhenguo


    To study possible cadmium (Cd) resistance mechanisms in cabbage (Brassica oleracea L.), several parameters of metal uptake, distribution, and complexation were compared between two varieties Chunfeng [CF (Cd-tolerant)] and Lvfeng [LF (Cd-sensitive)]. Results showed that CF contained significantly lower Cd concentrations in leaves and higher Cd concentrations in roots than LF. Approximately 70 to 74 % and 66 to 68 % of Cd taken up by LF and CF, respectively, was transported to shoots. More Cd was bound to the cell walls of leaves, stems, and roots in CF than in LF. The higher capacity of CF to limit Cd uptake into shoots could be explained by immobilization of Cd in root cell walls. Compared with control groups, Cd treatment also significantly increased concentrations of nonprotein thiols, phytochelatins (PCs), and citric acid in the leaves and roots of the two varieties; the increases were more pronounced in CF than in LF. Taken together, the results suggest that the greater Cd resistance in CF than in LF may be attributable to the greater capacity of CF to limit Cd uptake into shoots and complex Cd in cell walls and metal binding ligands, such as PCs and citric acid. However, the contributions of PCs and citric acid to Cd detoxification might be smaller than those in cell walls.

  17. Biochemical, immunological and kinetic characterisation of thiol protease inhibitor (cystatin) from liver.

    Shah, Aaliya; Priyadarshini, Medha; Khan, Mohd Shahnawaz; Aatif, Mohammad; Amin, Fakhra; Bano, Bilqees


    Regulation of the cysteine protease activity is imperative for proper functioning of the various organ systems. Elevated activities of cysteine proteinases due to impaired regulation by the endogenous cysteine proteinase inhibitors (cystatins) have been linked to liver malignancies. To gain an insight into these regulatory processes, it is essential to purify and characterise the inhibitors, cystatins. Present study was undertaken to purify the inhibitor from the liver. The purification was accomplished in four steps: alkaline treatment, ammonium sulphate fractionation, acetone precipitation and gel filtration column (Sephacryl S-100 HR). The eluted protein exhibited inhibitory activity towards papain, and its purity was further reaffirmed using western blotting and immunodiffusion. The purified inhibitor (liver cystatin (LC)) was stable in the pH range of 6-8 and temperature up to 45 °C. In view of the significance of kinetics parameters for drug delivery, the kinetic parameters of liver cystatin were also determined. LC showed the greatest affinity for papain followed by ficin and bromelain. UV and fluorescence spectroscopy results showed that binding of LC with thiol proteases induced changes in the environment of aromatic residues. Recent advances in the field of proteinase inhibitors have drawn attention to the possible use of this collected knowledge to control pathologies.

  18. Chemical derivatization for electrospray ionization mass spectrometry. 1. Alkyl halides, alcohols, phenols, thiols, and amines

    Quirke, J.M.E.; Adams, C.L.; Van Berkel, G.J. (Oak Ridge National Lab., TN (United States))


    Derivatization strategies and specific derivatization reactions for conversion of simple alkyl halides, alcohols, phenols, thiols, and amines to ionic or solution-ionizable derivatives, that is [open quotes]electrospray active[close quotes] (ES-active) forms of the analyte, are presented. Use of these reactions allows detection of analytes among those listed that are not normally amenable to analysis by electrospray ionization mass spectrometry (ES-MS). In addition, these reactions provide for analysis specificity and flexibility through functional group specific derivatization and through the formation of derivatives that can be detected in positive ion or in negative ion mode. For a few of the functional groups, amphoteric derivatives are formed that can be analyzed in either positive or negative ion modes. General synthetic strategies for transformation of members of these five compound classes to ES-active species are presented along with illustrative examples of suitable derivatives. Selected derivatives were prepared using model compounds and the ES mass spectra obtained for these derivatives are discussed. The analytical utility of derivatization for ES-MS analysis is illustrated in three experiments: (1) specific detection of the major secondary alcohol in oil of peppermint, (2) selective detection of phenols within a synthetic mixture of phenols, and (3) identification of the medicinal amines within a commercially available cold medication as primary, secondary or tertiary. 65 refs., 3 figs., 3 tabs.

  19. Plasma brominated polymer particles as grafting substrate for thiol-terminated telomers.

    Byström, Emil; Nordborg, Anna; Limé, Fredrik; Dinh, Ngoc Phuoc; Irgum, Knut


    A combined surface activation and "grafting to" strategy was developed to convert divinylbenzene particles into weak cation exchangers suitable for protein separation. The initial activation step was based on plasma modification with bromoform, which rendered the particles amenable to further reaction with nucleophiles by introducing Br to a surface content of 11.2 atom-%, as determined by X-ray photoelectron spectroscopy. Grafting of thiol-terminated glydicyl methacrylate telomers to freshly plasma activated surfaces was accomplished without the use of added initiator, and the grafting was verified both by reduction in bromine content and the appearance of sulfur-carbon linkages, showing that the surface grafts were covalently bonded. Following grafting the attached glydicyl methacrylate telomer tentacles were further modified by a two-step procedure involving hydrolysis to 2,3-hydroxypropyl groups and conversion of hydroxyl groups to carboxylate functionality by succinic anhydride. The final material was capable of baseline separating four model proteins in 3 min by gradient cation exchange chromatography in a fully aqueous eluent.

  20. Gold electrode modified by self-assembled monolayers of thiols to determine DNA sequences hybridization

    Mízia M S Silva; Igor T Cavalcanti; M Fátima Barroso; M Goreti F Sales; Rosa Fireman Dutra


    The process of immobilization of biological molecules is one of the most important steps in the construction of a biosensor. In the case of DNA, the way it exposes its bases can result in electrochemical signals to acceptable levels. The use of self-assembled monolayer that allows a connection to the gold thiol group and DNA binding to an aldehydic ligand resulted in the possibility of determining DNA hybridization. Immobilized single strand of DNA (ssDNA) from calf thymus pre-formed from alkanethiol film was formed by incubating a solution of 2-aminoethanothiol (Cys) followed by glutaraldehyde (Glu). Cyclic voltammetry (CV) was used to characterize the self-assembled monolayer on the gold electrode and, also, to study the immobilization of ssDNA probe and hybridization with the complementary sequence (target ssDNA). The ssDNA probe presents a well-defined oxidation peak at +0.158 V. When the hybridization occurs, this peak disappears which confirms the efficacy of the annealing and the DNA double helix performing without the presence of electroactive indicators. The use of SAM resulted in a stable immobilization of the ssDNA probe, enabling the hybridization detection without labels. This study represents a promising approach for molecular biosensor with sensible and reproducible results.

  1. Thiol redox requirements and substrate specificities of recombinant cytochrome c assembly systems II and III.

    Richard-Fogal, Cynthia L; San Francisco, Brian; Frawley, Elaine R; Kranz, Robert G


    The reconstitution of biosynthetic pathways from heterologous hosts can help define the minimal genetic requirements for pathway function and facilitate detailed mechanistic studies. Each of the three pathways for the assembly of cytochrome c in nature (called systems I, II, and III) has been shown to function recombinantly in Escherichia coli, covalently attaching heme to the cysteine residues of a CXXCH motif of a c-type cytochrome. However, recombinant systems I (CcmABCDEFGH) and II (CcsBA) function in the E. coli periplasm, while recombinant system III (CCHL) attaches heme to its cognate receptor in the cytoplasm of E. coli, which makes direct comparisons between the three systems difficult. Here we show that the human CCHL (with a secretion signal) attaches heme to the human cytochrome c (with a signal sequence) in the E. coli periplasm, which is bioenergetically (p-side) analogous to the mitochondrial intermembrane space. The human CCHL is specific for the human cytochrome c, whereas recombinant system II can attach heme to multiple non-cognate c-type cytochromes (possessing the CXXCH motif.) We also show that the recombinant periplasmic systems II and III use components of the natural E. coli periplasmic DsbC/DsbD thiol-reduction pathway. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

  2. Mitochondrial Sulfide Detoxification Requires a Functional Isoform O-Acetylserine(thiol)lyase C in Arabidopsis thaliana

    Consolación (A)lvarez; Irene García; Luis C.Romero; Cecilia Gotor


    In non-cyanogenic species,the main source of cyanide derives from ethylene and camalexin biosyntheses.In mitochondria,cyanide is a potent inhibitor of the cytochrome c oxidase and is metabolized bythe β-cyanoalanine synthase CYS-C1,catalyzing the conversion of cysteine and cyanide to hydrogen sulfide and β-cyanoalanine.The hydrogen sulfide released also inhibits the cytochrome c oxidase and needs to be detoxified by the O-acetylserine(thiol)lyase mitochondrial isoform,OAS-C,which catalyzes the incorporation of sulfide to O-acetylserine to produce cysteine,thus generating a cyclic pathway in the mitochondria.The loss of functional OAS-C isoforms causes phenotypic characteristics very similar to the loss of the CYS-C1 enzyme,showing defects in root hair formation.Genetic complementation with the OAS-C gene rescues the impairment of root hair elongation,restoring the wild-type phenotype.The mitochondria compromise their capacity to properly detoxify cyanide and the resulting sulfide because the latter cannot re-assimilate into cysteine in the oas-c null mutant.Consequently,we observe an accumulation of sulfide and cyanide and of the alternative oxidase,which is unable to prevent the production of reactive oxygen species probably due to the accumulation of both toxic molecules.Our results allow us to suggest that the significance of OAS-C is related to its role in the proper sulfide and cyanide detoxification in mitochondria.

  3. Toxicity induced by cumene hydroperoxide in PC12 cells: protective role of thiol donors.

    Vimard, F; Saucet, M; Nicole, O; Feuilloley, M; Duval, D


    Oxidative shock and production of reactive oxygen species are known to play a major role in situations leading to neuron degeneration, but the precise mechanisms responsible for cell degeneration remain uncertain. In the present article, we have studied in PC 12 cells the effect of cumene hydroxyperoxide on both cell metabolism and morphology. We observed that relatively low concentrations of the drug (100 μM) led to a significant decrease in the cellular content of ATP and reduced glutathione as well as to a decreased mitochondrial potential. These metabolic alterations were followed by an important increase in intracellular free calcium and membrane disruption and death. In parallel, we observed profound changes in cell morphology with a shortening of cell extensions, the formation of ruffles and blebs at the cell surface, and a progressive detachment of the cells from the surface of the culture flasks. We also showed that addition of thiol donors such as N-acetylcysteine or β-mercaptoethanol, which were able to enhance cell glutathione content, almost completely protected PC 12 cells from the toxic action of cumene hydroperoxide whereas pretreatment by buthionine sulfoximine, a selective inhibitor of GSH synthesis, enhanced its action.

  4. Photolithographic fabrication of solid-liquid core waveguides by thiol-ene chemistry

    Sagar, Kaushal; Gopalakrishnan, Nimi; Brøkner Christiansen, Mads; Kristensen, Anders; Ndoni, Sokol


    In this work we demonstrate an efficient and cleanroom compatible method for the fabrication of solid-liquid core waveguides based on nanoporous polymers. We have used thiol-ene photo-grafting to tune and pattern the hydrophilicity of an originally hydrophobic nanoporous 1, 2-polybutadiene. The generated refractive index contrast between the patterned water-filled volume and the surrounding empty hydrophobic porous polymer allows for light confinement within the water-filled volume—the solid-liquid core. The presented fabrication process is simple and fast. It allows a high degree of flexibility on the type and grade of surface chemistry imparted to the large nanoporous area depending upon the application. The fabrication does not need demanding chemical reaction conditions. Thus, it can be readily used on a standard silicon lithography bench. The propagation loss values reported in this work are comparable with literature values for state-of-the-art liquid-core waveguide devices. The demonstrated waveguide function added to the nanoporous polymer with a very high internal surface area makes the system interesting for many applications in different areas, such as diagnostics and bio-chemical sensing.

  5. Automated ARGET ATRP Accelerates Catalyst Optimization for the Synthesis of Thiol-Functionalized Polymers.

    Siegwart, Daniel J; Leiendecker, Matthias; Langer, Robert; Anderson, Daniel G


    Conventional synthesis of polymers by ATRP is relatively low throughput, involving iterative optimization of conditions in an inert atmosphere. Automated, high-throughput controlled radical polymerization was developed to accelerate catalyst optimization and production of disulfide-functionalized polymers without the need of an inert gas. Using ARGET ATRP, polymerization conditions were rapidly identified for eight different monomers, including the first ARGET ATRP of 2-(diethylamino)ethyl methacrylate and di(ethylene glycol) methyl ether methacrylate. In addition, butyl acrylate, oligo(ethylene glycol) methacrylate 300 and 475, 2-(dimethylamino)ethyl methacrylate, styrene, and methyl methacrylate were polymerized using bis(2-hydroxyethyl) disulfide bis(2-bromo-2-methylpropionate) as the initiator, tris(2-pyridylmethyl)amine as the ligand, and tin(II) 2-ethylhexanoate as the reducing agent. The catalyst and reducing agent concentration was optimized specifically for each monomer, and then a library of polymers was synthesized systematically using the optimized conditions. The disulfide-functionalized chains could be cleaved to two thiol-terminated chains upon exposure to dithiothreitol, which may have utility for the synthesis of polymer bioconjugates. Finally, we demonstrated that these new conditions translated perfectly to conventional batch polymerization. We believe the methods developed here may prove generally useful to accelerate the systematic optimization of a variety of chemical reactions and polymerizations.

  6. Methylenetetrahydrofolate reductase gene polymorphism in Indian stroke patients

    Kalita J


    Full Text Available Background and Aims: In view of the prevailing controversy about the role of Methylenetetrahydrofolate reductase (MTHFR C677T mutation in stroke and paucity of studies from India, this study has been undertaken to evaluate MTHFR C677T gene polymorphism in consecutive ischemic stroke patients and correlate these with folic acid, homocysteine (Hcy and conventional risk factors. Settings and Design: Ischemic stroke patients prospectively evaluated in a tertiary care teaching hospital. Materials and Methods: Computerized tomography proven ischemic stroke patients were prospectively evaluated including clinical, family history of stroke, dietary habits and addictions. Their fasting and postprandial blood sugar, lipid profile, vitamin B12, folic acid and MTHFR gene analysis were done. Statistical Analysis: MTHFR gene polymorphism was correlated with serum folic acid, Vitamin B12 and Hcy levels; family history of stroke in first-degree relatives; and dietary habits; employing Chi-square test. Results: There were 58 patients with ischemic stroke, whose mean age was 50 (4-79 years; among them, 10 were females. MTHFR gene polymorphism was present in 19 (32.8% patients, 3 were homozygous and 16 were heterozygous. Both serum folate and B12 levels were low in 29 (50% patients and Hcy in 48 (83%. Hypertension was present in 28 (48% patients, diabetes in 12 (21%, hyperlipidemia in 52 (90%, smoking in 17 (29%, obesity in 1 (1.7% and family history of stroke in first-degree relatives in 13 (22.4%. There was no significant relationship of MTHFR gene polymorphism with folic acid, B12, Hcy levels, dietary habits and number of risk factors. Vitamin B12 level was low in vegetarians ( P Conclusion: MTHFR gene polymorphism was found in one-third of patients with ischemic stroke and was insignificantly associated with higher frequency of elevated Hcy.

  7. Rational Design of a Structural and Functional Nitric Oxide Reductase

    Yeung, N.; Lin, Y; Gao, Y; Zhao, X; Russell, B; Lei, L; Miner, L; Robinson, H; Lu, Y


    Protein design provides a rigorous test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. Whereas progress has been made in designing proteins that mimic native proteins structurally, it is more difficult to design functional proteins. In comparison to recent successes in designing non-metalloproteins, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes. This is because protein metal-binding sites are much more varied than non-metal-containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal-binding site properties in silico, as many of the parameters, such as force fields, are ill-defined. Therefore, the successful design of a structural and functional metalloprotein would greatly advance the field of protein design and our understanding of enzymes. Here we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a haem/non-haem FeB centre that is remarkably similar to that in the crystal structure. This designed protein also exhibits NO reduction activity, and so models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.

  8. Aldo-Keto Reductases 1B in Endocrinology and Metabolism.

    Pastel, Emilie; Pointud, Jean-Christophe; Volat, Fanny; Martinez, Antoine; Lefrançois-Martinez, Anne-Marie


    The aldose reductase (AR; human AKR1B1/mouse Akr1b3) has been the focus of many research because of its role in diabetic complications. The starting point of these alterations is the massive entry of glucose in polyol pathway where it is converted into sorbitol by this enzyme. However, the issue of AR function in non-diabetic condition remains unresolved. AR-like enzymes (AKR1B10, Akr1b7, and Akr1b8) are highly related isoforms often co-expressed with bona fide AR, making functional analysis of one or the other isoform a challenging task. AKR1B/Akr1b members share at least 65% protein identity and the general ability to reduce many redundant substrates such as aldehydes provided from lipid peroxidation, steroids and their by-products, and xenobiotics in vitro. Based on these properties, AKR1B/Akr1b are generally considered as detoxifying enzymes. Considering that divergences should be more informative than similarities to help understanding their physiological functions, we chose to review specific hallmarks of each human/mouse isoforms by focusing on tissue distribution and specific mechanisms of gene regulation. Indeed, although the AR shows ubiquitous expression, AR-like proteins exhibit tissue-specific patterns of expression. We focused on three organs where certain isoforms are enriched, the adrenal gland, enterohepatic, and adipose tissues and tried to connect recent enzymatic and regulation data with endocrine and metabolic functions of these organs. We presented recent mouse models showing unsuspected physiological functions in the regulation of glucido-lipidic metabolism and adipose tissue homeostasis. Beyond the widely accepted idea that AKR1B/Akr1b are detoxification enzymes, these recent reports provide growing evidences that they are able to modify or generate signal molecules. This conceptually shifts this class of enzymes from unenviable status of scavenger to upper class of messengers.

  9. Prokaryotic arsenate reductase enhances arsenate resistance in Mammalian cells.

    Wu, Dan; Tao, Xuanyu; Wu, Gaofeng; Li, Xiangkai; Liu, Pu


    Arsenic is a well-known heavy metal toxicant in the environment. Bioremediation of heavy metals has been proposed as a low-cost and eco-friendly method. This article described some of recent patents on transgenic plants with enhanced heavy metal resistance. Further, to test whether genetic modification of mammalian cells could render higher arsenic resistance, a prokaryotic arsenic reductase gene arsC was transfected into human liver cancer cell HepG2. In the stably transfected cells, the expression level of arsC gene was determined by quantitative real-time PCR. Results showed that arsC was expressed in HepG2 cells and the expression was upregulated by 3 folds upon arsenate induction. To further test whether arsC has function in HepG2 cells, the viability of HepG2-pCI-ArsC cells exposed to arsenite or arsenate was compared to that of HepG2-pCI cells without arsC gene. The results indicated that arsC increased the viability of HepG2 cells by 25% in arsenate, but not in arsenite. And the test of reducing ability of stably transfected cells revealed that the concentration of accumulated trivalent arsenic increased by 25% in HepG2-pCI-ArsC cells. To determine the intracellular localization of ArsC, a fusion vector with fluorescent marker pEGFP-N1-ArsC was constructed and transfected into.HepG2. Laser confocal microscopy showed that EGFP-ArsC fusion protein was distributed throughout the cells. Taken together, these results demonstrated that prokaryotic arsenic resistant gene arsC integrated successfully into HepG2 genome and enhanced arsenate resistance of HepG2, which brought new insights of arsenic detoxification in mammalian cells.

  10. Short-chain dehydrogenases/reductases in cyanobacteria.

    Kramm, Anneke; Kisiela, Michael; Schulz, Rüdiger; Maser, Edmund


    The short-chain dehydrogenases/reductases (SDRs) represent a large superfamily of enzymes, most of which are NAD(H)-dependent or NADP(H)-dependent oxidoreductases. They display a wide substrate spectrum, including steroids, alcohols, sugars, aromatic compounds, and xenobiotics. On the basis of characteristic sequence motifs, the SDRs are subdivided into two main (classical and extended) and three smaller (divergent, intermediate, and complex) families. Despite low residue identities in pairwise comparisons, the three-dimensional structure among the SDRs is conserved and shows a typical Rossmann fold. Here, we used a bioinformatics approach to determine whether and which SDRs are present in cyanobacteria, microorganisms that played an important role in our ecosystem as the first oxygen producers. Cyanobacterial SDRs could indeed be identified, and were clustered according to the SDR classification system. Furthermore, because of the early availability of its genome sequence and the easy application of transformation methods, Synechocystis sp. PCC 6803, one of the most important cyanobacterial strains, was chosen as the model organism for this phylum. Synechocystis sp. SDRs were further analysed with bioinformatics tools, such as hidden Markov models (HMMs). It became evident that several cyanobacterial SDRs show remarkable sequence identities with SDRs in other organisms. These so-called 'homologous' proteins exist in plants, model organisms such as Drosophila melanogaster and Caenorhabditis  elegans, and even in humans. As sequence identities of up to 60% were found between Synechocystis and humans, it was concluded that SDRs seemed to have been well conserved during evolution, even after dramatic terrestrial changes such as the conversion of the early reducing atmosphere to an oxidizing one by cyanobacteria.

  11. Increased 5. cap alpha. -reductase activity in idiopathic hirsutism

    Serafini, P.; Lobo, R.A.


    In vitro, genital skin 5..cap alpha..-reductase activity (5..cap alpha..-RA) was measured in ten hirsute women with normal androgen levels (idiopathic hirsutism (IH)) and in ten hirsute women with elevated androgen levels (polycystic ovary syndrome (PCO)) in order to determine the influence of secreted androgens on 5..cap alpha..-RA. In vitro 5..cap alpha..-RA was assessed by incubations of skin with /sup 14/C-testosterone (T) for 2 hours, after which steroids were separated and the radioactivity of dihydrotestosterone (DHT) and 5..cap alpha..-androstane 3..cap alpha..-17..beta..-estradiol (3..cap alpha..-diol) in specific eluates were determined. All androgens were normal in IH with the exception of higher levels of 3..cap alpha..-diol glucuronide which were similar to the levels of PCO. The conversion ratio (CR) of T to DHT in IH and PCO were similar, yet significantly greater than the CR of control subjects. The CR of T to 3..cap alpha..-diol in IH and PCO were similar, yet higher than in control subjects. Serum androgens showed no correlation with 5..cap alpha..-RA, while the CR of T to DHT showed a significant positive correlation with the Ferriman and Gallwey score. The increased 5..cap alpha..-RA in IH appears to be independent of serum androgen levels and is, therefore, an inherent abnormality. The term idiopathic is a misnomer, because hirsutism in these patients may be explained on the basis of increased skin 5..cap alpha..-RA.

  12. Metabolism of bupropion by carbonyl reductases in liver and intestine.

    Connarn, Jamie N; Zhang, Xinyuan; Babiskin, Andrew; Sun, Duxin


    Bupropion's metabolism and the formation of hydroxybupropion in the liver by cytochrome P450 2B6 (CYP2B6) has been extensively studied; however, the metabolism and formation of erythro/threohydrobupropion in the liver and intestine by carbonyl reductases (CR) has not been well characterized. The purpose of this investigation was to compare the relative contribution of the two metabolism pathways of bupropion (by CYP2B6 and CR) in the subcellular fractions of liver and intestine and to identify the CRs responsible for erythro/threohydrobupropion formation in the liver and the intestine. The results showed that the liver microsome generated the highest amount of hydroxybupropion (Vmax = 131 pmol/min per milligram, Km = 87 μM). In addition, liver microsome and S9 fractions formed similar levels of threohydrobupropion by CR (Vmax = 98-99 pmol/min per milligram and Km = 186-265 μM). Interestingly, the liver has similar capability to form hydroxybupropion (by CYP2B6) and threohydrobupropion (by CR). In contrast, none of the intestinal fractions generate hydroxybupropion, suggesting that the intestine does not have CYP2B6 available for metabolism of bupropion. However, intestinal S9 fraction formed threohydrobupropion to the extent of 25% of the amount of threohydrobupropion formed by liver S9 fraction. Enzyme inhibition and Western blots identified that 11β-dehydrogenase isozyme 1 in the liver microsome fraction is mainly responsible for the formation of threohydrobupropion, and in the intestine AKR7 may be responsible for the same metabolite formation. These quantitative comparisons of bupropion metabolism by CR in the liver and intestine may provide new insight into its efficacy and side effects with respect to these metabolites.

  13. PEG hydrogels formed by thiol-ene photo-click chemistry and their effect on the formation and recovery of insulin-secreting cell spheroids.

    Lin, Chien-Chi; Raza, Asad; Shih, Han


    Hydrogels provide three-dimensional frameworks with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. While recent research efforts have created diverse macromer chemistry to form hydrogels, the mechanisms of hydrogel polymerization for in situ cell encapsulation remain limited. Hydrogels prepared from chain-growth photopolymerization of poly(ethylene glycol) diacrylate (PEGDA) are commonly used to encapsulate cells. However, free radical associated cell damage poses significant limitation for this gel platform. More recently, PEG hydrogels formed by thiol-ene photo-click chemistry have been developed for cell encapsulation. While both chain-growth and step-growth photopolymerizations offer spatial-temporal control over polymerization kinetics, step-growth thiol-ene hydrogels offer more diverse and preferential properties. Here, we report the superior properties of step-growth thiol-ene click hydrogels, including cytocompatibility of the reactions, improved hydrogel physical properties, and the ability for 3D culture of pancreatic β-cells. Cells encapsulated in thiol-ene hydrogels formed spherical clusters naturally and were retrieved via rapid chymotrypsin-mediated gel erosion. The recovered cell spheroids released insulin in response to glucose treatment, demonstrating the cytocompatibility of thiol-ene hydrogels and the enzymatic mechanism of cell spheroids recovery. Thiol-ene click reactions provide an attractive means to fabricate PEG hydrogels with superior gel properties for in situ cell encapsulation, as well as to generate and recover 3D cellular structures for regenerative medicine applications.

  14. A novel thiol-affinity micropipette tip method using zinc(II)-cyclen-attached agarose beads for enrichment of cysteine-containing molecules.

    Kusamoto, Hiroshi; Shiba, Akio; Koretake, Norinao; Fujioka, Haruto; Hieda, Yuhzo; Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Koike, Tohru


    Cysteine-containing biomolecules are attractive targets in the study of thiol biology. Here we introduce a novel method for the selective enrichment of thiol-containing molecules using a thiol-capture zinc(II) complex of 1,4,7,10-tetraazacyclododecane (Zn(2+)-cyclen). Recognition of N-acetylcysteine amide by Zn(2+)-cyclen has been studied by potentiometric pH titration, revealing formation of a 1:1 thiolate-bound Zn(2+)-cyclen complex with a large thiolate-affinity constant of 10(6.2)M(-1) at 25°C and I=0.10M (NaCl). The Zn(2+)-bound thiolate anion is unexpectedly stable in aqueous solution at pH 7.8 under atmospheric conditions for a few days. These findings have contributed to the development of a convenient method for separation of thiol compounds by using a micropipette tip. A 200μL micropipette tip containing 10μL of hydrophilic cross-linked agarose beads attached to Zn(2+)-cyclen moieties was prepared. All steps for thiol-affinity separation (binding, washing, and eluting) are conducted using aqueous buffers at room temperature. The entire separation protocol requires less than 15min per sample. We demonstrate practical example separations of cysteine-containing molecules. This micropipette tip method would be used preferentially as an alternative to existing tools for reliable enrichment of thiol-containing molecules.

  15. The Characteristics and Regulatory Mechanisms of Superoxide Generation from eNOS Reductase Domain.

    Hu Peng

    Full Text Available In addition to superoxide (O2.- generation from nitric oxide synthase (NOS oxygenase domain, a new O2.- generation site has been identified in the reductase domain of inducible NOS (iNOS and neuronal NOS (nNOS. Cysteine S-glutathionylation in eNOS reductase domain also induces O2.- generation from eNOS reductase domain. However, the characteristics and regulatory mechanism of the O2.- generation from NOS reductase domain remain unclear. We cloned and purified the wild type bovine eNOS (WT eNOS, a mutant of Serine 1179 replaced with aspartic acid eNOS (S1179D eNOS, which mimics the negative charge caused by phosphorylationand truncated eNOS reductase domain (eNOS RD. Both WT eNOS and S1179D eNOS generated significant amount of O2.- in the absence of BH4 and L-arginine. The capacity of O2.- generation from S1179D eNOS was significantly higher than that of WT eNOS (1.74:1. O2.- generation from both WT eNOS and S1179D eNOS were not completely inhibited by 100nM tetrahydrobiopterin(BH4. This BH4 un-inhibited O2.- generation from eNOS was blocked by 10mM flavoprotein inhibitor, diphenyleneiodonium (DPI. Purified eNOS reductase domain protein confirmed that this BH4 un-inhibited O2.- generation originates at the FMN or FAD/NADPH binding site of eNOS reductase domain. DEPMPO-OOH adduct EPR signals and NADPH consumptions analyses showed that O2.- generation from eNOS reductase domain was regulated by Serine 1179 phosphorylation and DPI, but not by L-arginine, BH4 or calmodulin (CaM. In addition to the heme center of eNOS oxygenase domain, we confirmed another O2.- generation site in the eNOS reductase domain and characterized its regulatory properties.

  16. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase.

    Montalvetti, A; Peña-Díaz, J; Hurtado, R; Ruiz-Pérez, L M; González-Pacanowska, D


    In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control.

  17. Expression of 5α-Reductase Type 2 Gene in Human Testis, Epididymis and Vas Deferens

    刘德瑜; 吴燕婉; 罗宏志; 张桂元


    Objectives To study the expression pattern of 5α-reductase type 2 gene in human malereproductive organsMethods The expression level of 5α-reductase type 2 gene inhuman testis, epididymisand vas deferens tissues was determined by in situ hybridization using Digoxin labeled5α-reductase type 2 cRNA probe.Results The brown granules of hybridizing signals distributed in the cytoplasm ofSertoli and Leydig cells of the testis, the principle cells of epididymis and the epithe-lial cells of vas deferens, but there was no positive signal in the nuclei of above-men-tioned cells. No positive signal was observed in germ cells, basement of the testis,interstium of epididymis and basement, as well as smooth muscle of vas deferens.Conclusion This study confirmed that the 5α-reductase type 2 gene expressed in Ser-toli, Leydig cells of the testis, and the principle cells of epididymis. The expressionpattern of the gene in these cells in human was similar to that of rat and monkey. Thepresence of 5a-reductase type 2 gene in epithelial cells of the vas deferens suggested itmight possess an important physiological role in human reproduction.

  18. Recombinant pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) catalyze opposite enantiospecific conversions.

    Fujita, M; Gang, D R; Davin, L B; Lewis, N G


    Although the heartwood of woody plants represents the main source of fiber and solid wood products, essentially nothing is known about how the biological processes leading to its formation are initiated and regulated. Accordingly, a reverse transcription-polymerase chain reaction-guided cloning strategy was employed to obtain genes encoding pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) as a means to initiate the study of its heartwood formation. (+)-Pinoresinol-(+)-lariciresinol reductase from Forsythia intermedia was used as a template for primer construction for reverse transcription-polymerase chain reaction amplifications, which, when followed by homologous hybridization cloning, resulted in the isolation of two distinct classes of putative pinoresinol-lariciresinol reductase cDNA clones from western red cedar. A representative of each class was expressed as a fusion protein with beta-galactosidase and assayed for enzymatic activity. Using both deuterated and radiolabeled (+/-)-pinoresinols as substrates, it was established that each class of cDNA encoded a pinoresinol-lariciresinol reductase of different (opposite) enantiospecificity. Significantly, the protein from one class converted (+)-pinoresinol into (-)-secoisolariciresinol, whereas the other utilized the opposite (-)-enantiomer to give the corresponding (+)-form. This differential substrate specificity raises important questions about the role of each of these individual reductases in heartwood formation, such as whether they are expressed in different cells/tissues or at different stages during heartwood development.

  19. Cloning of thioredoxin h reductase and characterization of the thioredoxin reductase-thioredoxin h system from wheat.

    Serrato, Antonio J; Pérez-Ruiz, Juan M; Cejudo, Francisco J


    Thioredoxins h are ubiquitous proteins reduced by NADPH- thioredoxin reductase (NTR). They are able to reduce disulphides in target proteins. In monocots, thioredoxins h accumulate at high level in seeds and show a predominant localization in the nucleus of seed cells. These results suggest that the NTR-thioredoxin h system probably plays an important role in seed physiology. To date, the study of this system in monocots is limited by the lack of information about NTR. In the present study, we describe the cloning of a full-length cDNA encoding NTR from wheat ( Triticum aestivum ). The polypeptide deduced from this cDNA shows close similarity to NTRs from Arabidopsis, contains FAD- and NADPH-binding domains and a disulphide probably interacting with the disulphide at the active site of thioredoxin h. Wheat NTR was expressed in Escherichia coli as a His-tagged protein. The absorption spectrum of the purified recombinant protein is typical of flavoenzymes. Furthermore, it showed NADPH-dependent thioredoxin h reduction activity, thus confirming that the cDNA clone reported in the present study encodes wheat NTR. Using the His-tagged NTR and TRXhA (wheat thioredoxin h ), we successfully reconstituted the wheat NTR-thioredoxin h system in vitro, as shown by the insulin reduction assay. A polyclonal antibody was raised against wheat NTR after immunization of rabbits with the purified His-tagged protein. This antibody efficiently detected a single polypeptide of the corresponding molecular mass in seed extracts and it allowed the analysis of the pattern of accumulation of NTR in different wheat organs and developmental stages. NTR shows a wide distribution in wheat, but, surprisingly, its accumulation in seeds is low, in contrast with the level of thioredoxins h.

  20. Side chain conformational averaging in human dihydrofolate reductase.

    Tuttle, Lisa M; Dyson, H Jane; Wright, Peter E


    The three-dimensional structures of the dihydrofolate reductase enzymes from Escherichia coli (ecDHFR or ecE) and Homo sapiens (hDHFR or hE) are very similar, despite a rather low level of sequence identity. Whereas the active site loops of ecDHFR undergo major conformational rearrangements during progression through the reaction cycle, hDHFR remains fixed in a closed loop conformation in all of its catalytic intermediates. To elucidate the structural and dynamic differences between the human and E. coli enzymes, we conducted a comprehensive analysis of side chain flexibility and dynamics in complexes of hDHFR that represent intermediates in the major catalytic cycle. Nuclear magnetic resonance relaxation dispersion experiments show that, in marked contrast to the functionally important motions that feature prominently in the catalytic intermediates of ecDHFR, millisecond time scale fluctuations cannot be detected for hDHFR side chains. Ligand flux in hDHFR is thought to be mediated by conformational changes between a hinge-open state when the substrate/product-binding pocket is vacant and a hinge-closed state when this pocket is occupied. Comparison of X-ray structures of hinge-open and hinge-closed states shows that helix αF changes position by sliding between the two states. Analysis of χ1 rotamer populations derived from measurements of (3)JCγCO and (3)JCγN couplings indicates that many of the side chains that contact helix αF exhibit rotamer averaging that may facilitate the conformational change. The χ1 rotamer adopted by the Phe31 side chain depends upon whether the active site contains the substrate or product. In the holoenzyme (the binary complex of hDHFR with reduced nicotinamide adenine dinucleotide phosphate), a combination of hinge opening and a change in the Phe31 χ1 rotamer opens the active site to facilitate entry of the substrate. Overall, the data suggest that, unlike ecDHFR, hDHFR requires minimal backbone conformational rearrangement as

  1. Crystallization and preliminary X-ray diffraction analysis of maize aldose reductase

    Kiyota, Eduardo [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil); Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas-SP (Brazil); Sousa, Sylvia Morais de [Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas-SP (Brazil); Santos, Marcelo Leite dos; Costa Lima, Aline da [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil); Menossi, Marcelo [Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas, Campinas-SP (Brazil); Yunes, José Andrés [Laboratório de Biologia Molecular, Centro Infantil Boldrini, Campinas-SP (Brazil); Aparicio, Ricardo, E-mail: [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil)


    Preliminary X-ray diffraction studies of apo maize aldose reductase at 2.0 Å resolution are reported. Maize aldose reductase (AR) is a member of the aldo-keto reductase superfamily. In contrast to human AR, maize AR seems to prefer the conversion of sorbitol into glucose. The apoenzyme was crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 47.2, b = 54.5, c = 100.6 Å and one molecule in the asymmetric unit. Synchrotron X-ray diffraction data were collected and a final resolution limit of 2.0 Å was obtained after data reduction. Phasing was carried out by an automated molecular-replacement procedure and structural refinement is currently in progress. The refined structure is expected to shed light on the functional/enzymatic mechanism and the unusual activities of maize AR.

  2. Cuminaldehyde: Aldose Reductase and alpha-Glucosidase Inhibitor Derived from Cuminum cyminum L. Seeds.

    Lee, Hoi-Seon


    The inhibitory activity of Cuminum cyminum seed-isolated component was evaluated against lens aldose reductase and alpha-glucosidase isolated from Sprague-Dawley male rats and compared to that of 11 commercially available components derived from C. cyminum seed oil, as well as quercitrin as an aldose reductase inhibitor and acarbose as an alpha-glucosidase inhibitor. The biologically active constituent of C. cyminum seed oil was characterized as cuminaldehyde by various spectral analyses. The IC(50) value of cuminaldehyde is 0.00085 mg/mL against aldose reductase and 0.5 mg/mL against alpha-glucosidase, respectively. Cuminaldehyde was about 1.8 and 1.6 times less in inhibitory activity than acarbose and quercitin, respectively. Nonetheless, cuminaldehyde may be useful as a lead compound and a new agent for antidiabetic therapeutics.

  3. [Progress in research of aldose reductase inhibitors in traditional medicinal herbs].

    Feng, Chang-Gen; Zhang, Lin-Xia; Liu, Xia


    The traditional medicinal herbs are natural product, and have no obviously toxic action and side effect, and their resources are extensive. The adverse effects produced by aldose reductase inhibitors in traditional medicinal herbs are less than those from chemical synthesis and micro-organism, they can effectively prevent and delay diabetic complication, such as diabetic nephropathy, vasculopathy, retinopathy, peripheral neuropathy, and so on. They will have a wonderful respect. Flavonoid compounds and their derivates from traditional medicinal herbs are active inhibitors to aldose reductase, such as quercetin, silymarin, puerarin, baicalim, berberine and so on. In addition, some compound preparations show more strongly activity in inhibiting aldose reductase and degrading sorbitol contents, such as Shendan in traditional medicinal herbs being active inhibitors and Jianyi capsule, Jinmaitong composita, Liuwei Di-huang pill, et al. The progresses definite functions of treating diabetes complications have been reviewed.

  4. Circadian variation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in swine liver and ileum.

    Rogers, D H; Kim, D N; Lee, K T; Reiner, J M; Thomas, W A


    The temporal variation of HMG-CoA reductase activity in the liver and intestine of swine was investigated. The thin-layer chromatographic method widely used in the assay of the reductase was successfully applied to the porcine enzymes. Parallel circadian rhythms were demonstrated in both hepatic and ileal reductases from mash-fed animals. Peak activity occurred approximately 6 hr after feeding, 2.7-fold over the basal level in the liver, and 1.6-fold in the ileum. A milk-cholesterol diet caused a marked depression of both rhythms (90% in liver, 50% in ileum); however, the hourly variation in activity persisted in both organs. Cholestyramine was found to elevate hepatic activity (2.7-fold throughout the rhythm) without affecting that of the intestine. Clofibrate had no effect on either enzyme at any time during the cycle despite a 34% reduction in serum cholesterol concentrations.

  5. A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi

    Pena Diaz, Javier; Montalvetti, A; Camacho, A;


    We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest...... sensitive to proteolytic inactivation. Furthermore the enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. Thus Trypanosoma cruzi HMG-CoA reductase is unique in the sense that it totally lacks the membrane-spanning sequences present in all eukaryotic HMG...... cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts. Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly...

  6. Denitrification by plant roots? New aspects of plant plasma membrane-bound nitrate reductase.

    Eick, Manuela; Stöhr, Christine


    A specific form of plasma membrane-bound nitrate reductase in plants is restricted to roots. Two peptides originated from plasma membrane integral proteins isolated from Hordeum vulgare have been assigned as homologues to the subunit NarH of respiratory nitrate reductase of Escherichia coli. Corresponding sequences have been detected for predicted proteins of Populus trichocarpa with high degree of identities for the subunits NarH (75%) and NarG (65%), however, with less accordance for the subunit NarI. These findings coincide with biochemical properties, particularly in regard to the electron donors menadione and succinate. Together with the root-specific and plasma membrane-bound nitrite/NO reductase, nitric oxide is produced under hypoxic conditions in the presence of nitrate. In this context, a possible function in nitrate respiration of plant roots and an involvement of plants in denitrification processes are discussed.

  7. Directed Molecular Evolution of Nitrite Oxido-reductase by DNA-shuffling



    Objective To develtop directly molecular evolution of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremely slow and are unable to nitrify effectively inorganic nitrogen in wastewater treatment. Methods The norB gene coding the nitrite oxido-reductase in nitrobacteria was cloned and sequenced. Then, directed molecular evolution of nitrite oxido-reductase was developed by DNA-shuffling of 15 norB genes from different nitrobacteria. Results After DNA-shuffling with sexual PCR and staggered extension process PCR, the sequence was different from its parental DNA fragments and the homology ranged from 98% to 99%. The maximum nitrification rate of the modified bacterium of X16 by modified bacterium had the same characteristics of its parental bacteria of E. coli and could grow rapidly in normal cultures.Conclusion DNA-shuffling was successfully used to engineer E. coli, which had norB gene and could degrade inorganic nitrogen effectively.

  8. A substrate-bound structure of cyanobacterial biliverdin reductase identifies stacked substrates as critical for activity

    Takao, Haruna; Hirabayashi, Kei; Nishigaya, Yuki; Kouriki, Haruna; Nakaniwa, Tetsuko; Hagiwara, Yoshinori; Harada, Jiro; Sato, Hideaki; Yamazaki, Toshimasa; Sakakibara, Yoichi; Suiko, Masahito; Asada, Yujiro; Takahashi, Yasuhiro; Yamamoto, Ken; Fukuyama, Keiichi; Sugishima, Masakazu; Wada, Kei


    Biliverdin reductase catalyses the last step in haem degradation and produces the major lipophilic antioxidant bilirubin via reduction of biliverdin, using NAD(P)H as a cofactor. Despite the importance of biliverdin reductase in maintaining the redox balance, the molecular details of the reaction it catalyses remain unknown. Here we present the crystal structure of biliverdin reductase in complex with biliverdin and NADP+. Unexpectedly, two biliverdin molecules, which we designated the proximal and distal biliverdins, bind with stacked geometry in the active site. The nicotinamide ring of the NADP+ is located close to the reaction site on the proximal biliverdin, supporting that the hydride directly attacks this position of the proximal biliverdin. The results of mutagenesis studies suggest that a conserved Arg185 is essential for the catalysis. The distal biliverdin probably acts as a conduit to deliver the proton from Arg185 to the proximal biliverdin, thus yielding bilirubin. PMID:28169272

  9. A DFT-based QSAR study on inhibition of human dihydrofolate reductase.

    Karabulut, Sedat; Sizochenko, Natalia; Orhan, Adnan; Leszczynski, Jerzy


    Diaminopyrimidine derivatives are frequently used as inhibitors of human dihydrofolate reductase, for example in treatment of patients whose immune system are affected by human immunodeficiency virus. Forty-seven dicyclic and tricyclic potential inhibitors of human dihydrofolate reductase were analyzed using the quantitative structure-activity analysis supported by DFT-based and DRAGON-based descriptors. The developed model yielded an RMSE deviation of 1.1 a correlation coefficient of 0.81. The prediction set was characterized by R(2)=0.60 and RMSE=3.59. Factors responsible for inhibition process were identified and discussed. The resulting model was validated via cross validation and Y-scrambling procedure. From the best model, we found several mass-related descriptors and Sanderson electronegativity-related descriptors that have the best correlations with the investigated inhibitory concentration. These descriptors reflect results from QSAR studies based on characteristics of human dihydrofolate reductase inhibitors.

  10. DFT characterization of key intermediates in thiols oxidation catalyzed by amavadin.

    Bertini, Luca; Barbieri, Valentina; Fantucci, Piercarlo; De Gioia, Luca; Zampella, Giuseppe


    Amavadin is an unusual octa-coordinated V(IV) complex isolated from Amanita muscaria mushrooms. The outer-sphere catalytic properties of such a complex toward several oxidation reactions are well known. Nevertheless, a remarkable example exists, in which the V(V) (d(0)) oxidized form of amavadin is able to electro-catalyze the oxidation of some thiols to the corresponding disulfides through an inner-sphere mechanism (Guedes da Silva et al. J. Am. Chem. Soc.1996, 118, 7568-7573.) The reaction mechanism implies the formation of an amavadin-substrate intermediate, whose half-life is about 0.3 s. By means of Density Functional Theory (DFT) computations and Quantum Theory of Atoms in Molecules (QTAIM) analysis of the electron density, we have first characterized the stereoelectronic features of the V(IV) (inactive) and V(V) (active) states of amavadin. Then, the formation of the V(V) complex with methyl mercaptoacetate (MMA), which has been chosen as a prototypical substrate, has been characterized both thermodynamically and kinetically. DFT results reveal that protonation of V(V) amavadin at a carboxylate oxygen not directly involved in the V coordination, favors MMA binding into the first coordination sphere of vanadium, by substitution of the amavadin carboxylate oxygen with that of the substrate and formation of an S-HO hydrogen bond interaction. The latter interaction can promote SH deprotonation and binding of the thiolate group to vanadium. The kinetic and thermodynamic feasibility of the V(V)-MMA intermediates formation is in agreement, along with electrochemical experimental data, also with the biological role exerted by amavadin. Finally, the presence of an ester functional group as an essential requisite for MMA oxidation has been rationalized.

  11. Silane-coated magnetic nanoparticles with surface thiol functions for conjugation with gold nanostars

    Pallavicini, Piersandro


    Small (d ∼ 8 nm) magnetite nanoparticles, FeONP, are prepared and coated with mercaptopropyl trimethoxysilane (MPTS) to form FeONP@MPTS. In the coating step controlled MPTS/FeONP molar ratios are used, ranging from 1 to 7.8 × 10. The total quantity of MPTS per FeONP is determined by SEM-EDS analysis and the average number of free, reactive -SH groups per FeONP is calculated by a colorimetric method. At very low molar ratios MPTS forms a submonolayer on the FeONP surface with all -SH free to react, while on increasing the MPTS/FeONP molar ratio the (CHO)Si- groups of MPTS polymerize, forming a progressively thicker shell, in which only a small fraction of the -SH groups, positioned on the shell surface, is available for further reaction. The MPTS shell reduces the magnetic interactions occurring between the magnetite cores, lowering the occurrence and strength of collective magnetic states, with FeONP@MPTS showing the typical behaviour expected for a sample with a mono-modal size distribution of superparamagnetic nanoparticles. Interaction of FeONP@MPTS with gold nanostars (GNS) was tested, using both FeONP@MPTS with a MPTS submonolayer and with increasing shell thickness. Provided that a good balance is used between the number of available -SH and the overall size of FeONP@MPTS, the free thiols of such nanoparticles bind GNS decorating their surface, as shown by UV-Vis spectroscopy and TEM imaging.

  12. Cadmium sensitivity, uptake, subcellular distribution and thiol induction in a marine diatom: Recovery from cadmium exposure

    Wang Mengjiao [State Key Laboratory in Marine Pollution, Section of Marine Ecology and Biotechnology, Division of Life Science, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong); Wang Wenxiong, E-mail: [State Key Laboratory in Marine Pollution, Section of Marine Ecology and Biotechnology, Division of Life Science, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong)


    Studies in the recovery from metal stress and the tolerance development to metal exposure of aquatic organisms are important for the understanding of epidemic pollution. In this study, the responses of a marine diatom, Thalassiosira nordenskioeldii, following recovery from environmental cadmium (Cd) stress were investigated. The diatoms were exposed to different concentrations of Cd for 7 days, and were then allowed different periods of time to recover. The Cd sensitivity increased after recovery from Cd stress, followed by a gradual restoration. The extent of restoration depended on both the recovery time and the environmental Cd stress during the exposure period. A complete restoration of Cd tolerance proved to be impossible for cells pre-exposed to High-Cd. The Cd cellular burden and subcellular Cd concentration decreased to the control level within the first day of recovery, indicating that the elevated sensitivity may have been due to the accumulation of functional damage caused by Cd exposure instead of a result of physical Cd accumulation. The rapid change in phytochelatins (PC) to both the increase in and the withdrawal of environmental Cd stress made it a good quantitative bioindicator of environmental Cd contamination. However, the relationships between Cd distribution in the metal sensitive fraction (MSF-Cd) or intracellular Cd to thiol ratio (intra-Cd/PC-SH) and the relative change in the median inhibition [Cd{sup 2+}] ([Cd{sup 2+}]-based-IC{sub 50}, i.e., Cd sensitivity) differed for the various exposure and recovery periods tested. Our study suggests that more attention should be given to the recovery of aquatic organisms from episodic metal exposure.

  13. Cadmium sensitivity, uptake, subcellular distribution and thiol induction in a marine diatom: Recovery from cadmium exposure.

    Wang, Meng-Jiao; Wang, Wen-Xiong


    Studies in the recovery from metal stress and the tolerance development to metal exposure of aquatic organisms are important for the understanding of epidemic pollution. In this study, the responses of a marine diatom, Thalassiosira nordenskioeldii, following recovery from environmental cadmium (Cd) stress were investigated. The diatoms were exposed to different concentrations of Cd for 7 days, and were then allowed different periods of time to recover. The Cd sensitivity increased after recovery from Cd stress, followed by a gradual restoration. The extent of restoration depended on both the recovery time and the environmental Cd stress during the exposure period. A complete restoration of Cd tolerance proved to be impossible for cells pre-exposed to High-Cd. The Cd cellular burden and subcellular Cd concentration decreased to the control level within the first day of recovery, indicating that the elevated sensitivity may have been due to the accumulation of functional damage caused by Cd exposure instead of a result of physical Cd accumulation. The rapid change in phytochelatins (PC) to both the increase in and the withdrawal of environmental Cd stress made it a good quantitative bioindicator of environmental Cd contamination. However, the relationships between Cd distribution in the metal sensitive fraction (MSF-Cd) or intracellular Cd to thiol ratio (intra-Cd/PC-SH) and the relative change in the median inhibition [Cd(2+)] ([Cd(2+)]-based-IC(50), i.e., Cd sensitivity) differed for the various exposure and recovery periods tested. Our study suggests that more attention should be given to the recovery of aquatic organisms from episodic metal exposure.

  14. Thiol antioxidant-functionalized CdSe/ZnS quantum dots: synthesis, characterization, cytotoxicity.

    Zheng, Hong; Mortensen, Luke J; DeLouise, Lisa A


    Nanotechnology is a growing industry with wide ranging applications in consumer product and technology development. In the biomedical field, nanoparticles are finding increasing use as imaging agents for biomolecular labeling and tumor targeting. The nanoparticle physiochemical properties must be tailored for the specific application. For example, nanoparticle chemical and physical stability in the biological milieu (no oxidation, aggregation, agglomeration or toxicity) are often required. Nanoparticles used for biomolecular fluorescent imaging should also have high quantum yield (QY). The aim of this paper is to examine the QY, stability, and cell toxicity of a series of positive, negative and neutral surface charge quantum dot (QD) nanoparticles. Simple protocols are described to prepare water soluble QDs by modifying the surface with thiol containing antioxidant ligands and polymers keeping the QD core/shell composition constant. The ligands used to produce negatively charged QDs include glutathione (GSH), N-acetyl-L-cysteine (NAC), dihydrolipoic acid (DHLA), tiopronin (TP), bucilliamine (BUC), and mercaptosuccinic acid (MSA). Ligands used to produce positively charged QDs include cysteamine (CYS) and polyethylenimine (PEI). Dithiothreitol (DTT) was used to produce neutral charged QDs. Commercially available nonaqueous octadecylamine (ODA) capped QDs served as the starting material. Our results suggest that QD uptake and cytotoxicity are both dependent on surface ligand coating composition. The negative charged GSH coated QDs show superior performance exhibiting low cytotoxicity, high stability, high QY and therefore are best suited for bioimaging applications. PEI coated QD also show superior performance exhibiting high QY and stability. However, they are considerably more cytotoxic due to their high positive charge which is an advantageous property that can be exploited for gene transfection and/or tumor targeting applications. The synthetic procedures

  15. Surface plasmon resonance and magnetism of thiol-capped gold nanoparticles

    Guerrero, E; Munoz-Marquez, M A; Fernandez, A [Instituto de Ciencia de Materiales de Sevilla, CSIC-US, Avenida Americo Vespucio 49, E-41092 Sevilla (Spain); Garcia, M A; Crespo, P; Fernandez-Pinel, E; Hernando, A [Instituto de Magnetismo Aplicado (UCM-ADIF-CSIC), PO Box 155, E-28230 Las Rozas, Madrid (Spain)], E-mail:


    Surface plasmon resonance measurements and magnetic characterization studies have been carried out for two types of thiol-capped gold nanoparticles (NPs) with similar diameters between 2.0 and 2.5 nm and different organic molecules linked to the sulfur atom: dodecanethiol and tiopronin. In addition, Au NPs capped with tetraoctyl ammonium bromide have also been included in the investigation since such capping molecules weakly interact with the gold surface atoms and, therefore, this system can be used as a model for naked gold NPs; such particles presented a bimodal size distribution with diameters around 1.5 and 5 nm. The plasmon resonance is non-existent for tiopronin-capped NPs, whereas a trace of such a feature is observed for NPs covered with dodecanethiol molecules and a bulk-like feature is measured for NPs capped with tetralkyl ammonium salts. These differences would indicate that the modification of the surface electronic structure of the Au NPs depends on the geometry and self-assembling capabilities of the capping molecules and on the electric charge transferred between Au and S atoms. Regarding the magnetization, dodecanethiol-capped NPs have a ferromagnetic-like behaviour, while the NPs capped with tiopronin exhibit a paramagnetic behaviour and tetralkyl ammonium-protected NPs are diamagnetic across the studied temperature range; straight chains with a well-defined symmetry axis can induce orbital momentum on surface electrons close to the binding atoms. The orbital momentum not only contributes to the magnetization but also to the local anisotropy, giving rise to permanent magnetism. Due to the domain structure of the adsorbed molecules, orbital momentum is not induced for tiopronin-capped NPs and the charge transfer only induces a paramagnetic spin component.

  16. Characterisation of a desmosterol reductase involved in phytosterol dealkylation in the silkworm, Bombyx mori.

    Leonora F Ciufo

    Full Text Available Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C(29 and C(28 yielding cholesterol (C(27. The final step of this dealkylation pathway involves desmosterol reductase (DHCR24-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735. Following PCR-based cloning of the cDNA (1.6 kb and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD-dependent reaction.Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250 kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation.

  17. Putative Role of the Aldo-Keto Reductase from Trypanosoma cruzi in Benznidazole Metabolism.

    Garavaglia, Patricia Andrea; Laverrière, Marc; Cannata, Joaquín J B; García, Gabriela Andrea


    Benznidazole (Bz), the drug used for treatment of Chagas' disease (caused by the protozoan Trypanosoma cruzi), is activated by a parasitic NADH-dependent type I nitroreductase (NTR I). However, several studies have shown that other enzymes are involved. The aim of this study was to evaluate whether the aldo-keto reductase from T. cruzi (TcAKR), a NADPH-dependent oxido-reductase previously described by our group, uses Bz as the substrate. We demonstrated that both recombinant and native TcAKR enzymes reduce Bz by using NADPH, but not NADH, as a cofactor. TcAKR-overexpressing epimastigotes showed higher NADPH-dependent Bz reductase activity and a 50% inhibitory concentration (IC50) value for Bz 1.8-fold higher than that of the controls, suggesting that TcAKR is involved in Bz detoxification instead of activation. To understand the role of TcAKR in Bz metabolism, we studied TcAKR expression and NADPH/NADH-dependent Bz reductase activities in two T. cruzi strains with differential susceptibility to Bz: CL Brener and Nicaragua. Taking into account the results obtained with TcAKR-overexpressing epimastigotes, we expected the more resistant strain, Nicaragua, to have higher TcAKR levels than CL Brener. However, the results were the opposite. CL Brener showed 2-fold higher TcAKR expression and 5.7-fold higher NADPH-Bz reduction than the Nicaragua strain. In addition, NADH-dependent Bz reductase activity, characteristic of NTR I, was also higher in CL Brener than in Nicaragua. We conclude that although TcAKR uses Bz as the substrate, TcAKR activity is not a determinant of Bz resistance in wild-type strains and may be overcome by other enzymes involved in Bz activation, such as NADPH- and NADH-dependent reductases.

  18. 5{alpha}-reductase expression by prostate cancer cell lines and benign prostatic hyperplasia in vitro

    Smith, C.M.; Masters, J.R.W. [Univ. College of London (United Kingdom)]|[Pfizer Central Research, Kent (United Kingdom); Ballard, S.A.; Worman, N. [Pfizer Central Research, Sandwich (United Kingdom)


    5{alpha}-Reductase (5{alpha}R) activity in two human prostate cancer cell lines was compared to that in benign prostatic hyperplasia (BPH) tissue and COS cells transfected with and expressing the human genes for 5{alpha}-reductase type 1 (5{alpha}R1) and type 2 (5{alpha}R2). Comparisons were based on pH profiles and sensitivities to selective inhibitors of 5{alpha}-reductase. In the cancer lines, activity was greatest over the pH range 7-8, compared to a sharp peak of activity between pH 5-5.5 in BPH tissue and COS cells expressing 5{alpha}R2. Finasteride and SKF105,657 were potent inhibitors of 5{alpha}-reductase activity in BPH tissue and COS cells expressing 5{alpha}R2, but weak inhibitors in the cancer lines and in COS cells expressing 5{alpha}R1. In contrast, LTK1 17,026 was a more potent inhibitor of 5{alpha}-reductase activity in the prostate cancer cell lines and in COS cells expressing 5{alpha}R1. These data indicate that human prostate cancer cell lines express 5{alpha}-reductase activity similar to that in COS cells transfected with 5{alpha}R1, but different from that in BPH tissue. This may be a consequence of in vitro culture. Alternatively, it may reflect a change occurring as a result of neoplastic transformation, in which case it will be important to select appropriate inhibitors in the clinic. 29 refs., 3 figs., 2 tabs.

  19. Adverse Effects and Safety of 5-alpha Reductase Inhibitors (Finasteride, Dutasteride): A Systematic Review

    Hirshburg, Jason M.; Kelsey, Petra A.; Therrien, Chelsea A.; Gavino, A. Carlo; Reichenberg, Jason S.


    Finasteride and dutasteride, both 5-alpha reductase inhibitors, are considered first-line treatment for androgenetic hair loss in men and used increasingly in women. In each case, patients are expected to take the medications indefinitely despite the lack of research regarding long-term adverse effects. Concerns regarding the adverse effects of these medications has led the United States National Institutes of Health to add a link for post-finasteride syndrome to its Genetic and Rare Disease Information Center. Herein, the authors report the results of a literature search reviewing adverse events of 5-alpha reductase inhibitors as they relate to prostate cancer, psychological effects, sexual health, and use in women. Several large studies found no increase in incidence of prostate cancer, a possible increase of high-grade cancer when detected, and no change in survival rate with 5-alpha reductase inhibitor use. Currently, there is no direct link between 5-alpha reductase inhibitor use and depression; however, several small studies have led to depression being listed as a side effect on the medication packaging. Sexual effects including erectile dysfunction and decreased libido and ejaculate were reported in as many as 3.4 to 15.8 percent of men. To date, there are very few studies evaluating 5-alpha reductase inhibitor use in women. Risks include birth defects in male fetuses if used in pregnancy, decreased libido, headache, gastrointestinal discomfort, and isolated reports of changes in menstruation, acne, and dizziness. Overall, 5-alpha reductase inhibitors were well-tolerated in both men and women, but not without risk, highlighting the importance of patient education prior to treatment. PMID:27672412

  20. Nitrosative stress, cellular stress response, and thiol homeostasis in patients with Alzheimer's disease.

    Calabrese, Vittorio; Sultana, Rukhsana; Scapagnini, Giovanni; Guagliano, Eleonora; Sapienza, Maria; Bella, Rita; Kanski, Jaroslaw; Pennisi, Giovanni; Mancuso, Cesare; Stella, Anna Maria Giuffrida; Butterfield, D A


    Alzheimer's disease (AD) is a neurodegenerative disorder with cognitive and memory decline, personality changes, and synapse loss. Increasing evidence indicates that factors such as oxidative and nitrosative stress, glutathione depletion, and impaired protein metabolism can interact in a vicious cycle, which is central to AD pathogenesis. In the present study, we demonstrate that brains of AD patients undergo oxidative changes classically associated with a strong induction of the so-called vitagenes, including the heat shock proteins (HSPs) heme oxygenase-1 (HO-1), HSP60, and HSP72, as well as thioredoxin reductase (TRXr). In inferior parietal brain of AD patients, a significant increase in the expression of HO-1 and TRXr was observed, whereas HO-2 expression was decreased, compared with controls. TRHr was not increased in AD cerebellum. Plasma GSH was decreased in AD patients, compared with the control group, and was associated with a significant increase in oxidative stress markers (i.e., GSSG, hydroxynonenal, protein carbonyl content, and nitrotyrosine). In AD lymphocytes, we observed an increased expression of inducible nitric oxide synthase, HO-1, Hsp72, HSP60, and TRXr. Our data support a role for nitrative stress in the pathogenesis of AD and indicate that the stress-responsive genes, such as HO-1 and TRXr, may represent important targets for novel cytoprotective strategies.

  1. Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism

    Kirkensgaard, Kristine Groth; Hägglund, Per; Finnie, Christine


    Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs...... relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead...

  2. Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

    Nascimento, Alessandro S.; Ferrarezi, Thiago [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil); Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A. [Facultad de Ciencias Bioquímicas y Farmacéuticas, Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario (Argentina); Polikarpov, Igor, E-mail: [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil)


    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP{sup +} reductase. Ferredoxin-NADP{sup +} reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source.

  3. Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone

    Dow, J. M.; Grahl, S.; Ward, R; Evans, R.; Byron, O; Norman, D. G.; Palmer, T; Sargent, F


    Escherichia coli is a Gram‐negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the periplasmic nitrate reductase NapA contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin‐arginine protein transport pathway. NapD is a small cytoplasmic protein that is essential for the activity of the periplasmic nitrate reductase and binds tightly to the twin‐arginine signal peptide of NapA. Here we show, using spin labelli...

  4. Alpha 1-blockers vs 5 alpha-reductase inhibitors in benign prostatic hyperplasia. A comparative review

    Andersen, J T


    During recent years, pharmacological treatment of symptomatic benign prostatic hyperplasia (BPH) has become the primary treatment choice for an increasing number of patients. The 2 principal drug classes employed are alpha 1-blockers and 5 alpha-reductase inhibitors. Current information from...... of patients who will respond well to alpha 1-blockers have yet to be identified, and data concerning the long term effects of these drugs are not yet available. 5 alpha-Reductase inhibitors have a slow onset of effect, but treatment leads to improvement in symptoms, reduction of the size of the prostate gland...... or unwilling to undergo surgical resection of the prostate will benefit from such therapy....

  5. Feedback regulation of cholesterol synthesis:sterol-accelerated ubiquitination and degradation of HMG CoA reductase

    Russell A DeBose-Boyd


    3Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate,an important intermediate in the synthesis of cholesterol and essential nonsterol isoprenoids.The reductase is subject to an exorbitant amount of feedback control through multiple mechanisms that are mediated by sterol and nonsterol end-products of mevalonate metabolism.Here,Ⅰwill discuss recent advances that shed light on one mechanism for control of reductase,which involves rapid degradation of the enzyme.Accumulation of certain sterols triggers binding of reductase to endoplasmic reticulum (ER) membrane proteins called Insig-1 and Insig-2.Reductase-Insig binding results in recruitment of a membrane-associated ubiquitin ligase called gp78,which initiates ubiquitination of reductase.This ubiquitination is an obligatory reaction for recognition and degradation of reductase from ER membranes by cytosolic 26S proteasomes.Thus,sterol-accelerated degradation of reductase represents an example of how a general cellular process (ER-associated degradation) is used to control an important metabolic pathway (cholesterol synthesis).

  6. Studies on some characteristics of nitrate reductase from sugar beet (Beta vulgaris L.)leaves

    LiWenhua; YanGuiping; 等


    Some characteristics of nitrate reductase from sugar beet leaves shown in this paper were as follows:The nitrate reductase from sugar beet leaves required NADH as an electron donor.Accordingly,the nitrate reductase was classified as NADH-dependent(E.C.1.6.61).The Km value of the nitrate reductase for NADH and NO3- were 0.86m mol and 0.18μ mol respectively.The optimum pH in reaction mixture solution for nitrate reduction activity was 7.5.The effect of variable concentrations of inorganic phosphorus in the reaction buffer on nitrate reductase activity was investigated.When the inorganic phosphorus concentration was below 35m mol,the nitrate reductase activity was increased with increase of inorganic phosphorus concentration.Conversely,when the inorganic phosphorus concentration was over 35m mol,the nitrate reductase activity was inhibited.The nitrate reductase activity assayed in vitro was 3.2 and 5.6times of that assayed in vivo under the condition of exogenous and endogenous ground substance respectively.

  7. NADPH-Thioredoxin Reductase C Mediates the Response to Oxidative Stress and Thermotolerance in the Cyanobacterium Anabaena sp PCC7120

    Sanchez-Riego, Ana M.; Mata-Cabana, Alejandro; Galmozzi, CarlaV.; Florencio, Francisco J.


    NADPH-thioredoxin reductase C (NTRC) is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thiioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of t

  8. Rubredoxin Reductase of Pseudomonas oleovorans. Structural Relationship to Other Flavoprotein Oxidoreductases Based on One NAD and Two FAD Fingerprints

    Eggink, Gerrit; Engel, Henk; Vriend, Gert; Terpstra, Peter; Witholt, Bernard


    The oxidation of alkanes to alkanols by Pseudomonas oleovorans involves a three-component enzyme system: alkane hydroxylase, rubredoxin and rubredoxin reductase. Alkane hydroxylase and rubredoxin are encoded by the alkBPGHJKL operon, while previous studies indicated that rubredoxin reductase is most

  9. Pinpointing a Mechanistic Switch Between Ketoreduction and “Ene” Reduction in Short‐Chain Dehydrogenases/Reductases

    Lygidakis, Antonios; Karuppiah, Vijaykumar; Hoeven, Robin; Ní Cheallaigh, Aisling; Leys, David; Gardiner, John M.; Toogood, Helen S.


    Abstract Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (−)‐menthone:(−)‐menthol reductase and (−)‐menthone:(+)‐neomenthol reductase, and the “ene” reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue‐swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β‐unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases. PMID:27411040

  10. Arsenite tolerance in rice (Oryza sativa L.) involves coordinated role of metabolic pathways of thiols and amino acids.

    Tripathi, Preeti; Tripathi, Rudra Deo; Singh, Rana Pratap; Dwivedi, Sanjay; Chakrabarty, Debasis; Trivedi, Prabodh K; Adhikari, Bijan


    Thiolic ligands and several amino acids (AAs) are known to build up in plants against heavy metal stress. In the present study, alteration of various AAs in rice and its synchronized role with thiolic ligand was explored for arsenic (As) tolerance and detoxification. To understand the mechanism of As tolerance and stress response, rice seedlings of one tolerant (Triguna) and one sensitive (IET-4786) cultivar were exposed to arsenite (0-25 μM) for 7 days for various biochemical analyses using spectrophotometer, HPLC and ICPMS. Tolerant and sensitive cultivars respond differentially in terms of thiol metabolism, essential amino acids (EEAs) and nonessential amino acids (NEEAs) vis-á-vis As accumulation. Thiol biosynthesis-related enzymes were positively correlated to As accumulation in Triguna. Conversely, these enzymes, cysteine content and GSH/GSSG ratio declined significantly in IET-4786 upon As exposure. The level of identified phytochelatin (PC) species (PC(2), PC(3) and PC(4)) and phytochelatin synthase activity were also more pronounced in Triguna than IET-4786. Nearly all EAAs were negatively affected by As-induced oxidative stress (except phenylalanine in Triguna), but more significantly in IET-4786 than Triguna. However, most of the stress-responsive NEAAs like glutamic acid, histidine, alanine, glycine, tyrosine, cysteine and proline were enhanced more prominently in Triguna than IET-4786 upon As exposure. The study suggests that IET-4786 appears sensitive to As due to reduction of AAs and thiol metabolic pathway. However, a coordinated response of thiolic ligands and stress-responsive AAs seems to play role for As tolerance in Triguna to achieve the effective complexation of As by PCs.

  11. On sulfur core level binding energies in thiol self-assembly and alternative adsorption sites: An experimental and theoretical study

    Jia, Juanjuan [Institut des Sciences Moléculaires d’Orsay, Université-Paris Sud, 91405 Orsay (France); CNRS, UMR 8214, Institut des Sciences Moléculaires d’Orsay, Orsay ISMO, Bâtiment 351, Université Paris Sud, 91405 Orsay (France); Kara, Abdelkader, E-mail:, E-mail: [Department of Physics, University of Central Florida, Orlando, Florida 32816 (United States); Pasquali, Luca [Dipartimento di Ingegneria “E. Ferrari,” Università di Modena e Reggio Emilia, Via Vignolese 905, 41125 Modena (Italy); IOM-CNR, s.s. 14, Km. 163.5 in AREA Science Park, 34149 Basovizza, Trieste (Italy); Department of Physics, University of Johannesburg, P.O. Box 524, Auckland Park 2006 (South Africa); Bendounan, Azzedine; Sirotti, Fausto [Synchrotron SOLEIL, L’Orme des Merisiers, Saint-Aubin, BP 48, F-91192 Gif-sur-Yvette Cedex (France); Esaulov, Vladimir A., E-mail:, E-mail: [Institut des Sciences Moléculaires d’Orsay, Université-Paris Sud, 91405 Orsay (France); CNRS, UMR 8214, Institut des Sciences Moléculaires d’Orsay, Orsay ISMO, Bâtiment 351, Université Paris Sud, 91405 Orsay (France); IOM-CNR, s.s. 14, Km. 163.5 in AREA Science Park, 34149 Basovizza, Trieste (Italy)


    Characteristic core level binding energies (CLBEs) are regularly used to infer the modes of molecular adsorption: orientation, organization, and dissociation processes. Here, we focus on a largely debated situation regarding CLBEs in the case of chalcogen atom bearing molecules. For a thiol, this concerns the case when the CLBE of a thiolate sulfur at an adsorption site can be interpreted alternatively as due to atomic adsorption of a S atom, resulting from dissociation. Results of an investigation of the characteristics of thiol self-assembled monolayers (SAMs) obtained by vacuum evaporative adsorption are presented along with core level binding energy calculations. Thiol ended SAMs of 1,4-benzenedimethanethiol (BDMT) obtained by evaporation on Au display an unconventional CLBE structure at about 161.25 eV, which is close to a known CLBE of a S atom on Au. Adsorption and CLBE calculations for sulfur atoms and BDMT molecules are reported and allow delineating trends as a function of chemisorption on hollow, bridge, and atop sites and including the presence of adatoms. These calculations suggest that the 161.25 eV peak is due to an alternative adsorption site, which could be associated to an atop configuration. Therefore, this may be an alternative interpretation, different from the one involving the adsorption of atomic sulfur resulting from the dissociation process of the S–C bond. Calculated differences in S(2p) CLBEs for free BDMT molecules, SH group sulfur on top of the SAM, and disulfide are also reported to clarify possible errors in assignments.

  12. Simple quantitative determination of potent thiols at ultratrace levels in wine by derivatization and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis.

    Capone, Dimitra L; Ristic, Renata; Pardon, Kevin H; Jeffery, David W


    Volatile sulfur compounds contribute characteristic aromas to foods and beverages and are widely studied, because of their impact on sensory properties. Certain thiols are particularly important to the aromas of roasted coffee, cooked meat, passion fruit, grapefruit, and guava. These same thiols enhance the aroma profiles of different wine styles, imparting pleasant aromas reminiscent of citrus and tropical fruits (due to 3-mercaptohexan-1-ol, 3-mercaptohexyl acetate, 4-mercapto-4-methylpentan-2-one), roasted coffee (2-furfurylthiol), and struck flint (benzyl mercaptan), at nanogram-per-liter levels. In contrast to the usual gas chromatography (GC) approaches, a simple and unique high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for routine analysis of five wine thiols, using 4,4'-dithiodipyridine (DTDP) as a derivatizing agent and polydeuterated internal standards for maximum accuracy and precision. DTDP reacted rapidly with thiols at wine pH and provided stable derivatives, which were enriched by solid-phase extraction (SPE) prior to analysis by HPLC-MS/MS. All steps were optimized and the method was validated in different wine matrices, with method performance being comparable to a well-optimized but more cumbersome gas chromatography-mass spectrometry (GC-MS) method. A range of commercial wines was analyzed with the new method, revealing the distribution of the five thiols in white, red, rosé, and sparkling wine styles.

  13. The Nonenzymatic Reactivity of the Acyl-Linked Metabolites of Mefenamic Acid toward Amino and Thiol Functional Group Bionucleophiles


    Mefenamic acid (MFA), a carboxylic acid–containing nonsteroidal anti-inflammatory drug, is metabolized into the chemically-reactive MFA-1-O-acyl-glucuronide (MFA-1-O-G), MFA-acyl-adenylate (MFA-AMP), and the MFA-S-acyl-coenzyme A (MFA-CoA), all of which are electrophilic and capable of acylating nucleophilic sites on biomolecules. In this study, we investigate the nonenzymatic ability of each MFA acyl-linked metabolite to transacylate amino and thiol functional groups on the acceptor biomolec...

  14. Alkenyl/thiol-derived metal-organic frameworks (MOFs) by means of postsynthetic modification for effective mercury adsorption.

    Liu, Tao; Che, Jin-Xin; Hu, Yong-Zhou; Dong, Xiao-Wu; Liu, Xin-Yuan; Che, Chi-Ming


    The synthesis of new functionally diverse alkenyl-derived Cr-MIL-101s (MIL=material of Institute Lavoisier) was realized by a novel and convenient postsynthetic modification (PSM) protocol by means of the carbon-carbon bond-forming Mizoroki-Heck reaction. The new PSM protocol demonstrates a broad scope of substrates with excellent tolerance of functionality under mild reaction conditions. Moreover, a new metal-organic framework (MOF) that bears both alkenyl and thiol side chains prepared by means of the tandem PSM method has shown excellent adsorbent ability in removing mercury ions from water.

  15. Thiol-ene and photo-cleavage chemistry for controlled presentation of biomolecules in hydrogels.

    Grim, Joseph C; Marozas, Ian A; Anseth, Kristi S


    photochemical methods have led to important advances in the tissue regeneration community, namely the thiol-ene photo-click reaction for bioconjugation and photocleavage reactions that allow for the removal of protecting groups. Specific examples will be highlighted where these methodologies have been used to engineer hydrogels that control and direct cell function with the aim of inspiring their use in regenerative medicine.

  16. Part of respiratory nitrate reductase of Klebsiella aerogenes is intimately associated with the peptidoglycan.

    Abraham, P R; Wientjes, F B; Nanninga, N; Van't Riet, J


    Lysozyme digestion and sonication of sodium dodecyl sulfate (SDS)-purified Klebsiella aerogenes murein sacculi resulted in the quantitative release of both subunits of nitrate reductase, as well as a number of other cytoplasmic membrane polypeptides (5.2%, by weight, of the total membrane proteins). Similar results were obtained after lysozyme digestion of SDS-prepared peptidoglycan fragments, which excluded the phenomenon of simple trapping of the polypeptides by the surrounding peptidoglycan matrix. About 28% of membrane-bound nitrate reductase appears to be tightly associated with the peptidoglycan. Additional evidence for this association was demonstrated by positive immunogold labeling of SDS-murein sacculi and thin sections of plasmolyzed bacteria. Qualitative amino acid analysis of trypsin-treated sacculi, a tryptic product of holo-nitrate reductase, and amino- and carboxypeptidase digests of both nitrate reductase subunits indicated the possible existence of a terminal anchoring peptide containing the following amino acids: (Gly)n, Trp, Ser, Pro, Ile, Leu, Phe, Cys, Tyr, Asp, and Lys.

  17. Primary △4-3-oxosteroid 5β-reductase deficiency: Two cases in China

    Jing Zhao; Ling-Juan Fang; Kenneth DR Setchell; Rui Chen; Li-Ting Li; Jian-She Wang


    Aldo-keto reductase 1D1 (AKR1D1) deficiency,a rare but life-threatening form of bile acid deficiency,has not been previously described in China.Here,we describe the first two primary △4-3-oxosteroid 5β-reductase deficiency patients in Mainland China diagnosed by fast atom bombardment-mass spectroscopy of urinary bile acids and confirmed by genetic analysis.A high proportion of atypical 3-oxo-A4-bile acids in the urine indicated a deficiency in A4-3-oxosteroid 5β-reductase.All of the coding exons and adjacent intronic sequence of the AKR1D1 gene were sequenced using peripheral lymphocyte genomic DNA of two patients and one of the patient's parents.One patient exhibited compound heterozygous mutations:c.396C>A and c.722A>T,while the other was heterozygous for the mutation c.797G>A.Based on these mutations,a diagnosis of primary △4-3-oxosteroid 5β-reductase deficiency could be confirmed.With ursodeoxycholic acid treatment and fat-soluble vitamin supplements,liver function tests normalized rapidly,and the degree of hepatomegaly was markedly reduced in both patients.

  18. Sensing nitrite through a pseudoazurin-nitrite reductase electron transfer relay

    Astier, Y; Canters, GW; Davis, JJ; Hill, HAO; Verbeet, MP; Wijma, HJ


    Nitrite is converted to nitric oxide by haem or copper-containing enzymes in denitrifying bacteria during the process of denitrification. In designing an efficient biosensor, this enzymic turnover must be quantitatively assessed. The enzyme nitrite reductase from Alcaligenes faecalis contains a redo

  19. Structural and docking studies of Leucaena leucocephala Cinnamoyl CoA reductase.

    Prasad, Nirmal K; Vindal, Vaibhav; Kumar, Vikash; Kabra, Ashish; Phogat, Navneet; Kumar, Manoj


    Lignin, a major constituent of plant call wall, is a phenolic heteropolymer. It plays a major role in the development of plants and their defense mechanism against pathogens. Therefore Lignin biosynthesis is one of the critical metabolic pathways. In lignin biosynthesis, the Cinnamoyl CoA reductase is a key enzyme which catalyzes the first step in the pathway. Cinnamoyl CoA reductase provides the substrates which represent the main transitional molecules of lignin biosynthesis pathway, exhibits a high in vitro kinetic preference for feruloyl CoA. In present study, the three-dimensional model of cinnamoyl CoA reductase was constructed based on the crystal structure of Grape Dihydroflavonol 4-Reductase. Furthermore, the docking studies were performed to understand the substrate interactions to the active site of CCR. It showed that residues ARG51, ASN52, ASP54 and ASN58 were involved in substrate binding. We also suggest that residue ARG51 in CCR is the determinant residue in competitive inhibition of other substrates. This structural and docking information have prospective implications to understand the mechanism of CCR enzymatic reaction with feruloyl CoA, however the approach will be applicable in prediction of substrates and engineering 3D structures of other enzymes as well.

  20. In silico docking studies of aldose reductase inhibitory activity of commercially available flavonoids

    Arumugam Madeswaran


    Full Text Available The primary objective of this study was to investigate the aldose reductase inhibitory activity of flavonoids using in silico docking studies. In this perspective, flavonoids like biochanin, butein, esculatin, fisetin and herbacetin were selected. Epalrestat, a known aldose reductase inhibitor was used as the standard. In silico docking studies were carried out using AutoDock 4.2, based on the Lamarckian genetic algorithm principle. The results showed that all the selected flavonoids showed binding energy ranging between -9.33 kcal/mol to -7.23 kcal/mol when compared with that of the standard (-8.73 kcal/mol. Inhibition constant (144.13 µM to 4.98 µM and intermolecular energy (-11.42 kcal/mol to -7.83 kcal/mol of the flavonoids also coincide with the binding energy. All the selected flavonoids contributed aldose reductase inhibitory activity because of its structural properties. These molecular docking analyses could lead to the further development of potent aldose reductase inhibitors for the treatment of diabetes.