Ketopantoyl lactone reductase is a conjugated polyketone reductase.

Science.gov (United States)

Hata, H; Shimizu, S; Hattori, S; Yamada, H

1989-03-01

Ketopantoyl lactone reductase (EC 1.1.1.168) of Saccharomyces cerevisiae was found to catalyze the reduction of a variety of natural and unnatural conjugated polyketone compounds and quinones, such as isatin, ninhydrin, camphorquinone and beta-naphthoquinone in the presence of NADPH. 5-Bromoisatin is the best substrate for the enzyme (Km = 3.1 mM; Vmax = 650 mumol/min/mg). The enzyme is inhibited by quercetin, and several polyketones. These results suggest that ketopantoyl lactone reductase is a carbonyl reductase which specifically catalyzes the reduction of conjugated polyketones.

Biochemical parameters as biomarkers for the early recognition of environmental pollution on Scots pine trees. II. The antioxidative metabolites ascorbic acid, glutathione, {alpha}-tocopherol and the enzymes superoxide dismutase and glutathione reductase

Energy Technology Data Exchange (ETDEWEB)

Schulz, H.; Haertling, S. [UFZ Centre for Environmental Research Leipzig-Halle, Halle (Germany). Dept. of Soil Sciences

2001-10-01

Field investigations with Scots pine trees (Pinus sylvestris L.) were performed in eastern Germany, where ambient SO{sub 2}, NO{sub x} and O{sub 3} concentrations differed significantly in 1992-99 at three sites, namely Neuglobsow (yearly mean SO{sub 2} in 1992: 9 {mu}g m{sup -3}), Taura (yearly mean SO{sub 2} in 1992: 54 {mu}g m{sup -3}) and Roesa (yearly mean SO{sub 2} in 1992: 73 {mu}g m{sup -3}). To investigate the effects of SO{sub 2}, NO{sub x} and O{sub 3} on antioxidants (superoxide dismutase, ascorbic acid, glutathione, glutathione reductase, {alpha}-tocopherol) and pigments including chlorophyll fluorescence as well as visible damage symptoms in the form of needle yellowing and tip necroses, needles of the 1st and 2nd age class from young and mature trees were collected at the sites every October. Eight years after the start of the field study in 1992, the ambient SO{sub 2} concentrations had decreased significantly at Neuglobsow (yearly mean SO{sub 2} in 1999: 4 {mu}g m{sup -3}), Taura (yearly mean SO{sub 2} in 1999: 5 {mu}g m{sup -3}) and Roesa (yearly mean SO{sub 2} in 1999: 5 {mu}g m{sup -3}). NO{sub x} and O{sub 3} differed less at the three sites and showed no temporal variations. Whole needle glutathione continuously decreased, although concentrations were higher in needles of the 1st and 2nd age class from the polluted sites Taura and Roesa than the unpolluted site Neuglobsow. The activities of glutathione reductase exhibited the same site-related differences and temporal variations and were correlated with concentrations of oxidized glutathione (GSSG). In contrast, the activities of the enzyme superoxide dismutase and the concentrations of whole needle ascorbic acid remained unchanged over the period. Only at the end of the investigation period did the concentrations of oxidized ascorbic acid (dehydroascorbate) increase in six-month-old needles at the polluted sites Taura and Roesa. Despite the clear decreases in SO{sub 2}, the visible symptoms

Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae.

Science.gov (United States)

Moon, Jaewoong; Liu, Z Lewis

2015-04-01

The aldehyde reductase gene ARI1 is a recently characterized member of an intermediate subfamily within the short-chain dehydrogenase/reductase (SDR) superfamily that clarified mechanisms of in situ detoxification of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde by Saccharomyces cerevisiae. Uncharacterized open reading frames (ORFs) are common among tolerant candidate genes identified for lignocellulose-to-advanced biofuels conversion. This study presents partially purified proteins of two ORFs, YDR541C and YGL039W, and direct enzyme assay evidence against aldehyde-inhibitory compounds commonly encountered during lignocellulosic biomass fermentation processes. Each of the partially purified proteins encoded by these ORFs showed a molecular mass of approximately 38 kDa, similar to Ari1p, a protein encoded by aldehyde reductase gene. Both proteins demonstrated strong aldehyde reduction activities toward 14 aldehyde substrates, with high levels of reduction activity for Ydr541cp toward both aromatic and aliphatic aldehydes. While Ydr541cp was observed to have a significantly higher specific enzyme activity at 20 U/mg using co-factor NADPH, Ygl039wp displayed a NADH preference at 25 U/mg in reduction of butylaldehyde. Amino acid sequence analysis identified a characteristic catalytic triad, Ser, Tyr and Lys; a conserved catalytic motif of Tyr-X-X-X-Lys; and a cofactor-binding sequence motif, Gly-X-X-Gly-X-X-Ala, near the N-terminus that are shared by Ydr541cp, Ygl039wp, Yol151wp/GRE2 and Ari1p. Findings of aldehyde reductase genes contribute to the yeast gene annotation and aids development of the next-generation biocatalyst for advanced biofuels production. Copyright © 2015 John Wiley & Sons, Ltd.

The Antitumor Effect of Single-domain Antibodies Directed Towards Membrane-associated Catalase and Superoxide Dismutase.

Science.gov (United States)

Bauer, Georg; Motz, Manfred

2016-11-01

Neutralizing single-domain antibodies directed towards catalase or superoxide dismutase (SOD) caused efficient reactivation of intercellular reactive oxygen species/reactive nitrogen species (ROS/RNS)-dependent apoptosis-inducing signaling specifically in human tumor cells. Single-domain antibodies targeted tumor cell-specific membrane-associated SOD and catalase, but not the corresponding intracellular enzymes. They were shown to be about 200-fold more effective than corresponding classical recombinant antigen-binding fragments and more than four log steps more efficient than monoclonal antibodies. Combined addition of single-domain antibodies against catalase and SOD caused a remarkable synergistic effect. Proof-of-concept experiments in immunocompromised mice using human tumor xenografts and single-domain antibodies directed towards SOD showed an inhibition of tumor growth. Neutralizing single-domain antibodies directed to catalase and SOD also caused a very strong synergistic effect with the established chemotherapeutic agent taxol, indicating an overlap of signaling pathways. This effect might also be useful in order to avoid unwanted side-effects and to drastically lower the costs for taxol-based therapy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Superoxide anion production and superoxide dismutase and catalase activities in Coxiella burnetii.

OpenAIRE

Akporiaye, E T; Baca, O G

1983-01-01

Coxiella burnetii was examined for superoxide anion (O2-) production and superoxide dismutase and catalase activities. The organism generated O2- at pH 4.5 but not at pH 7.4. The rickettsia displayed superoxide dismutase activity distinguishable from that of the host cell (L-929 mouse fibroblast). Catalase activity was maximal at pH 7.0 and diminished at pH 4.5. These enzymes may account, in part, for the ability of this obligate intracellular parasite to survive within phagocytes.

The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

Science.gov (United States)

Wang, Jinling; de Montellano, Paul R Ortiz

2003-05-30

Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

Species-specific control of external superoxide levels by the coral holobiont during a natural bleaching event

Science.gov (United States)

Diaz, Julia M.; Hansel, Colleen M.; Apprill, Amy; Brighi, Caterina; Zhang, Tong; Weber, Laura; McNally, Sean; Xun, Liping

2016-12-01

The reactive oxygen species superoxide (O2.-) is both beneficial and detrimental to life. Within corals, superoxide may contribute to pathogen resistance but also bleaching, the loss of essential algal symbionts. Yet, the role of superoxide in coral health and physiology is not completely understood owing to a lack of direct in situ observations. By conducting field measurements of superoxide produced by corals during a bleaching event, we show substantial species-specific variation in external superoxide levels, which reflect the balance of production and degradation processes. Extracellular superoxide concentrations are independent of light, algal symbiont abundance and bleaching status, but depend on coral species and bacterial community composition. Furthermore, coral-derived superoxide concentrations ranged from levels below bulk seawater up to ~120 nM, some of the highest superoxide concentrations observed in marine systems. Overall, these results unveil the ability of corals and/or their microbiomes to regulate superoxide in their immediate surroundings, which suggests species-specific roles of superoxide in coral health and physiology.

Sources of superoxide/H2O2 during mitochondrial proline oxidation

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Renata L.S. Goncalves

2014-01-01

Full Text Available p53 Inducible gene 6 (PIG6 encodes mitochondrial proline dehydrogenase (PRODH and is up-regulated several fold upon p53 activation. Proline dehydrogenase is proposed to generate radicals that contribute to cancer cell apoptosis. However, there are at least 10 mitochondrial sites that can produce superoxide and/or H2O2, and it is unclear whether proline dehydrogenase generates these species directly, or instead drives production by other sites. Amongst six cancer cell lines, ZR75-30 human breast cancer cells had the highest basal proline dehydrogenase levels, and mitochondria isolated from ZR75-30 cells consumed oxygen and produced H2O2 with proline as sole substrate. Insects use proline oxidation to fuel flight, and mitochondria isolated from Drosophila melanogaster were even more active with proline as sole substrate than ZR75-30 mitochondria. Using mitochondria from these two models we identified the sites involved in formation of superoxide/H2O2 during proline oxidation. In mitochondria from Drosophila the main sites were respiratory complexes I and II. In mitochondria from ZR75-30 breast cancer cells the main sites were complex I and the oxoglutarate dehydrogenase complex. Even with combinations of substrates and respiratory chain inhibitors designed to minimize the contributions of other sites and maximize any superoxide/H2O2 production from proline dehydrogenase itself, there was no significant direct contribution of proline dehydrogenase to the observed H2O2 production. Thus proline oxidation by proline dehydrogenase drives superoxide/H2O2 production, but it does so mainly or exclusively by providing anaplerotic carbon for other mitochondrial dehydrogenases and not by producing superoxide/H2O2 directly.

Efficiency of superoxide anions in the inactivation of selected dehydrogenases

International Nuclear Information System (INIS)

Rodacka, Aleksandra; Serafin, Eligiusz; Puchala, Mieczyslaw

2010-01-01

The most ubiquitous of the primary reactive oxygen species, formed in all aerobes, is the superoxide free radical. It is believed that the superoxide anion radical shows low reactivity and in oxidative stress it is regarded mainly as an initiator of more reactive species such as · OH and ONOO - . In this paper, the effectiveness of inactivation of selected enzymes by radiation-generated superoxide radicals in comparison with the effectiveness of the other products of water radiolysis is examined. We investigate three enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH). We show that the direct contribution of the superoxide anion radical to GAPDH and ADH inactivation is significant. The effectiveness of the superoxide anion in the inactivation of GAPDH and ADG was only 2.4 and 2.8 times smaller, respectively, in comparison with hydroxyl radical. LDH was practically not inactivated by the superoxide anion. Despite the fact that the studied dehydrogenases belong to the same class of enzymes (oxidoreductases), all have a similar molecular weight and are tetramers, their susceptibility to free-radical damage varies. The differences in the radiosensitivity of the enzymes are not determined by the basic structural parameters analyzed. A significant role in inactivation susceptibility is played by the type of amino acid residues and their localization within enzyme molecules.

Efficiency of superoxide anions in the inactivation of selected dehydrogenases

Energy Technology Data Exchange (ETDEWEB)

Rodacka, Aleksandra, E-mail: olakow@biol.uni.lodz.p [Department of Molecular Biophysics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland); Serafin, Eligiusz, E-mail: serafin@biol.uni.lodz.p [Laboratory of Computer and Analytical Techniques, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland); Puchala, Mieczyslaw, E-mail: puchala@biol.uni.lodz.p [Department of Molecular Biophysics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland)

2010-09-15

The most ubiquitous of the primary reactive oxygen species, formed in all aerobes, is the superoxide free radical. It is believed that the superoxide anion radical shows low reactivity and in oxidative stress it is regarded mainly as an initiator of more reactive species such as {sup {center_dot}}OH and ONOO{sup -}. In this paper, the effectiveness of inactivation of selected enzymes by radiation-generated superoxide radicals in comparison with the effectiveness of the other products of water radiolysis is examined. We investigate three enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH). We show that the direct contribution of the superoxide anion radical to GAPDH and ADH inactivation is significant. The effectiveness of the superoxide anion in the inactivation of GAPDH and ADG was only 2.4 and 2.8 times smaller, respectively, in comparison with hydroxyl radical. LDH was practically not inactivated by the superoxide anion. Despite the fact that the studied dehydrogenases belong to the same class of enzymes (oxidoreductases), all have a similar molecular weight and are tetramers, their susceptibility to free-radical damage varies. The differences in the radiosensitivity of the enzymes are not determined by the basic structural parameters analyzed. A significant role in inactivation susceptibility is played by the type of amino acid residues and their localization within enzyme molecules.

Prevalence of methylenetetrahydrofolate reductase ( MTHFR ) and ...

African Journals Online (AJOL)

Methylenetetrahydrofolate reductase (MTHFR) and Cytosolic serine hydroxymethyltransferase (cSHMT) are enzymes involve in folate regulation in human. The C to T transition of the cSHMT and MTHFR genes at the 1420 as well as 677 nucleotides both carries TT genotype respectively. These enzymes have direct and ...

Constraints on superoxide mediated formation of manganese oxides

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Deric R. Learman

2013-09-01

Full Text Available Manganese (Mn oxides are among the most reactive sorbents and oxidants within the environment, where they play a central role in the cycling of nutrients, metals, and carbon. Recent discoveries have identified superoxide (O2- (both of biogenic and abiogenic origin as an effective oxidant of Mn(II leading to the formation of Mn oxides. Here we examined the conditions under which abiotically produced superoxide led to oxidative precipitation of Mn and the solid-phases produced. Oxidized Mn, as both aqueous Mn(III and Mn(III/IV oxides, was only observed in the presence of active catalase, indicating that hydrogen peroxide, a product of the reaction of O2- with Mn(II, inhibits the oxidation process presumably through the reduction of Mn(III. Citrate and pyrophosphate increased the yield of oxidized Mn but decreased the amount of Mn oxide produced via formation of Mn(III-ligand complexes. While complexing ligands played a role in stabilizing Mn(III, they did not eliminate the inhibition of net Mn(III formation by H2O2. The Mn oxides precipitated were highly disordered colloidal hexagonal birnessite, similar to those produced by biotically generated superoxide. Yet, in contrast to the large particulate Mn oxides formed by biogenic superoxide, abiotic Mn oxides did not ripen to larger, more crystalline phases. This suggests that the deposition of crystalline Mn oxides within the environment requires a biological, or at least organic, influence. This work provides the first direct evidence that, under conditions relevant to natural waters, oxidation of Mn(II by superoxide can occur and lead to formation of Mn oxides. For organisms that oxidize Mn(II by producing superoxide, these findings may also point to other microbially mediated processes, in particular enzymatic hydrogen peroxide degradation and/or production of organic ligand metabolites, that allow for Mn oxide formation.

Direct Comparison of the Enzymatic Characteristics and Superoxide Production of the Four Aldehyde Oxidase Enzymes Present in Mouse.

Science.gov (United States)

Kücükgöze, Gökhan; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke

2017-08-01

Aldehyde oxidases (AOXs) are molybdoflavoenzymes with an important role in the metabolism and detoxification of heterocyclic compounds and aliphatic as well as aromatic aldehydes. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. Four different enzymes, mAOX1, mAOX3, mAOX4, and mAOX2, which are the products of distinct genes, are present in the mouse. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes has never been performed. In this report, the four catalytically active mAOX enzymes were purified after heterologous expression in Escherichia coli The kinetic parameters of the four mouse AOX enzymes were determined and compared with the use of six predicted substrates of physiologic and toxicological interest, i.e., retinaldehyde, N 1 -methylnicotinamide, pyridoxal, vanillin, 4-(dimethylamino)cinnamaldehyde ( p- DMAC), and salicylaldehyde. While retinaldehyde, vanillin, p- DMAC, and salycilaldehyde are efficient substrates for the four mouse AOX enzymes, N 1 -methylnicotinamide is not a substrate of mAOX1 or mAOX4, and pyridoxal is not metabolized by any of the purified enzymes. Overall, mAOX1, mAOX2, mAOX3, and mAOX4 are characterized by significantly different K M and k cat values for the active substrates. The four mouse AOXs are also characterized by quantitative differences in their ability to produce superoxide radicals. With respect to this last point, mAOX2 is the enzyme generating the largest rate of superoxide radicals of around 40% in relation to moles of substrate converted, and mAOX1, the homolog to the human enzyme, produces a rate of approximately 30% of superoxide radicals with the same substrate. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Comparative Analyses of Cu-Zn Superoxide Dismutase (SOD1) and Thioredoxin Reductase (TrxR) at the mRNA Level between Apis mellifera L. and Apis cerana F. (Hymenoptera: Apidae) Under Stress Conditions.

Science.gov (United States)

Koo, Hyun-Na; Lee, Soon-Gyu; Yun, Seung-Hwan; Kim, Hyun Kyung; Choi, Yong Soo; Kim, Gil-Hah

2016-01-01

This study compared stress-induced expression of Cu-Zn superoxide dismutase (SOD1) and thioredoxin reductase (TrxR) genes in the European honeybee Apis mellifera L. and Asian honeybee Apis cerana F. Expression of both SOD1 and TrxR rapidly increased up to 5 h after exposure to cold (4 °C) or heat (37 °C) treatment and then gradually decreased, with a stronger effect induced by cold stress in A. mellifera compared with A. cerana. Injection of stress-inducing substances (methyl viologen, [MV] and H2O2) also increased SOD1 and TrxR expression in both A. mellifera and A. cerana, and this effect was more pronounced with MV than H2O2. Additionally, we heterologously expressed the A. mellifera and A. cerana SOD1 and TrxR proteins in an Escherichia coli expression system, and detection by SDS-PAGE, confirmed by Western blotting using anti-His tag antibodies, revealed bands at 16 and 60 kDa, respectively. Our results show that the expression patterns of SOD1 and TrxR differ between A. mellifera and A. cerana under conditions of low or high temperature as well as oxidative stress. © The Author 2016. Published by Oxford University Press on behalf of the Entomological Society of America.

Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants.

Science.gov (United States)

Echtay, Karim S; Murphy, Michael P; Smith, Robin A J; Talbot, Darren A; Brand, Martin D

2002-12-06

Superoxide activates nucleotide-sensitive mitochondrial proton transport through the uncoupling proteins UCP1, UCP2, and UCP3 (Echtay, K. S., et al. (2002) Nature 415, 1482-1486). Two possible mechanisms were proposed: direct activation of the UCP proton transport mechanism by superoxide or its products and a cycle of hydroperoxyl radical entry coupled to UCP-catalyzed superoxide anion export. Here we provide evidence for the first mechanism and show that superoxide activates UCP2 in rat kidney mitochondria from the matrix side of the mitochondrial inner membrane: (i) Exogenous superoxide inhibited matrix aconitase, showing that external superoxide entered the matrix. (ii) Superoxide-induced uncoupling was abolished by low concentrations of the mitochondrially targeted antioxidants 10-(6'-ubiquinonyl)decyltriphenylphosphonium (mitoQ) or 2-[2-(triphenylphosphonio)ethyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol bromide (mitoVit E), which are ubiquinone (Q) or tocopherol derivatives targeted to the matrix by covalent attachment to triphenylphosphonium cation. However, superoxide-induced uncoupling was not affected by similar concentrations of the nontargeted antioxidants Q(o), Q(1), decylubiquinone, vitamin E, or 6-hydroxy-2,5,7,8-tetramethylchroman 2-carboxylic acid (TROLOX) or of the mitochondrially targeted but redox-inactive analogs decyltriphenylphosphonium or 4-chlorobutyltriphenylphosphonium. Thus matrix superoxide appears to be necessary for activation of UCP2 by exogenous superoxide. (iii) When the reduced to oxidized ratio of mitoQ accumulated by mitochondria was increased by inhibiting cytochrome oxidase, it induced nucleotide-sensitive uncoupling that was not inhibited by external superoxide dismutase. Under these conditions quinols are known to produce superoxide, and because mitoQ is localized within the mitochondrial matrix this suggests that production of superoxide in the matrix was sufficient to activate UCP2. Furthermore, the superoxide

Immunocytochemical localization of APS reductase and bisulfite reductase in three Desulfovibrio species

NARCIS (Netherlands)

Kremer, D.R.; Veenhuis, M.; Fauque, G.; Peck Jr., H.D.; LeGall, J.; Lampreia, J.; Moura, J.J.G.; Hansen, T.A.

1988-01-01

The localization of APS reductase and bisulfite reductase in Desulfovibrio gigas, D. vulgaris Hildenborough and D. thermophilus was studied by immunoelectron microscopy. Polyclonal antibodies were raised against the purified enzymes from each strain. Cells fixed with formaldehyde/glutaraldehyde were

Overexpression of Iron Superoxide Dismutase in Transformed Poplar Modifies the Regulation of Photosynthesis at Low CO2 Partial Pressures or Following Exposure to the Prooxidant Herbicide Methyl Viologen1

Science.gov (United States)

Arisi, Ana-Carolina M.; Cornic, Gabriel; Jouanin, Lise; Foyer, Christine H.

1998-01-01

Chloroplast-targeted overexpression of an Fe superoxide dismutase (SOD) from Arabidopsis thaliana resulted in substantially increased foliar SOD activities. Ascorbate peroxidase, glutathione reductase, and monodehydroascorbate reductase activities were similar in the leaves from all of the lines, but dehydroascorbate reductase activity was increased in the leaves of the FeSOD transformants relative to untransformed controls. Foliar H2O2, ascorbate, and glutathione contents were comparable in all lines of plants. Irradiance-dependent changes in net CO2 assimilation and chlorophyll a fluorescence quenching parameters were similar in all lines both in air (21% O2) and at low (1%) O2. CO2-response curves for photosynthesis showed similar net CO2-exchange characteristics in all lines. In contrast, values of photochemical quenching declined in leaves from untransformed controls at intercellular CO2 (Ci) values below 200 μL L−1 but remained constant with decreasing Ci in leaves of FeSOD transformants. When the O2 concentration was decreased from 21 to 1%, the effect of FeSOD overexpression on photochemical quenching at limiting Ci was abolished. At high light (1000 μmol m−2 s−1) a progressive decrease in the ratio of variable (Fv) to maximal (Fm) fluorescence was observed with decreasing temperature. At 6oC the high-light-induced decrease in the Fv/Fm ratio was partially prevented by low O2 but values were comparable in all lines. Methyl viologen caused decreased Fv/Fm ratios, but this was less marked in the FeSOD transformants than in the untransformed controls. These observations suggest that the rate of superoxide dismutation limits flux through the Mehler-peroxidase cycle in certain conditions. PMID:9625709

Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae

Science.gov (United States)

Aldehyde reductase gene ARI1 is a recently characterized member of intermediate subfamily under SDR (short-chain dehydrogenase/reductase) superfamily that revealed mechanisms of in situ detoxification of furfural and HMF for tolerance of Saccharomyces cerevisiae. Uncharacterized open reading frames ...

Participation of superoxide generating system, superoxide dismutase and vitamin E in the radiation hazards

International Nuclear Information System (INIS)

Aono, Kaname; Yamamoto, Michio; Iida, Sosuke; Utsumi, Kozo

1978-01-01

In relation to the mechanism by which hemolysis was induced in radiated human erythrocytes in vitro, several inducements of membrane lipid peroxidation and protective effects of vitamin E (V.E) and superoxide dismutase (SOD) were investigated. (1) K + -release from erythrocytes was accelerated by radiation prior to hemolysis. These accelerated hemolysis and K + -release were protected remarkably by V.E and evidently by SOD. (2) Mitochondrial Fe 2+ induced and Fe 3+ -superoxide generating system -- ADP induced lipid peroxidation, and microsomal superoxide generating system -- induced lipid peroxidation were also protected by V.E and SOD. (3) Radiation of x-ray or 60 Co γ-ray accelerated lipid peroxidation of liver homogenate, microsome and liposome. Some of these accelerated lipid peroxidations were protected effectively by V.E and SOD. These results suggest that superoxide and/or OH generation by radiation induces of membrane lipid peroxidation, which leads deterioration of membrane resulting in the change of ion permeability and then hemolysis. (author)

Peptide methionine sulfoxide reductase A (MsrA): direct electrochemical oxidation on carbon electrodes.

Science.gov (United States)

Enache, T A; Oliveira-Brett, A M

2013-02-01

The direct electrochemical behaviour of peptide methionine sulfoxide reductase A (MsrA) adsorbed on glassy carbon and boron doped diamond electrodes surface, was studied over a wide pH range by cyclic and differential pulse voltammetry. MsrA oxidation mechanism occurs in three consecutive, pH dependent steps, corresponding to the oxidation of tyrosine, tryptophan and histidine amino acid residues. At the glassy carbon electrode, the first step corresponds to the oxidation of tyrosine and tryptophan residues and occurs for the same potential. The advantage of boron doped diamond electrode was to enable the separation of tyrosine and tryptophan oxidation peaks. On the second step occurs the histidine oxidation, and on the third, at higher potentials, the second tryptophan oxidation. MsrA adsorbs on the hydrophobic carbon electrode surface preferentially through the three hydrophobic domains, C1, C2 and C3, which contain the tyrosine, tryptophan and histidine residues, and tryptophan exists only in these regions, and undergo electrochemical oxidation. Copyright © 2012 Elsevier B.V. All rights reserved.

Biological Superoxide In Manganese Oxide Formation

Science.gov (United States)

Hansel, C.; Learman, D.; Zeiner, C.; Santelli, C. M.

2011-12-01

Manganese (Mn) oxides are among the strongest sorbents and oxidants within the environment, controlling the fate and transport of numerous elements and the degradation of recalcitrant carbon. Both bacteria and fungi mediate the oxidation of Mn(II) to Mn(III/IV) oxides but the genetic and biochemical mechanisms responsible remain poorly understood. Furthermore, the physiological basis for microbial Mn(II) oxidation remains an enigma. We have recently reported that a common marine bacterium (Roseobacter sp. AzwK-3b) oxidizes Mn(II) via reaction with extracellular superoxide (O2-) produced during exponential growth. Here we expand this superoxide-mediated Mn(II) oxidation pathway to fungi, introducing a surprising homology between prokaryotic and eukaryotic metal redox processes. For instance, Stibella aciculosa, a common soil Ascomycete filamentous fungus, precipitates Mn oxides at the base of asexual reproductive structures (synnemata) used to support conidia (Figure 1). This distribution is a consequence of localized production of superoxide (and it's dismutation product hydrogen peroxide, H2O2), leading to abiotic oxidation of Mn(II) by superoxide. Disruption of NADPH oxidase activity using the oxidoreductase inhibitor DPI leads to diminished cell differentiation and subsequent Mn(II) oxidation inhibition. Addition of Cu(II) (an effective superoxide scavenger) leads to a concentration dependent decrease in Mn oxide formation. We predict that due to the widespread production of extracellular superoxide within the fungal and likely bacterial kingdoms, biological superoxide may be an important contributor to the cycling of Mn, as well as other metals (e.g., Hg, Fe). Current and future explorations of the genes and proteins involved in superoxide production and Mn(II) oxidation will ideally lend insight into the physiological and biochemical basis for these processes.

Selective 15N labeling and direct observation by NMR of the active-site glutamine of Fe-containing superoxide dismutase

International Nuclear Information System (INIS)

Vance, Carrie K.; Kang, Young M.; Miller, Anne-Frances

1997-01-01

The glutamine in position 69 is one of only three conserved active-site amino acid differences between Fe- and Mn-containing superoxide dismutases (SODs). We have refined the conditions for extremely selective labeling of the side chains of glutamine with 15N, and thus obtained dramatically simplified spectra, despite the large size of Fe-SOD. The improved resolution afforded by such highly specific labeling permits the use of direct 15N detection to observe and assign Gln 69, even though its distance to the paramagnetic Fe2+ is only 5A. Selective glutamine side-chain labeling is inexpensive and has general utility for large (and paramagnet-containing) proteins

Exogenous melatonin suppresses dark-induced leaf senescence by activating the superoxide dismutase-catalase antioxidant pathway and down-regulating chlorophyll degradation in excised leaves of perennial ryegrass (Lolium perenne L.

Directory of Open Access Journals (Sweden)

Jing Zhang

2016-10-01

Full Text Available Leaf senescence is a typical symptom in plants exposed to dark and may be regulated by plant growth regulators. The objective of this study was to determine whether exogenous application of melatonin (N-acetyl-5-methoxytryptamine suppresses dark-induced leaf senescence and the effects of melatonin on reactive oxygen species (ROS scavenging system and chlorophyll degradation pathway in perennial grass species. Mature perennial ryegrass (Lolium perenne L. cv. ‘Pinnacle’ leaves were excised and incubated in 3 mM 2-(N-morpholino ethanesulfonic buffer (pH 5.8 supplemented with melatonin or water (control and exposed to dark treatment for 8 d. Leaves treated with melatonin maintained significantly higher endogenous melatonin level, chlorophyll content, photochemical efficiency, and cell membrane stability expressed by lower electrolyte leakage and malondialdehyde (MDA content compared to the control. Exogenous melatonin treatment also reduced the transcript level of chlorophyll degradation-associated genes and senescence marker genes (LpSAG12.1, Lph36, and Lpl69 during the dark treatment. The endogenous O2- production rate and H2O2 content were significantly lower in these excised leaves treated with melatonin compared to the water control. Exogenous melatonin treatment caused increases in enzymatic activity and transcript levels of superoxide dismutase and catalase but had no significant effects on ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monohydroascorbate reductase. The content of non-enzymatic antioxidants, such as ascorbate and dehydroascorbate, were decreased by melatonin treatment, while the content of glutathione and oxidized glutathione was not affected by melatonin. These results suggest that the suppression of dark-induced leaf senescence by exogenous melatonin may be associated with its roles in regulating ROS scavenging through activating the superoxide dismutase-catalase enzymatic antioxidant

Direct antioxidant properties of bilirubin andbiliverdin. Is there a role for biliverdin reductase?

Directory of Open Access Journals (Sweden)

Thomas eJansen

2012-03-01

Full Text Available Reactive oxygen species (ROS and signaling events are involved in the pathogenesis of endothelial dysfunction and represent a major contribution to vascular regulation. Molecular signaling is highly dependent on reactive oxygen species. But depending on the amount of ROS production it might have toxic or protective effects. Despite a large number of negative outcomes in large clinical trials (e.g. HOPE, HOPE-TOO, antioxidant molecules and agents are important players to influence the critical balance between production and elimination of RONS. However, chronic systemic antioxidant therapy lacks clinical efficacy, probably by interfering with important physiological redox signaling pathways. Therefore, it may be a much more promising attempt to induce intrinsic antioxidant pathways in order to increase the antioxidants not systemically but at the place of oxidative stress and complications. Among others, heme oxygenase (HO has been shown to be important for attenuating the overall production of ROS in a broad range of disease states through its ability to degrade heme and to produce carbon monoxide (CO, biliverdin/bilirubin, and the release of free iron with subsequent ferritin induction. With the present review we would like to highlight the important antioxidant role of the heme oxygenase system and especially discuss the contribution of the biliverdin, bilirubin and biliverdin reductase to these beneficial effects. The bilierdin reductase was reported to confer an antioxidant redox amplification cycle by which low, physiological bilirubin concentrations confer potent antioxidant protection via recycling of biliverdin from oxidized bilirubin by the biliverdin reductase, linking this sink for oxidants to the NADPH pool. To date the existence and role of this antioxidant redox cycle is still under debate and we present and discuss the pros and cons as well as our own findings on this topic.

Ketopantoyl-lactone reductase from Candida parapsilosis: purification and characterization as a conjugated polyketone reductase.

Science.gov (United States)

Hata, H; Shimizu, S; Hattori, S; Yamada, H

1989-02-24

Ketopantoyl-lactone reductase (2-dehydropantoyl-lactone reductase, EC 1.1.1.168) was purified and crystallized from cells of Candida parapsilosis IFO 0708. The enzyme was found to be homogeneous on ultracentrifugation, high-performance gel-permeation liquid chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of the native and SDS-treated enzyme is approximately 40,000. The isoelectric point of the enzyme is 6.3. The enzyme was found to catalyze specifically the reduction of a variety of natural and unnatural polyketones and quinones other than ketopantoyl lactone in the presence of NADPH. Isatin and 5-methylisatin are rapidly reduced by the enzyme, the Km and Vmax values for isatin being 14 microM and 306 mumol/min per mg protein, respectively. Ketopantoyl lactone is also a good substrate (Km = 333 microM and Vmax = 481 mumol/min per mg protein). Reverse reaction was not detected with pantoyl lactone and NADP+. The enzyme is inhibited by quercetin, several polyketones and SH-reagents. 3,4-Dihydroxy-3-cyclobutene-1,2-dione, cyclohexenediol-1,2,3,4-tetraone and parabanic acid are uncompetitive inhibitors for the enzyme, the Ki values being 1.4, 0.2 and 3140 microM, respectively, with isatin as substrate. Comparison of the enzyme with the conjugated polyketone reductase of Mucor ambiguus (S. Shimizu, H. Hattori, H. Hata and H. Yamada (1988) Eur. J. Biochem. 174, 37-44) and ketopantoyl-lactone reductase of Saccharomyces cerevisiae suggested that ketopantoyl-lactone reductase is a kind of conjugated polyketone reductase.

Purification of nitrate reductase from Nicotiana plumbaginifolia by affinity chromatography using 5'AMP-sepharose and monoclonal antibodies.

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Moureaux, T; Leydecker, M T; Meyer, C

1989-02-15

Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5'AMP-Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose-1,5-bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000-fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21,000-fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107-kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotiana tabacum cv Xanthi).

The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis.

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Kavanagh, Kathryn L; Klimacek, Mario; Nidetzky, Bernd; Wilson, David K

2002-07-16

Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.

The aldo-keto reductase superfamily homepage.

Science.gov (United States)

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

2003-02-01

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 www.med.upenn.edu/akr). 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.

Tetrathionate reductase of Salmonella thyphimurium: a molybdenum containing enzyme

International Nuclear Information System (INIS)

Hinojosa-Leon, M.; Dubourdieu, M.; Sanchez-Crispin, J.A.; Chippaux, M.

1986-01-01

Use of radioactive molybdenum demonstrates that the tetrathionate reductase of Salmonella typhimurium is a molydenum containing enzyme. It is proposed that this enzyme shares with other molybdo-proteins, such as nitrate reductase, a common molybdenum containing cofactor the defect of which leads to the loss of the tetrathionate reductase and nitrate reductase activities

[Involvement of carbonate/bicarbonate ions in the superoxide-generating reaction of adrenaline autoxidation].

Science.gov (United States)

Sirota, T V

2015-01-01

An important role of carbonate/bicarbonate ions has been recognized in the superoxide generating reaction of adrenaline autooxidation in an alkaline buffer (a model of quinoid adrenaline oxidation in the body). It is suggested that these ions are directly involved not only in formation of superoxide anion radical (О(2)(-)) but also other radicals derived from the carbonate/bicarbonate buffer. Using various buffers it was shown that the rate of accumulation of adrenochrome, the end product of adrenaline oxidation, and the rate of О(2)(-)· formation depend on concentration of carbonate/bicarbonate ions in the buffer and that these ions significantly accelerate adrenaline autooxidation thus demonstrating prooxidant properties. The detectable amount of diformazan, the product of nitro blue tetrazolium (NBT) reduction, was significantly higher than the amount of adrenochrome formed; taking into consideration the literature data on О(2)(-)· detection by NBT it is suggested that adrenaline autooxidation is accompanied by one-electron reduction not only of oxygen dissolved in the buffer and responsible for superoxide formation but possible carbon dioxide also dissolved in the buffer as well as carbonate/bicarbonate buffer components leading to formation of corresponding radicals. The plots of the dependence of the inhibition of adrenochrome and diformazan formation on the superoxide dismutase concentration have shown that not only superoxide radicals are formed during adrenaline autooxidation. Since carbonate/bicarbonate ions are known to be universally present in the living nature, their involvement in free radical processes proceeding in the organism is discussed.

Potentiating antibiotics in drug-resistant clinical isolates via stimuli-activated superoxide generation.

Science.gov (United States)

Courtney, Colleen M; Goodman, Samuel M; Nagy, Toni A; Levy, Max; Bhusal, Pallavi; Madinger, Nancy E; Detweiler, Corrella S; Nagpal, Prashant; Chatterjee, Anushree

2017-10-01

The rise of multidrug-resistant (MDR) bacteria is a growing concern to global health and is exacerbated by the lack of new antibiotics. To treat already pervasive MDR infections, new classes of antibiotics or antibiotic adjuvants are needed. Reactive oxygen species (ROS) have been shown to play a role during antibacterial action; however, it is not yet understood whether ROS contribute directly to or are an outcome of bacterial lethality caused by antibiotics. We show that a light-activated nanoparticle, designed to produce tunable flux of specific ROS, superoxide, potentiates the activity of antibiotics in clinical MDR isolates of Escherichia coli , Salmonella enterica , and Klebsiella pneumoniae . Despite the high degree of antibiotic resistance in these isolates, we observed a synergistic interaction between both bactericidal and bacteriostatic antibiotics with varied mechanisms of action and our superoxide-producing nanoparticles in more than 75% of combinations. As a result of this potentiation, the effective antibiotic concentration of the clinical isolates was reduced up to 1000-fold below their respective sensitive/resistant breakpoint. Further, superoxide-generating nanoparticles in combination with ciprofloxacin reduced bacterial load in epithelial cells infected with S. enterica serovar Typhimurium and increased Caenorhabditis elegans survival upon infection with S. enterica serovar Enteriditis, compared to antibiotic alone. This demonstration highlights the ability to engineer superoxide generation to potentiate antibiotic activity and combat highly drug-resistant bacterial pathogens.

The essential dynamics of Cu, Zn superoxide dismutase : Suggestion of intersubunit communication

NARCIS (Netherlands)

Chillemi, G; Falconi, M; Amadei, A; Zimatore, G; Desideri, A; DiNola, A

A 300-ps molecular dynamics simulation of the whole Cu, Zn superoxide dismutase dimer has been carried out in water, and the trajectory has been analyzed by the essential dynamics method. The results indicate that the motion is defined by few preferred directions identified by the first four to six

Structure and mechanism of dimethylsulfoxide reductase, a molybdopterin-containing enzyme of DMSO reductase family

International Nuclear Information System (INIS)

McEwan, A.G.; Ridge, J.P.; McDevitt, C.A.; Hanson, G.R.

2001-01-01

Full text: Apart from nitrogenase, enzymes containing molybdenum are members of a superfamily, the molybdopterin-containing enzymes. Most of these enzymes catalyse an oxygen atom transfer and two electron transfer reaction. During catalysis the Mo at the active site cycles between the Mo(VI) and Mo(IV) states. The DMSO reductase family of molybdopterin-containing enzymes all contain a bis(molybdopterin guanine dinucleotide)Mo cofactor and over thirty examples have now been described. Over the last five years crystal structures of dimethylsulfoxide (DMSO) reductase and four other enzymes of the DMSO reductase family have revealed that enzymes of this family have a similar tertiary structure. The Mo atom at the active site is coordinated by four thiolate ligands provided by the dithiolene side chains of the two MGD molecules of the bis(MGD)Mo cofactor as well as a ligand provided by an amino acid side chain. In addition, an oxygen atom in the form of an oxo, hydroxo or aqua group is also coordinated to the Mo atom. In the case of dimethylsulfoxide reductase X-ray crystallography of the product-reduced species and Raman spectroscopy has demonstrated that the enzyme contains a single exchangeable oxo group that is H-bonded to W116

The Drosophila carbonyl reductase sniffer prevents oxidative stress-induced neurodegeneration.

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Botella, Jose A; Ulschmid, Julia K; Gruenewald, Christoph; Moehle, Christoph; Kretzschmar, Doris; Becker, Katja; Schneuwly, Stephan

2004-05-04

A growing body of evidence suggests that oxidative stress is a common underlying mechanism in the pathogenesis of neurodegenerative disorders such as Alzheimer's, Huntington's, Creutzfeld-Jakob and Parkinson's diseases. Despite the increasing number of reports finding a causal relation between oxidative stress and neurodegeneration, little is known about the genetic elements that confer protection against the deleterious effects of oxidation in neurons. We have isolated and characterized the Drosophila melanogaster gene sniffer, whose function is essential for preventing age-related neurodegeneration. In addition, we demonstrate that oxidative stress is a direct cause of neurodegeneration in the Drosophila central nervous system and that reduction of sniffer activity leads to neuronal cell death. The overexpression of the gene confers neuronal protection against oxygen-induced apoptosis, increases resistance of flies to experimental normobaric hyperoxia, and improves general locomotor fitness. Sniffer belongs to the family of short-chain dehydrogenase/reductase (SDR) enzymes and exhibits carbonyl reductase activity. This is the first in vivo evidence of the direct and important implication of this enzyme as a neuroprotective agent in the cellular defense mechanisms against oxidative stress.

BIOLOGICAL ROLE OF ALDO-KETO REDUCTASES IN RETINOIC ACID BIOSYNTHESIS AND SIGNALING

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F. Xavier eRuiz

2012-04-01

Full Text Available Several aldo-keto reductase (AKR enzymes from subfamilies 1B and 1C show retinaldehyde reductase activity, having low Km and kcat values. Only AKR1B10 and 1B12, with all-trans-retinaldehyde, and AKR1C3, with 9-cis-retinaldehyde, display high catalytic efficiency. Major structural determinants for retinaldehyde isomer specificity are located in the external loops (A and C for AKR1B10, and B for AKR1C3, as assessed by site-directed mutagenesis and molecular dynamics. Cellular models have shown that AKR1B and 1C enzymes are well suited to work in vivo as retinaldehyde reductases and to regulate retinoic acid (RA biosynthesis at hormone pre-receptor level. An additional physiological role for the retinaldehyde reductase activity of these enzymes, consistent with their tissue localization, is their participation in β-carotene absorption. Retinaldehyde metabolism may be subjected to subcellular compartmentalization, based on enzyme localization. While retinaldehyde oxidation to RA takes place in the cytosol, reduction to retinol could take place in the cytosol by AKRs or in the membranes of endoplasmic reticulum by microsomal retinaldehyde reductases. Upregulation of some AKR1 enzymes in different cancer types may be linked to their induction by oxidative stress and to their participation in different signaling pathways related to cell proliferation. AKR1B10 and AKR1C3, through their retinaldehyde reductase activity, trigger a decrease in the RA biosynthesis flow, resulting in RA deprivation and consequently lower differentiation, with an increased cancer risk in target tissues. Rational design of selective AKR inhibitors could lead to development of novel drugs for cancer treatment as well as reduction of chemotherapeutic drug resistance.

Expression, purification and molecular structure modeling of thioredoxin (Trx) and thioredoxin reductase (TrxR) from Acidithiobacillus ferrooxidans.

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Wang, Yiping; Zhang, Xiaojian; Liu, Qing; Ai, Chenbing; Mo, Hongyu; Zeng, Jia

2009-07-01

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, which plays several key roles in maintaining the redox environment of the cell. In Acidithiobacillus ferrooxidans, thioredoxin system may play important functions in the activity regulation of periplasmic proteins and energy metabolism. Here, we cloned thioredoxin (trx) and thioredoxin reductase (trxR) genes from Acidithiobacillus ferrooxidans, and expressed the genes in Escherichia coli. His-Trx and His-TrxR were purified to homogeneity with one-step Ni-NTA affinity column chromatography. Site-directed mutagenesis results confirmed that Cys33, Cys36 of thioredoxin, and Cys142, Cys145 of thioredoxin reductase were active-site residues.

Direct antioxidant properties of methotrexate: Inhibition of malondialdehyde-acetaldehyde-protein adduct formation and superoxide scavenging

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Matthew C. Zimmerman

2017-10-01

Full Text Available Methotrexate (MTX is an immunosuppressant commonly used for the treatment of autoimmune diseases. Recent observations have shown that patients treated with MTX also exhibit a reduced risk for the development of cardiovascular disease (CVD. Although MTX reduces systemic inflammation and tissue damage, the mechanisms by which MTX exerts these beneficial effects are not entirely known. We have previously demonstrated that protein adducts formed by the interaction of malondialdehyde (MDA and acetaldehyde (AA, known as MAA-protein adducts, are present in diseased tissues of individuals with rheumatoid arthritis (RA or CVD. In previously reported studies, MAA-adducts were shown to be highly immunogenic, supporting the concept that MAA-adducts not only serve as markers of oxidative stress but may have a direct role in the pathogenesis of inflammatory diseases. Because MAA-adducts are commonly detected in diseased tissues and are proposed to mitigate disease progression in both RA and CVD, we tested the hypothesis that MTX inhibits the generation of MAA-protein adducts by scavenging reactive oxygen species. Using a cell free system, we found that MTX reduces MAA-adduct formation by approximately 6-fold, and scavenges free radicals produced during MAA-adduct formation. Further investigation revealed that MTX directly scavenges superoxide, but not hydrogen peroxide. Additionally, using the Nrf2/ARE luciferase reporter cell line, which responds to intracellular redox changes, we observed that MTX inhibits the activation of Nrf2 in cells treated with MDA and AA. These studies define previously unrecognized mechanisms by which MTX can reduce inflammation and subsequent tissue damage, namely, scavenging free radicals, reducing oxidative stress, and inhibiting MAA-adduct formation.

A Suppressor of the Menadione-Hypersensitive Phenotype of a Xanthomonas campestris pv. phaseoli oxyR Mutant Reveals a Novel Mechanism of Toxicity and the Protective Role of Alkyl Hydroperoxide Reductase

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Vattanaviboon, Paiboon; Whangsuk, Wirongrong; Mongkolsuk, Skorn

2003-01-01

We isolated menadione-resistant mutants of Xanthomonas campestris pv. phaseoli oxyR (oxyRXp). The oxyRR2Xp mutant was hyperresistant to the superoxide generators menadione and plumbagin and was moderately resistant to H2O2 and tert-butyl hydroperoxide. Analysis of enzymes involved in oxidative-stress protection in the oxyRR2Xp mutant revealed a >10-fold increase in AhpC and AhpF levels, while the levels of superoxide dismutase (SOD), catalase, and the organic hydroperoxide resistance protein (Ohr) were not significantly altered. Inactivation of ahpC in the oxyRR2Xp mutant resulted in increased sensitivity to menadione killing. Moreover, high levels of expression of cloned ahpC and ahpF in the oxyRXp mutant complemented the menadione hypersensitivity phenotype. High levels of other oxidant-scavenging enzymes such as catalase and SOD did not protect the cells from menadione toxicity. These data strongly suggest that the toxicity of superoxide generators could be mediated via organic peroxide production and that alkyl hydroperoxide reductase has an important novel function in the protection against the toxicity of these compounds in X. campestris. PMID:12591894

Endogenous superoxide is a key effector of the oxygen sensitivity of a model obligate anaerobe.

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Lu, Zheng; Sethu, Ramakrishnan; Imlay, James A

2018-04-03

It has been unclear whether superoxide and/or hydrogen peroxide play important roles in the phenomenon of obligate anaerobiosis. This question was explored using Bacteroides thetaiotaomicron , a major fermentative bacterium in the human gastrointestinal tract. Aeration inactivated two enzyme families-[4Fe-4S] dehydratases and nonredox mononuclear iron enzymes-whose homologs, in contrast, remain active in aerobic Escherichia coli Inactivation-rate measurements of one such enzyme, B. thetaiotaomicron fumarase, showed that it is no more intrinsically sensitive to oxidants than is an E. coli fumarase. Indeed, when the E. coli enzymes were expressed in B. thetaiotaomicron , they no longer could tolerate aeration; conversely, the B. thetaiotaomicron enzymes maintained full activity when expressed in aerobic E. coli Thus, the aerobic inactivation of the B. thetaiotaomicron enzymes is a feature of their intracellular environment rather than of the enzymes themselves. B. thetaiotaomicron possesses superoxide dismutase and peroxidases, and it can repair damaged enzymes. However, measurements confirmed that the rate of reactive oxygen species production inside aerated B. thetaiotaomicron is far higher than in E. coli Analysis of the damaged enzymes recovered from aerated B. thetaiotaomicron suggested that they had been inactivated by superoxide rather than by hydrogen peroxide. Accordingly, overproduction of superoxide dismutase substantially protected the enzymes from aeration. We conclude that when this anaerobe encounters oxygen, its internal superoxide levels rise high enough to inactivate key catabolic and biosynthetic enzymes. Superoxide thus comprises a major element of the oxygen sensitivity of this anaerobe. The extent to which molecular oxygen exerts additional direct effects remains to be determined.

Overexpression of chloroplast NADPH-dependent thioredoxin reductase in Arabidopsis enhances leaf growth and elucidates in-vivo function of reductase and thioredoxin domains

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Jouni eToivola

2013-10-01

Full Text Available Plant chloroplasts have versatile thioredoxin systems including two thioredoxin reductases and multiple types of thioredoxins. Plastid-localized NADPH-dependent thioredoxin reductase (NTRC contains both reductase (NTRd and thioredoxin (TRXd domains in a single polypeptide and forms homodimers. To study the action of NTRC and NTRC domains in vivo, we have complemented the ntrc knockout line of Arabidopsis with the wild type and full-length NTRC genes, in which 2-Cys motifs either in NTRd, or in TRXd were inactivated. The ntrc line was also transformed either with the truncated NTRd or TRXd alone. Overexpression of wild-type NTRC promoted plant growth by increasing leaf size and biomass yield of the rosettes. Complementation of the ntrc line with the full-length NTRC gene containing an active reductase but an inactive thioredoxin domain, or vice versa, recovered wild-type chloroplast phenotype and, partly, rosette biomass production, indicating that the NTRC domains are capable of interacting with other chloroplast thioredoxin systems. Overexpression of truncated NTRd or TRXd in ntrc background did not restore wild-type phenotype. Modelling of the 3-dimensional structure of the NTRC dimer indicates extensive interactions between the NTR domains and the TRX domains further stabilize the dimeric structure. The long linker region between the NTRd and TRXd, however, allows flexibility for the position of the TRXd in the dimer. Supplementation of the TRXd in the NTRC homodimer model by free chloroplast thioredoxins indicated that TRXf is the most likely partner to interact with NTRC. We propose that overexpression of NTRC promotes plant biomass yield both directly by stimulation of chloroplast biosynthetic and protected pathways controlled by NTRC and indirectly via free chloroplast thioredoxins. Our data indicate that overexpression of chloroplast thiol redox-regulator has a potential to increase biofuel yield in plant and algal species suitable for

Manumycin A Is a Potent Inhibitor of Mammalian Thioredoxin Reductase-1 (TrxR-1).

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Tuladhar, Anupama; Rein, Kathleen S

2018-04-12

The anticancer effect of manumycin A (Man A) has been attributed to the inhibition of farnesyl transferase (FTase), an enzyme that is responsible for post-translational modification of Ras proteins. However, we have discovered that Man A inhibits mammalian cytosolic thioredoxin reductase 1 (TrxR-1) in a time-dependent manner, with an IC 50 of 272 nM with preincubation and 1586 nM without preincubation. The inhibition of TrxR-1 by Man A is irreversible and is the result of a covalent interaction between Man A and TrxR-1. Evidence presented herein demonstrates that Man A forms a Michael adduct with the selenocysteine residue, which is located in the C-terminal redox center of TrxR-1. Inhibitors of TrxR-1, which act through this mechanism, convert TrxR-1 into a SecTRAP, which utilizes NADPH to reduce oxygen to superoxide radical anion (O 2 -• ).

Synthesis and Activity of a New Series of(Z-3-Phenyl-2-benzoylpropenoic Acid Derivatives as Aldose Reductase Inhibitors

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Shao-Jie Wang

2007-04-01

Full Text Available During the course of studies directed towards the discovery of novel aldose reductase inhibitors for the treatment of diabetic complications, we synthesized a series of new (Z-3-phenyl-2-benzoylpropenoic acid derivatives and tested their in vitro inhibitory activities on rat lens aldose reductase. Of these compounds, (Z-3-(3,4-dihydroxyphenyl-2-(4-methylbenzoylpropenoicacid(3k was identified as the most potent inhibitor, with an IC50 of 0.49μM. The theoretical binding mode of 3k was obtained by simulation of its docking into the active site of the human aldose reductase crystal structure.

Cloning and sequence of the human adrenodoxin reductase gene

International Nuclear Information System (INIS)

Lin, Dong; Shi, Y.; Miller, W.L.

1990-01-01

Adrenodoxin reductase is a flavoprotein mediating electron transport to all mitochondrial forms of cytochrome P450. The authors cloned the human adrenodoxin reductase gene and characterized it by restriction endonuclease mapping and DNA sequencing. The entire gene is approximately 12 kilobases long and consists of 12 exons. The first exon encodes the first 26 of the 32 amino acids of the signal peptide, and the second exon encodes the remainder of signal peptide and the apparent FAD binding site. The remaining 10 exons are clustered in a region of only 4.3 kilobases, separated from the first two exons by a large intron of about 5.6 kilobases. Two forms of human adrenodoxin reductase mRNA, differing by the presence or absence of 18 bases in the middle of the sequence, arise from alternate splicing at the 5' end of exon 7. This alternately spliced region is directly adjacent to the NADPH binding site, which is entirely contained in exon 6. The immediate 5' flanking region lacks TATA and CAAT boxes; however, this region is rich in G+C and contains six copies of the sequence GGGCGGG, resembling promoter sequences of housekeeping genes. RNase protection experiments show that transcription is initiated from multiple sites in the 5' flanking region, located about 21-91 base pairs upstream from the AUG translational initiation codon

A quantitative and direct PCR assay for the subspecies-specific detection of Clavibacter michiganensis subsp. michiganensis based on a ferredoxin reductase gene.

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Cho, Min Seok; Lee, Jang Ha; Her, Nam Han; Kim, Changkug; Seol, Young-Joo; Hahn, Jang Ho; Baeg, Ji Hyoun; Kim, Hong Gi; Park, Dong Suk

2012-06-01

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis is the causal agent of canker disease in tomato. Because it is very important to control newly introduced inoculum sources from commercial materials, the specific detection of this pathogen in seeds and seedlings is essential for effective disease control. In this study, a novel and efficient assay for the detection and quantitation of C. michiganensis subsp. michiganensis in symptomless tomato and red pepper seeds was developed. A pair of polymerase chain reaction (PCR) primers (Cmm141F/R) was designed to amplify a specific 141 bp fragment on the basis of a ferredoxin reductase gene of C. michiganensis subsp. michiganensis NCPPB 382. The specificity of the primer set was evaluated using purified DNA from 16 isolates of five C. michiganensis subspecies, one other Clavibacter species, and 17 other reference bacteria. The primer set amplified a single band of expected size from the genomic DNA obtained from the C. michiganensis subsp. michiganensis strains but not from the other C. michiganensis subspecies or from other Clavibacter species. The detection limit was a single cloned copy of the ferredoxin reductase gene of C. michiganensis subsp. michiganensis. In conclusion, this quantitative direct PCR assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of seeds and seedlings with a low level or latent infection of C. michiganensis subsp. michiganensis.

Mitochondrial fumarate reductase as a target of chemotherapy: from parasites to cancer cells.

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Sakai, Chika; Tomitsuka, Eriko; Esumi, Hiroyasu; Harada, Shigeharu; Kita, Kiyoshi

2012-05-01

Recent research on respiratory chain of the parasitic helminth, Ascaris suum has shown that the mitochondrial NADH-fumarate reductase system (fumarate respiration), which is composed of complex I (NADH-rhodoquinone reductase), rhodoquinone and complex II (rhodoquinol-fumarate reductase) plays an important role in the anaerobic energy metabolism of adult parasites inhabiting hosts. The enzymes in these parasite-specific pathways are potential target for chemotherapy. We isolated a novel compound, nafuredin, from Aspergillus niger, which inhibits NADH-fumarate reductase in helminth mitochondria at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep indicating that mitochondrial complex I is a promising target for chemotherapy. In addition to complex I, complex II is a good target because its catalytic direction is reverse of succinate-ubiquionone reductase in the host complex II. Furthermore, we found atpenin and flutolanil strongly and specifically inhibit mitochondrial complex II. Interestingly, fumarate respiration was found not only in the parasites but also in some types of human cancer cells. Analysis of the mitochondria from the cancer cells identified an anthelminthic as a specific inhibitor of the fumarate respiration. Role of isoforms of human complex II in the hypoxic condition of cancer cells and fetal tissues is a challenge. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010. Copyright © 2011 Elsevier B.V. All rights reserved.

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

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Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

2001-04-03

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.

Unraveling the role of animal heme peroxidases in superoxide mediated Mn oxide formation

Science.gov (United States)

Learman, D. R.; Hansel, C. M.

2013-12-01

Manganese(III,IV) oxides are important in the environment as they can impact the fate of a broad range of nutrients (e.g. carbon and phosphate) and contaminates (e.g. lead and chromium). Bacteria play a valuable role in the production of Mn oxides, yet the mechanisms and physiological reasons remain unclear. Roseobacter sp. AzwK-3b, an organism within the abundant and ubiquitous Roseobacter clade, has recently been shown to oxidize Mn(II) via a novel pathway that involves enzymatic extracellular superoxide production. However, in reactions with only Mn(II) and abiotically generated superoxide, we find superoxide alone is not enough to produce Mn(III,IV) oxides. Scavenging of the byproduct hydrogen peroxide (via the addition of catalase) is required to generate Mn oxides via abiotic reaction of Mn(II) with superoxide. Thus, R. AzwK-3b must produce superoxide and also scavenge hydrogen peroxide to form Mn oxides. Further, in-gel Mn(II) oxidation assay revealed a protein band that could generate Mn oxides in the presence of soluble Mn(II). This Mn(II)-oxidizing protein band was excised from the gel and the peptides identified via mass spectrometry. An animal heme peroxidase (AHP) was the predominant protein found in this band. This protein is homologous to the AHPs previously implicated as a Mn(II)-oxidizing enzyme within the Alphaproteobacteria, Erythrobacter SD-21 and Aurantimonas manganoxydans strain SI85-9A1. Currently, protein expression of the AHPs in R. AzwK-3b is being examined to determine if expression is correlated with Mn(II) concentration or oxidative stress. Our data suggests that AHPs do not directly oxidize Mn(II) but rather plays a role in scavenging hydrogen peroxide and/or producing an organic Mn(III) ligand that complexes Mn(III) and likely aids in Mn oxide precipitation.

Bioinformatics approach of three partial polyprenol reductase genes in Kandelia obovata

Science.gov (United States)

Basyuni, M.; Wati, R.; Sagami, H.; Oku, H.; Baba, S.

2018-03-01

This present study describesthe bioinformatics approach to analyze three partial polyprenol reductase genes from mangrove plant, Kandeliaobovataas well aspredictedphysical and chemical properties, potential peptide, subcellular localization, and phylogenetic. The diversity was noted in the physical and chemical properties of three partial polyprenol reductase genes. The values of chloroplast were relatively high, showed that chloroplast transit peptide occurred in mangrove polyprenol reductase. The target peptide value of mitochondria varied from 0.088 to 0.198 indicated it was possible to be present. These results suggested the importance of understanding the diversity of physicochemical properties of the different amino acids in polyprenol reductase. The subcellular localization of two partial genes located in the plasma membrane. To confirm the homology among the polyprenol reductase in the database, a dendrogram was drawn. The phylogenetic tree depicts that there are three clusters, the partial genes of K. obovata joined the largest one: C23157 was close to Ricinus communis polyprenol reductase. Whereas, C23901 and C24171 were grouped with Ipomoea nil polyprenol reductase, suggested that these polyprenol reductase genes form distinct separation into tropical habitat plants.

Periplasmic Cu,Zn superoxide dismutase and cytoplasmic Dps concur in protecting Salmonella enterica serovar Typhimurium from extracellular reactive oxygen species.

Science.gov (United States)

Pacello, Francesca; Ceci, Pierpaolo; Ammendola, Serena; Pasquali, Paolo; Chiancone, Emilia; Battistoni, Andrea

2008-02-01

Several bacteria possess periplasmic Cu,Zn superoxide dismutases which can confer protection from extracellular reactive oxygen species. Thus, deletion of the sodC1 gene reduces Salmonella enterica serovar Typhimurium ability to colonize the spleens of wild type mice, but enhances virulence in p47phox mutant mice. To look into the role of periplamic Cu,Zn superoxide dismutase and into possible additive effects of the ferritin-like Dps protein involved in hydrogen peroxide detoxification, we have analyzed bacterial survival in response to extracellular sources of superoxide and/or hydrogen peroxide. Exposure to extracellular superoxide of Salmonella Typhimurium mutant strains lacking the sodC1 and sodC2 genes and/or the dps gene does not cause direct killing of bacteria, indicating that extracellular superoxide is poorly bactericidal. In contrast, all mutant strains display a sharp hydrogen peroxide-dependent loss of viability, the dps,sodC1,sodC2 mutant being less resistant than the dps or the sodC1,sodC2 mutants. These findings suggest that the role of Cu,Zn superoxide dismutase in bacteria is to remove rapidly superoxide from the periplasm to prevent its reaction with other reactive molecules. Moreover, the nearly additive effect of the sodC and dps mutations suggests that localization of antioxidant enzymes in different cellular compartments is required for bacterial resistance to extracytoplasmic oxidative attack.

Menadione enhances oxyradical formation in earthworm extracts: vulnerability of earthworms to quinone toxicity

International Nuclear Information System (INIS)

Osman, A.M.; Besten, P.J. den; Noort, P.C.M. van

2003-01-01

NAD(P)H-cytochrome c reductase activities have been determined in the earthworms, L. rubellus and A. chlorotica, extracts. Menadione (0.35 mM, maximum concentration tested) was found to stimulate the rates of NADPH- and NADH-dependent cytochrome c reduction by three- and twofold, respectively. Superoxide dismutase (SOD) inhibited completely this menadione-mediated stimulation, suggesting that ·O 2 - is involved in the redox cycling of menadione. However, SOD had no effect on the basal activity (activity in the absence of quinone) in the case of NADH-dependent cytochrome c reduction, whereas it partially inhibited the basal activity of NADPH-cytochrome c reduction. This indicates direct electron transfer in the former case and the formation of superoxide anion in the latter. DT-diaphorase, measured as the dicumarol-inhibitable part of menadione reductase activity, was not detectable in the earthworms' extracts. In contrast, it was found that DT-diaphorase represents about 70% of the menadione reductase activities in the freshwater mussel, Dreissena polymorpha. The results of this work suggest that earthworms, compared with mussels, could be more vulnerable to oxidative stress from quinones due to lack, or very low level of DT-diaphorase, an enzyme considered to play a significant role in the detoxification of quinones. On the contrary, mussels have efficient DT-diaphorase, which catalyzes two-electron reduction of menadione directly to hydroquinone, thus circumventing the formation of semiquinone

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

Science.gov (United States)

Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki , Gang; David R. , Sarkanen; Simo , Ford; Joshua D.

2003-10-21

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.

Superoxide anion production by human neutrophils activated by Trichomonas vaginalis.

Science.gov (United States)

Song, Hyun-Ouk; Ryu, Jae-Sook

2013-08-01

Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2 (.-)) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.

Direct nitrate reductase assay versus microscopic observation drug susceptibility test for rapid detection of MDR-TB in Uganda.

Directory of Open Access Journals (Sweden)

Freddie Bwanga

Full Text Available The most common method for detection of drug resistant (DR TB in resource-limited settings (RLSs is indirect susceptibility testing on Lowenstein-Jensen medium (LJ which is very time consuming with results available only after 2-3 months. Effective therapy of DR TB is therefore markedly delayed and patients can transmit resistant strains. Rapid and accurate tests suitable for RLSs in the diagnosis of DR TB are thus highly needed. In this study we compared two direct techniques--Nitrate Reductase Assay (NRA and Microscopic Observation Drug Susceptibility (MODS for rapid detection of MDR-TB in a high burden RLS. The sensitivity, specificity, and proportion of interpretable results were studied. Smear positive sputum was collected from 245 consecutive re-treatment TB patients attending a TB clinic in Kampala, Uganda. Samples were processed at the national reference laboratory and tested for susceptibility to rifampicin and isoniazid with direct NRA, direct MODS and the indirect LJ proportion method as reference. A total of 229 specimens were confirmed as M. tuberculosis, of these interpretable results were obtained in 217 (95% with either the NRA or MODS. Sensitivity, specificity and kappa agreement for MDR-TB diagnosis was 97%, 98% and 0.93 with the NRA; and 87%, 95% and 0.78 with the MODS, respectively. The median time to results was 10, 7 and 64 days with NRA, MODS and the reference technique, respectively. The cost of laboratory supplies per sample was low, around 5 USD, for the rapid tests. The direct NRA and MODS offered rapid detection of resistance almost eight weeks earlier than with the reference method. In the study settings, the direct NRA was highly sensitive and specific. We consider it to have a strong potential for timely detection of MDR-TB in RLS.

Extracellular production and degradation of superoxide in the coral Stylophora pistillata and cultured Symbiodinium.

Directory of Open Access Journals (Sweden)

Eldad Saragosti

2010-09-01

Full Text Available Reactive oxygen species (ROS are thought to play a major role in cell death pathways and bleaching in scleractinian corals. Direct measurements of ROS in corals are conspicuously in short supply, partly due to inherent problems with ROS quantification in cellular systems.In this study we characterized the dynamics of the reactive oxygen species superoxide anion radical (O(2(- in the external milieu of the coral Stylophora pistillata. Using a sensitive, rapid and selective chemiluminescence-based technique, we measured extracellular superoxide production and detoxification activity of symbiont (non-bleached and aposymbiont (bleached corals, and of cultured Symbiodinium (from clades A and C. Bleached and non-bleached Stylophora fragments were found to produce superoxide at comparable rates of 10(-11-10(-9 mol O(2(- mg protein(-1 min(-1 in the dark. In the light, a two-fold enhancement in O(2(- production rates was observed in non-bleached corals, but not in bleached corals. Cultured Symbiodinium produced superoxide in the dark at a rate of . Light was found to markedly enhance O(2(- production. The NADPH Oxidase inhibitor Diphenyleneiodonium chloride (DPI strongly inhibited O(2(- production by corals (and more moderately by algae, possibly suggesting an involvement of NADPH Oxidase in the process. An extracellular O(2(- detoxifying activity was found for bleached and non-bleached Stylophora but not for Symbiodinium. The O(2(- detoxifying activity was partially characterized and found to resemble that of the enzyme superoxide dismutase (SOD.The findings of substantial extracellular O(2(- production as well as extracellular O(2(- detoxifying activity may shed light on the chemical interactions between the symbiont and its host and between the coral and its environment. Superoxide production by Symbiodinium possibly implies that algal bearing corals are more susceptible to an internal build-up of O(2(-, which may in turn be linked to oxidative stress

Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription

DEFF Research Database (Denmark)

Cheng, Chi-Lien; Acedo, Gregoria N; Kristensen, Michael

1992-01-01

can replace light in eliciting an increase of nitrate reductase mRNA accumulation in dark-adapted green Arabidopsis plants. We show further that sucrose alone is sufficient for the full expression of nitrate reductase genes in etiolated Arabidopsis plants. Finally, using a reporter gene, we show......Nitrate reductase, the first enzyme in nitrate assimilation, is located at the crossroad of two energy-consuming pathways: nitrate assimilation and carbon fixation. Light, which regulates the expression of many higher-plant carbon fixation genes, also regulates nitrate reductase gene expression....... Located in the cytosol, nitrate reductase obtains its reductant not from photosynthesis but from carbohydrate catabolism. This relationship prompted us to investigate the indirect role that light might play, via photosynthesis, in the regulation of nitrate reductase gene expression. We show that sucrose...

Nitric oxide and superoxide anion production in monocytes from children exposed to arsenic and lead in region Lagunera, Mexico

International Nuclear Information System (INIS)

Pineda-Zavaleta, Ana Patricia; Garcia-Vargas, Gonzalo; Borja-Aburto, Victor H.; Acosta-Saavedra, Leonor C.; Vera Aguilar, Eunice; Gomez-Munoz, Aristides; Cebrian, Mariano E.; Calderon-Aranda, Emma S.

2004-01-01

We evaluated in Mexican children environmentally exposed to arsenic and lead monocyte nitric oxide (NO) and superoxide anion production in response to direct activation with interferon-γ (IFN-γ) + lipopolysaccharide (LPS). The integrity of Th1-regulated cellular immune response when monocytes were indirectly activated was also evaluated. Most children lived near a primary lead smelter. Lead and arsenic contamination in soil and dust by far exceeded background levels. As levels in water were between 10 and 30 ppb. Most children (93%) had urinary arsenic (AsU) concentrations above 50 μg/l (range 16.75-465.75) and 65% had lead blood levels (PbB) above 10 μg/dl (range 3.47-49.19). Multivariate analyses showed that NO production in monocytes activated indirectly was negatively associated with both PbB and AsU. Superoxide production in directly activated monocytes was negatively associated with AsU but positively associated with PbB. The models including the interaction term for AsU and PbB suggested the possibility of a negative interaction for NO production and a positive interaction for superoxide. There were indications of differential gender-based associations, NO production in indirectly activated monocytes obtained from girls was negatively associated with AsU but not with PbB. Superoxide production was positively associated with PbB in both directly and indirectly activated monocytes from boys but the latter was negatively associated with AsU. These effects are consistent with immune system abnormalities observed in human populations exposed to Pb or As. Further studies in larger populations are required to characterize As and Pb interactions and the mechanism(s) underlying the observed effects

Cloning and nitrate induction of nitrate reductase mRNA

OpenAIRE

Cheng, Chi-Lien; Dewdney, Julia; Kleinhofs, Andris; Goodman, Howard M.

1986-01-01

Nitrate is the major source of nitrogen taken from the soil by higher plants but requires reduction to ammonia prior to incorporation into amino acids. The first enzyme in the reducing pathway is a nitrate-inducible enzyme, nitrate reductase (EC 1.6.6.1). A specific polyclonal antiserum raised against purified barley nitrate reductase has been used to immunoprecipitate in vivo labeled protein and in vitro translation products, demonstrating that nitrate induction increases nitrate reductase p...

Dark production of extracellular superoxide by the coral Porites astreoides and representative symbionts

Directory of Open Access Journals (Sweden)

Tong Zhang

2016-11-01

Full Text Available The reactive oxygen species (ROS superoxide has been implicated in both beneficial and detrimental processes in coral biology, ranging from pathogenic disease resistance to coral bleaching. Despite the critical role of ROS in coral health, there is a distinct lack of ROS measurements and thus an incomplete understanding of underpinning ROS sources and production mechanisms within coral systems. Here, we quantified in situ extracellular superoxide concentrations at the surfaces of aquaria-hosted Porites astreoides during a diel cycle. High concentrations of superoxide (~10’s of nM were present at coral surfaces, and these levels did not change significantly as a function of time of day. These results indicate that the coral holobiont produces extracellular superoxide in the dark, independent of photosynthesis. As a short-lived anion at physiological pH, superoxide has a limited ability to cross intact biological membranes. Further, removing surface mucus layers from the P. astreoides colonies did not impact external superoxide concentrations. We therefore attribute external superoxide derived from the coral holobiont under these conditions to the activity of the coral host epithelium, rather than mucus-derived epibionts or internal sources such as endosymbionts (e.g., Symbiodinium. However, endosymbionts likely contribute to internal ROS levels via extracellular superoxide production. Indeed, common coral symbionts, including multiple strains of Symbiodinium (clades A to D and the bacterium Endozoicomonas montiporae LMG 24815, produced extracellular superoxide in the dark and at low light levels. Further, representative P. astreoides symbionts, Symbiodinium CCMP2456 (clade A and E. montiporae, produced similar concentrations of superoxide alone and in combination with each other, in the dark and low light, and regardless of time of day. Overall, these results indicate that healthy, non-stressed P. astreoides and representative symbionts produce

5α-reductase activity in rat adipose tissue

International Nuclear Information System (INIS)

Zyirek, M.; Flood, C.; Longcope, C.

1987-01-01

We measured the 5 α-reductase activity in isolated cell preparations of rat adipose tissue using the formation of [ 3 H] dihydrotestosterone from [ 3 H] testosterone as an endpoint. Stromal cells were prepared from the epididymal fat pad, perinephric fat, and subcutaneous fat of male rats and from perinephric fat of female rats. Adipocytes were prepared from the epididymal fat pad and perinephric fat of male rats. Stromal cells from the epididymal fat pad and perinephric fat contained greater 5α-reductase activity than did the adipocytes from these depots. Stromal cells from the epididymal fat pad contained greater activity than those from perinephric and subcutaneous depots. Perinephric stromal cells from female rats were slightly more active than those from male rats. Estradiol (10 -8 M), when added to the medium, caused a 90% decrease in 5α-reductase activity. Aromatase activity was minimal, several orders of magnitude less than 5α-reductase activity in each tissue studied

Characterization of mitochondrial thioredoxin reductase from C. elegans

International Nuclear Information System (INIS)

Lacey, Brian M.; Hondal, Robert J.

2006-01-01

Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a k cat of 610 min -1 and a K m of 610 μM using E. coli thioredoxin as substrate. The reported k cat is 25% of the k cat of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate

Dietary sources of aldose reductase inhibitors: prospects for alleviating diabetic complications.

Science.gov (United States)

Saraswat, Megha; Muthenna, P; Suryanarayana, P; Petrash, J Mark; Reddy, G Bhanuprakash

2008-01-01

Activation of polyol pathway due to increased aldose reductase activity is one of the several mechanisms that have been implicated in the development of various secondary complications of diabetes. Though numerous synthetic aldose reductase inhibitors have been tested, these have not been very successful clinically. Therefore, a number of common plant/ natural products used in Indian culinary have been evaluated for their aldose reductase inhibitory potential in the present study. The aqueous extracts of 22 plant-derived materials were prepared and evaluated for the inhibitory property against rat lens and human recombinant aldose reductase. Specificity of these extracts towards aldose reductase was established by testing their ability to inhibit a closely related enzyme viz, aldehyde reductase. The ex vivo incubation of erythrocytes in high glucose containing medium was used to underscore the significance in terms of prevention of intracellular sorbitol accumulation. Among the 22 dietary sources tested, 10 showed considerable inhibitory potential against both rat lens and human recombinant aldose reductase. Prominent inhibitory property was found in spinach, cumin, fennel, lemon, basil and black pepper with an approximate IC50 of 0.2 mg/mL with an excellent selectivity towards aldose reductase. As against this, 10 to 20 times higher concentrations were required for 50% inhibition of aldehyde reductase. Reduction in the accumulation of intracellular sorbitol by the dietary extracts further substantiated their in vivo efficacy. The findings reported here indicate the scope of adapting life-style modifications in the form of inclusion of certain common sources in the diet for the management of diabetic complications.

5α-reductases in human physiology: an unfolding story.

Science.gov (United States)

Traish, Abdulmaged M

2012-01-01

5α-reductases are a family of isozymes expressed in a wide host of tissues including the central nervous system (CNS) and play a pivotal role in male sexual differentiation, development and physiology. A comprehensive literature search from 1970 to 2011 was made through PubMed and the relevant information was summarized. 5α reductases convert testosterone, progesterone, deoxycorticosterone, aldosterone and corticosterone into their respective 5α-dihydro-derivatives, which serve as substrates for 3α-hydroxysteroid dehydrogenase enzymes. The latter transforms these 5α-reduced metabolites into a subclass of neuroactive steroid hormones with distinct physiological functions. The neuroactive steroid hormones modulate a multitude of functions in human physiology encompassing regulation of sexual differentiation, neuroprotection, memory enhancement, anxiety, sleep and stress, among others. In addition, 5α -reductase type 3 is also implicated in the N-glycosylation of proteins via formation of dolichol phosphate. The family of 5α-reductases was targeted for drug development to treat pathophysiological conditions, such as benign prostatic hyperplasia and androgenetic alopecia. While the clinical use of 5α-reductase inhibitors was well established, the scope and the magnitude of the adverse side effects of such drugs, especially on the CNS, is still unrecognized due to lack of knowledge of the various physiological functions of this family of enzymes, especially in the CNS. There is an urgent need to better understand the function of 5α-reductases and the role of neuroactive steroids in human physiology in order to minimize the potential adverse side effects of inhibitors targeting 5α-reductases to treat benign prostatic hyperplasia and androgenic alopecia.

Effect of Low Level Cadmium Exposure on Superoxide Dismutase ...

African Journals Online (AJOL)

Purpose: To investigate the effect of low level cadmium (Cd) exposure on the activity of superoxide dismutase ... cancer, aging and a diversity of diseases [5]. Superoxide .... responsible for the long biological half-life of cadmium [12]. ... indicator of the balance between the damaging effects and the ... Scand J Work Environ.

Histochemical Localization of Glutathione Dependent NBT-Reductase in Mouse Skin

Institute of Scientific and Technical Information of China (English)

无

2001-01-01

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.

Multiple antioxidant proteins protect Chlorobaculum tepidum against oxygen and reactive oxygen species

DEFF Research Database (Denmark)

Li, Hui; Jubelirer, Sara; Garcia Costas, Amaya M

2009-01-01

include cytochrome bd quinol oxidase, NADH oxidase, rubredoxin oxygen oxidoreductase, several thiol peroxidases, alkyl hydroperoxide reductase, superoxide dismutase, methionine sulfoxide reductase, and rubrerythrin. To test the physiological functions of some of these proteins, ten genes were...

Crystallization and diffraction analysis of thioredoxin reductase from Streptomyces coelicolor

International Nuclear Information System (INIS)

Koháryová, Michaela; Brynda, Jiří; Řezáčová, Pavlína; Kollárová, Marta

2011-01-01

Thioredoxin reductase from S. coelicolor was crystallized and diffraction data were collected to 2.4 Å resolution. Thioredoxin reductases are homodimeric flavoenzymes that catalyze the transfer of electrons from NADPH to oxidized thioredoxin substrate. Bacterial thioredoxin reductases represent a promising target for the development of new antibiotics. Recombinant thioredoxin reductase TrxB from Streptomyces coelicolor was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from cryocooled crystals to 2.4 Å resolution using a synchrotron-radiation source. The crystals belonged to the primitive monoclinic space group P2 1 , with unit-cell parameters a = 82.9, b = 60.6, c = 135.4 Å, α = γ = 90.0, β = 96.5°

Menadione enhances oxyradical formation in earthworm extracts: vulnerability of earthworms to quinone toxicity

Energy Technology Data Exchange (ETDEWEB)

Osman, A.M.; Besten, P.J. den; Noort, P.C.M. van

2003-10-08

NAD(P)H-cytochrome c reductase activities have been determined in the earthworms, L. rubellus and A. chlorotica, extracts. Menadione (0.35 mM, maximum concentration tested) was found to stimulate the rates of NADPH- and NADH-dependent cytochrome c reduction by three- and twofold, respectively. Superoxide dismutase (SOD) inhibited completely this menadione-mediated stimulation, suggesting that {center_dot}O{sub 2}{sup -} is involved in the redox cycling of menadione. However, SOD had no effect on the basal activity (activity in the absence of quinone) in the case of NADH-dependent cytochrome c reduction, whereas it partially inhibited the basal activity of NADPH-cytochrome c reduction. This indicates direct electron transfer in the former case and the formation of superoxide anion in the latter. DT-diaphorase, measured as the dicumarol-inhibitable part of menadione reductase activity, was not detectable in the earthworms' extracts. In contrast, it was found that DT-diaphorase represents about 70% of the menadione reductase activities in the freshwater mussel, Dreissena polymorpha. The results of this work suggest that earthworms, compared with mussels, could be more vulnerable to oxidative stress from quinones due to lack, or very low level of DT-diaphorase, an enzyme considered to play a significant role in the detoxification of quinones. On the contrary, mussels have efficient DT-diaphorase, which catalyzes two-electron reduction of menadione directly to hydroquinone, thus circumventing the formation of semiquinone.

Light Sensitivity of Lactococcus lactis Thioredoxin Reductase

DEFF Research Database (Denmark)

Skjoldager, Nicklas

The thioredoxin system has evolved in all kingdoms of life acting as a key antioxidant system in the defense against oxidative stress. The thioredoxin system utilizes reducing equivalents from NADPH to reduce protein disulfide targets. The reducing equivalents are shuttled via a flavin and redox...... active dithiol motif in thioredoxin reductase (TrxR) to reduce the small ubiquitous thioredoxin (Trx). Trx in turn regulates the protein dithiol/disulfide balance by reduction of protein disulfide targets in e.g. ribonucleotide reductase, peroxiredoxins and methionine sulfoxide reductase. The glutathione......, thus expected to rely mainly on the Trx system for thiol-disulfide control. L. lactis is an important industrial microorganism used as starter culture in the dairy production of cheese, buttermilk etc. and known to be sensitive to oxidative stress. The L. lactis TrxR (LlTrxR) is a homodimeric...

Effect of Oxidative Damage on the Stability and Dimerization of Superoxide Dismutase 1

OpenAIRE

Petrov, Drazen; Daura, Xavier; Zagrovic, Bojan

2016-01-01

During their life cycle, proteins are subject to different modifications involving reactive oxygen species. Such oxidative damage to proteins may lead to the formation of insoluble aggregates and cytotoxicity and is associated with age-related disorders including neurodegenerative diseases, cancer, and diabetes. Superoxide dismutase 1 (SOD1), a key antioxidant enzyme in human cells, is particularly susceptible to such modifications. Moreover, this homodimeric metalloenzyme has been directly l...

Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes.

Science.gov (United States)

Rodríguez-Dorantes, M; Lizano-Soberón, M; Camacho-Arroyo, I; Calzada-León, R; Morimoto, S; Téllez-Ascencio, N; Cerbón, M A

2002-03-01

The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.

The role of biliverdin reductase in colorectal cancer

International Nuclear Information System (INIS)

Bauer, M.

2010-01-01

In recent years, the effects of biliverdin and bilirubin have been studied extensively, and an inhibitory effect of bile pigments in cancer progression has been proposed. In this study we focused on the effects of biliverdin reductase, the enzyme that converts biliverdin to bilirubin, in colorectal cancer. For in vitro experiments we used a human colorectal carcinoma cell line and transfected it with an expression construct of shRNA specific for biliverdin reductase, to create cells with stable knock-down of enzyme expression. Cell proliferation was analyzed using the CASY model TT cell counting device. Western blot protein analysis was performed to study intracellular signaling cascades. Samples of human colorectal cancer were analyzed using immunohistochemistry. We were able to confirm the antiproliferative effects of bile pigments on cancer cells in vitro. However, this effect was attenuated in biliverdin reductase knock down cells. ERK and Akt activation seen under biliverdin and bilirubin treatment was also reduced in biliverdin reductase deficient cells. Immunohistochemical analysis of tumor samples from patients with colorectal cancer showed elevated biliverdin reductase levels. High enzyme expression was associated with lower overall and disease free patient survival. We conclude that BVR is required for bile pigment mediated effects regarding cancer cell proliferation and modulation of intracellular signaling cascades. The role of BVR overexpression in vivo and its exact influence on cancer progression and patient survival need to be further investigated. (author) [de

Superoxide dismutase amplifies organismal sensitivity to ionizing radiation

International Nuclear Information System (INIS)

Scott, M.D.; Meshnick, S.R.; Eaton, J.W.

1989-01-01

Although increased superoxide dismutase (SOD) activity is often associated with enhanced resistance of cells and organisms to oxidant challenges, few direct tests of the antioxidant importance of this enzyme have been carried out. To assess the importance of SOD in defending against gamma-radiation, we employed Escherichia coli with deficient, normal, and super-normal enzyme activities. Surprisingly, the radiation sensitivity of E. coli actually increases as bacterial SOD activity increases. Elevated intracellular SOD activity sensitizes E. coli to radiation-induced mortality, whereas SOD-deficient bacteria show normal or decreased radiosensitivity. Toxic effects of activated oxygen species are involved in this phenomenon; bacterial SOD activity has no effect on radiation sensitivity under anaerobic conditions or on the lethality of other, non-oxygen-dependent, toxins such as ultraviolet radiation

Non-selective regulation of peroxide and superoxide resistance genes by PerR in Campylobacter jejuni

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Jong-Chul eKim

2015-02-01

Full Text Available Campylobacter jejuni is an important foodborne pathogen. The molecular mechanisms for the regulation of oxidative stress resistance have not yet been understood fully in this bacterium. In this study, we investigated how PerR (peroxide stress regulator modulates the transcriptional regulation of both peroxide and superoxide resistance genes in C. jejuni, particularly under oxidative stress conditions. The transcriptional levels of ahpC, katA, and sodB were substantially increased by aeration and oxidant exposure. Interestingly, a perR mutation completely abrogated the transcriptional response of ahpC, katA and sodB to oxidants. Furthermore, we demonstrated that perR transcription was reduced by aeration and oxidant exposure. In contrast to the unique role of PerR homologs in peroxide stress regulation in other bacteria, interestingly, C. jejuni PerR directly regulates the transcription of sodB, the most important gene in superoxide defense, as evidenced by the alteration of sodB transcription by the perR mutation and direct binding of rPerR to the sodB promoter. In addition, we also observed notable morphological changes in C. jejuni from spiral rods to coccoid morphology under aerobic conditions. Based on the intracellular ATP levels, C. jejuni entered a viable-but-non-culturable state under aerobic conditions. These findings clearly demonstrate that C. jejuni possesses a unique regulatory mechanism of oxidative stress defense that does not specifically distinguish between peroxide and superoxide defense, and PerR plays a pivotal role in this non-selective regulation of oxidative stress resistance in C. jejuni.

Ab initio molecular dynamics of the reaction of quercetin with superoxide radical

International Nuclear Information System (INIS)

Lespade, Laure

2016-01-01

Highlights: • Ab initio molecular dynamics is performed to describe the reaction of quercetin and superoxide. • The reaction occurs near the sites 4′ and 7 when the system contains sufficiently water molecules. • The difference of reactivity of superoxide compared to commonly used radicals as DPPH · or ABTS ·+ is explained. - Abstract: Superoxide plays an important role in biology but in unregulated concentrations it is implicated in a lot of diseases such as cancer or atherosclerosis. Antioxidants like flavonoids are abundant in plant and are good scavengers of superoxide radical. The modeling of superoxide scavenging by flavonoids from the diet still remains a challenge. In this study, ab initio molecular dynamics of the reaction of the flavonoid quercetin toward superoxide radical has been carried out using Car–Parrinello density functional theory. The study has proven different reactant solvation by modifying the number of water molecules surrounding superoxide. The reaction consists in the gift of a hydrogen atom of one of the hydroxyl groups of quercetin to the radical. When it occurs, it is relatively fast, lower than 100 fs. Calculations show that it depends largely on the environment of the hydroxyl group giving its hydrogen atom, the geometry of the first water layer and the presence of a certain number of water molecules in the second layer, indicating a great influence of the solvent on the reactivity.

Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase

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Justin R. Prigge

2017-06-01

Full Text Available Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1 disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1 and glutathione reductase (Gsr, respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.

Kinetics of carbonyl reductase from human brain.

OpenAIRE

Bohren, K M; von Wartburg, J P; Wermuth, B

1987-01-01

Initial-rate analysis of the carbonyl reductase-catalysed reduction of menadione by NADPH gave families of straight lines in double-reciprocal plots consistent with a sequential mechanism being obeyed. The fluorescence of NADPH was increased up to 7-fold with a concomitant shift of the emission maximum towards lower wavelength in the presence of carbonyl reductase, and both NADPH and NADP+ caused quenching of the enzyme fluorescence, indicating formation of a binary enzyme-coenzyme complex. D...

Respiratory arsenate reductase as a bidirectional enzyme

Science.gov (United States)

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

2009-01-01

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.

YqhD. A broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals

Energy Technology Data Exchange (ETDEWEB)

Jarboe, Laura R. [Iowa State Univ., Ames, IA (United States). Dept. of Chemical and Biological Engineering

2011-01-15

The Escherichia coli NADPH-dependent aldehyde reductase YqhD has contributed to a variety of metabolic engineering projects for production of biorenewable fuels and chemicals. As a scavenger of toxic aldehydes produced by lipid peroxidation, YqhD has reductase activity for a broad range of short-chain aldehydes, including butyraldehyde, glyceraldehyde, malondialdehyde, isobutyraldehyde, methylglyoxal, propanealdehyde, acrolein, furfural, glyoxal, 3-hydroxypropionaldehyde, glycolaldehyde, acetaldehyde, and acetol. This reductase activity has proven useful for the production of biorenewable fuels and chemicals, such as isobutanol and 1,3- and 1,2-propanediol; additional capability exists for production of 1-butanol, 1-propanol, and allyl alcohol. A drawback of this reductase activity is the diversion of valuable NADPH away from biosynthesis. This YqhD-mediated NADPH depletion provides sufficient burden to contribute to growth inhibition by furfural and 5-hydroxymethyl furfural, inhibitory contaminants of biomass hydrolysate. The structure of YqhD has been characterized, with identification of a Zn atom in the active site. Directed engineering efforts have improved utilization of 3-hydroxypropionaldehyde and NADPH. Most recently, two independent projects have demonstrated regulation of yqhD by YqhC, where YqhC appears to function as an aldehyde sensor. (orig.)

Atomic Structure of Salutaridine Reductase from the Opium Poppy (Papaver somniferum)

Energy Technology Data Exchange (ETDEWEB)

Higashi, Yasuhiro; Kutchan, Toni M.; Smith, Thomas J. (Danforth)

2011-11-18

The opium poppy (Papaver somniferum L.) is one of the oldest known medicinal plants. In the biosynthetic pathway for morphine and codeine, salutaridine is reduced to salutaridinol by salutaridine reductase (SalR; EC 1.1.1.248) using NADPH as coenzyme. Here, we report the atomic structure of SalR to a resolution of {approx}1.9 {angstrom} in the presence of NADPH. The core structure is highly homologous to other members of the short chain dehydrogenase/reductase family. The major difference is that the nicotinamide moiety and the substrate-binding pocket are covered by a loop (residues 265-279), on top of which lies a large 'flap'-like domain (residues 105-140). This configuration appears to be a combination of the two common structural themes found in other members of the short chain dehydrogenase/reductase family. Previous modeling studies suggested that substrate inhibition is due to mutually exclusive productive and nonproductive modes of substrate binding in the active site. This model was tested via site-directed mutagenesis, and a number of these mutations abrogated substrate inhibition. However, the atomic structure of SalR shows that these mutated residues are instead distributed over a wide area of the enzyme, and many are not in the active site. To explain how residues distal to the active site might affect catalysis, a model is presented whereby SalR may undergo significant conformational changes during catalytic turnover.

Nitrate reductase activity and its relationship with applied nitrogen in soybean

International Nuclear Information System (INIS)

Ge Wenting; Jin Xijun; Ma Chunmei; Dong Shoukun; Gong Zhenping; Zhang Lei

2011-01-01

Field experiments were conducted to study the nitrate reductase activity and its relationship to nitrogen by using frame tests (pot without bottom), sand culture and 15 N-urea at transplanting in soybean variety Suinong 14. Results showed that the activity of nitrate reductase in leaf changed as a signal peak curve with the soybean growth, lower in vegetative growth phase, higher in reproductive growth period and reached the peak in blooming period, then decreased gradually. Nitrogen application showed obvious effect on the nitrate reductase activity. The activities of nitrate reductase in leaves followed the order of N 135 > N 90 > N 45 > N 0 in vegetative growth stage, no clear regularity was found during the whole reproductive growth period. The activities of nitrate reductase in leaves were accorded with the order of upper leaves > mid leaves > lower leaves, and it was very significant differences (P 15 N labeling method during beginning seed stage and full seed stage shown that 15 N abundance in various organs at different node position also followed the same order, suggesting that high level of nitrate reductase activity at upper leaves of soybean promoted the assimilation of NO 3 - . (authors)

Requirements for superoxide-dependent tyrosine hydroperoxide formation in peptides

DEFF Research Database (Denmark)

Winterbourn, Christine C; Parsons-Mair, Helena N; Gebicki, Silvia

2004-01-01

Superoxide reacts rapidly with other radicals, but these reactions have received little attention in the context of oxidative stress. For tyrosyl radicals, reaction with superoxide is 3-fold faster than dimerization, and forms the addition product tyrosine hydroperoxide. We have explored structural...... requirements for hydroperoxide formation using tyrosine analogues and di- and tri-peptides. Superoxide and phenoxyl radicals were generated using xanthine oxidase, peroxidase and the respective tyrosine derivative, or by gamma-radiation. Peroxides were measured using FeSO4/Xylenol Orange. Tyrosine and tyramine...... formed stable hydroperoxides, but N-acetyltyrosine and p-hydroxyphenylacetic acid did not, demonstrating a requirement for a free amino group. Using [14C]tyrosine, the hydroperoxide and dityrosine were formed at a molar ratio of 1.8:1. Studies with pre-formed hydroperoxides, and measurements of substrate...

Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala.

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Alberto Guevara-Flores

Full Text Available A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR, thioredoxin-glutathione reductase (TGR, and a putative thioredoxin reductase (TrxR was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.

Mitochondrial respiration scavenges extramitochondrial superoxide anion via a nonenzymatic mechanism.

OpenAIRE

Guidot, D M; Repine, J E; Kitlowski, A D; Flores, S C; Nelson, S K; Wright, R M; McCord, J M

1995-01-01

We determined that mitochondrial respiration reduced cytosolic oxidant stress in vivo and scavenged extramitochondrial superoxide anion (O2-.) in vitro. First, Saccharomyces cerevisiae deficient in both the cytosolic antioxidant cupro-zinc superoxide dismutase (Cu,Zn-SOD) and electron transport (Rho0 state) grew poorly (P 0.05) in all yeast. Seco...

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

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Jolanta Marciniak

2014-01-01

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.

Ab initio molecular dynamics of the reaction of quercetin with superoxide radical

Energy Technology Data Exchange (ETDEWEB)

Lespade, Laure, E-mail: l.lespade@ism.u-bordeaux1.fr

2016-08-22

Highlights: • Ab initio molecular dynamics is performed to describe the reaction of quercetin and superoxide. • The reaction occurs near the sites 4′ and 7 when the system contains sufficiently water molecules. • The difference of reactivity of superoxide compared to commonly used radicals as DPPH{sup ·} or ABTS{sup ·+} is explained. - Abstract: Superoxide plays an important role in biology but in unregulated concentrations it is implicated in a lot of diseases such as cancer or atherosclerosis. Antioxidants like flavonoids are abundant in plant and are good scavengers of superoxide radical. The modeling of superoxide scavenging by flavonoids from the diet still remains a challenge. In this study, ab initio molecular dynamics of the reaction of the flavonoid quercetin toward superoxide radical has been carried out using Car–Parrinello density functional theory. The study has proven different reactant solvation by modifying the number of water molecules surrounding superoxide. The reaction consists in the gift of a hydrogen atom of one of the hydroxyl groups of quercetin to the radical. When it occurs, it is relatively fast, lower than 100 fs. Calculations show that it depends largely on the environment of the hydroxyl group giving its hydrogen atom, the geometry of the first water layer and the presence of a certain number of water molecules in the second layer, indicating a great influence of the solvent on the reactivity.

Developmental and environmental regulation of the Nicotiana plumbaginifolia cytosolic Cu/Zn-superoxide dismutase promoter in transgenic tobacco.

Science.gov (United States)

Hérouart, D; Van Montagu, M; Inzé, D

1994-03-01

Superoxide dismutases (SODs) play a key role in the cellular defense against reactive oxygen species. To study the transcriptional regulation at the cellular level, the promoter of the Nicotiana plumbaginifolia cytosolic gene encoding Cu/ZnSOD (SODCc) was fused to the beta-glucuronidase (GUS) reporter gene (gusA) and analyzed in transgenic tobacco plants. The promoter was highly active in vascular bundles of leaves and stems, where it is confined to phloem cells. In flowers, GUS activity was detected in ovules and pollen grains, in pigmented tissues of petals, and in vascular tissue of ovaries and anthers. In response to treatment with the superoxide-generating herbicide paraquat, very strong GUS staining was observed in photosynthetically active cells of leaves and in some epidermal root cells of seedlings. The expression of the SODCc-gusA was also induced in seedlings after heat shock and chilling and after treatment with sulfhydryl antioxidants such as reduced glutathione and cysteine. It is postulated that SODCc expression is directly linked to a cell-specific production of excess superoxide radicals in the cytosol.

Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings

Science.gov (United States)

Chaki, Mounira; Valderrama, Raquel; Fernández-Ocaña, Ana M.; Carreras, Alfonso; Gómez-Rodríguez, Maria. V.; Pedrajas, José R.; Begara-Morales, Juan C.; Sánchez-Calvo, Beatriz; Luque, Francisco; Leterrier, Marina; Corpas, Francisco J.; Barroso, Juan B.

2011-01-01

Nitric oxide (NO) and related molecules such as peroxynitrite, S-nitrosoglutathione (GSNO), and nitrotyrosine, among others, are involved in physiological processes as well in the mechanisms of response to stress conditions. In sunflower seedlings exposed to five different adverse environmental conditions (low temperature, mechanical wounding, high light intensity, continuous light, and continuous darkness), key components of the metabolism of reactive nitrogen species (RNS) and reactive oxygen species (ROS), including the enzyme activities L-arginine-dependent nitric oxide synthase (NOS), S-nitrosogluthathione reductase (GSNOR), nitrate reductase (NR), catalase, and superoxide dismutase, the content of lipid hydroperoxide, hydrogen peroxide, S-nitrosothiols (SNOs), the cellular level of NO, GSNO, and GSNOR, and protein tyrosine nitration [nitrotyrosine (NO2-Tyr)] were analysed. Among the stress conditions studied, mechanical wounding was the only one that caused a down-regulation of NOS and GSNOR activities, which in turn provoked an accumulation of SNOs. The analyses of the cellular content of NO, GSNO, GSNOR, and NO2-Tyr by confocal laser scanning microscopy confirmed these biochemical data. Therefore, it is proposed that mechanical wounding triggers the accumulation of SNOs, specifically GSNO, due to a down-regulation of GSNOR activity, while NO2-Tyr increases. Consequently a process of nitrosative stress is induced in sunflower seedlings and SNOs constitute a new wound signal in plants. PMID:21172815

Differential Effects of Superoxide Dismutase Mimetics after Mechanical Overload of Articular Cartilage

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Mitchell C. Coleman

2017-11-01

Full Text Available Post-traumatic osteoarthritis can develop as a result of the initial mechanical impact causing the injury and also as a result of chronic changes in mechanical loading of the joint. Aberrant mechanical loading initiates excessive production of reactive oxygen species, oxidative damage, and stress that appears to damage mitochondria in the surviving chondrocytes. To probe the benefits of increasing superoxide removal with small molecular weight superoxide dismutase mimetics under severe loads, we applied both impact and overload injury scenarios to bovine osteochondral explants using characterized mechanical platforms with and without GC4403, MnTE-2-PyP, and MnTnBuOE-2-PyP. In impact scenarios, each of these mimetics provides some dose-dependent protection from cell death and loss of mitochondrial content while in repeated overloading scenarios only MnTnBuOE-2-PyP provided a clear benefit to chondrocytes. These results support the hypothesis that superoxide is generated in excess after impact injuries and suggest that superoxide production within the lipid compartment may be a critical mediator of responses to chronic overload. This is an important nuance distinguishing roles of superoxide, and thus superoxide dismutases, in mediating damage to cellular machinery in hyper-acute impact scenarios compared to chronic scenarios.

Studies on protective effects of superoxide dismutase on radiation induced-chromosomal aberrations

International Nuclear Information System (INIS)

Zheng Siying; Jiang Jiagui; Lin Xingcheng

1987-09-01

This study demonstrates that radiation induced-chromosomal aberrations are not only due to the direct effect of radiation h it , but the indirect effect of free radical as well. Therefore, chromosome damage induced by radiation may be reduced by adding exogenous SOD into the radiation exposed lymphocyte culture to eliminate the superoxide free radical which damages DNA. On the other hand, however, the radiosensitivity of lymphocytes can be raised by adding SOD inhibitor (DDC) into the lymphocyte culture, which makes radiation induced-chromosomal damages more severely

Effects of superoxide dismutase, dithiothreitol and formate ion on the inactivation of papain by hydroxyl and superoxide radicals in aerated solutions

International Nuclear Information System (INIS)

Lin, W.S.; Armstrong, D.A.

1978-01-01

Losses in enzyme activity and sulphydryl content have been studied in aerated papain solutions containing formate, superoxide dismutase and dithiothreitol. Both formate and dithiothreitol converted .OH to .0 2 -, whereas superoxide dismutase completely suppressed the inactivation by .0 2 -. Using results from all systems, the fraction of .0 2 - reactions with papain that caused inactivation of the enzyme was 0.33+-0.07. The results also showed that the fraction of .OH reactions, which cause inactivation of papain, is significantly higher in aerated than in oxygen-free solutions. (author)

In vivo photoinactivation of Escherichia coli ribonucleoside reductase by near-ultraviolet light

International Nuclear Information System (INIS)

Peters, J.

1977-01-01

Some experimental work is described showing that near-U.V. irradiation of E.coli cells selectively destroys RDP-reductase (ribonucleoside diphosphate reductase) activity in vivo are providing evidence relating the loss of RDP-reductase to loss of cellular visibility and the inactivity of irrdiated cells to support the replication of DNA phages. The data are consistent with the interpretation that the principal cause in the killing of exponentially growing E.coli cells by near-U.V., and the loss of ability of irradiated host cells to support the replication of DNA phages, is the photoinactivation of the RDP-reductase complex. (U.K.)

In vivo photoinactivation of Escherichia coli ribonucleoside reductase by near-ultraviolet light

Energy Technology Data Exchange (ETDEWEB)

Peters, J [California Univ., Irvine (USA)

1977-06-09

Some experimental work is described showing that near-uv irradiation of E.coli cells selectively destroys RDP-reductase (ribonucleoside diphosphate reductase) activity in vivo are providing evidence relating the loss of RDP-reductase to loss of cellular visibility and the inactivity of irrdiated cells to support the replication of DNA phages. The data are consistent with the interpretation that the principal cause in the killing of exponentially growing E.coli cells by near-uv, and the loss of ability of irradiated host cells to support the replication of DNA phages, is the photoinactivation of the RDP-reductase complex.

The influence of extracellular superoxide on iron redox chemistry and bioavailability to aquatic microorganisms

Directory of Open Access Journals (Sweden)

Andrew eRose

2012-04-01

Full Text Available Superoxide, the one-electron reduced form of dioxygen, is produced in the extracellular milieu of aquatic microbes through a range of abiotic chemical processes and also by microbes themselves. Due to its ability to promote both oxidative and reductive reactions, superoxide may have a profound impact on the redox state of iron, potentially influencing iron solubility, complex speciation and bioavailability. The interplay between iron, superoxide and oxygen may also produce a cascade of other highly reactive transients in oxygenated natural waters. For microbes, the overall effect of reactions between superoxide and iron may be deleterious or beneficial, depending on the organism and its chemical environment. Here I critically discuss recent advances in understanding: (i sources of extracellular superoxide in natural waters, with a particular emphasis on microbial generation; (ii the chemistry of reactions between superoxide and iron; and (iii the influence of these processes on iron bioavailability and microbial iron nutrition.

Effect of superoxide anion scavenger on rat hearts with chronic intermittent hypoxia.

Science.gov (United States)

Pai, Peiying; Lai, Ching Jung; Lin, Ching-Yuang; Liou, Yi-Fan; Huang, Chih-Yang; Lee, Shin-Da

2016-04-15

Only very limited information regarding the protective effects of the superoxide anion scavenger on chronic intermittent hypoxia-induced cardiac apoptosis is available. The purpose of this study is to evaluate the effects of the superoxide anion scavenger on cardiac apoptotic and prosurvival pathways in rats with sleep apnea. Forty-two Sprague-Dawley rats were divided into three groups, rats with normoxic exposure (Control, 21% O2, 1 mo), rats with chronic intermittent hypoxia exposure (Hypoxia, 3-7% O2vs. 21% O2per 40 s cycle, 8 h per day, 1 mo), and rats with pretreatment of the superoxide anion scavenger and chronic intermittent hypoxia exposure (Hypoxia-O2 (-)-Scavenger, MnTMPyP pentachloride, 1 mg/kg ip per day; 3-7% O2vs. 21% O2per 40 s cycle, 8 h per day, 1 mo) at 5-6 mo of age. After 1 mo, the protein levels and apoptotic cells of excised hearts from three groups were measured by Western blotting and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay. The superoxide anion scavenger decreased hypoxia-induced myocardial architecture abnormalities, left ventricular hypertrophy, and TUNEL-positive apoptosis. The superoxide anion scavenger decreased hypoxia-induced Fas ligand, Fas death receptors, Fas-associated death domain (FADD), activated caspase-8, and activated caspase-3 (Fas-dependent apoptotic pathway) as well as Bad, activated caspase-9 and activated caspase-3 (mitochondria-dependent apoptotic pathway), endonuclease G (EndoG), apoptosis-inducing factor (AIF), and TUNEL-positive apoptosis. The superoxide anion scavenger increased IGF-1, IGF-1R, p-PI3k, p-Akt, p-Bad, Bcl-2, and Bcl-xL (survival pathway). Our findings imply that the superoxide anion scavenger might prevent cardiac Fas-mediated and mitochondrial-mediated apoptosis and enhance the IGF-1-related survival pathway in chronic intermittent hypoxia. The superoxide anion scavenger may prevent chronic sleep apnea-enhanced cardiac apoptotic pathways and enhances

Association between methylenetetrahydrofolate reductase (MTHFR ...

African Journals Online (AJOL)

Association between methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and risk of ischemic stroke in North Indian population: A hospital based case–control study. Amit Kumar, Shubham Misra, Anjali Hazarika, Pradeep Kumar, Ram Sagar, Abhishek Pathak, Kamalesh Chakravarty, Kameshwar ...

Colour formation in fermented sausages by meat-associated staphylococci with different nitrite- and nitrate-reductase activities.

Science.gov (United States)

Gøtterup, Jacob; Olsen, Karsten; Knøchel, Susanne; Tjener, Karsten; Stahnke, Louise H; Møller, Jens K S

2008-04-01

Three Staphylococcus strains, S. carnosus, S. simulans and S. saprophyticus, selected due to their varying nitrite and/or nitrate-reductase activities, were used to initiate colour formation during sausage fermentation. During fermentation of sausages with either nitrite or nitrate added, colour was followed by L(∗)a(∗)b measurements and the content of nitrosylmyoglobin (MbFe(II)NO) quantified by electron spin resonance (ESR). MbFe(II)NO was rapidly formed in sausages with added nitrite independent of the presence of nitrite reducing bacteria, whereas the rate of MbFe(II)NO formation in sausages with added nitrate depended on the specific Staphylococcus strain. Strains with high nitrate-reductase activity showed a significantly faster rate of pigment formation, but other factors were of influence as well. Product stability for the sliced, packaged sausage was evaluated as surface colour and oxidation by autofluorescence and hexanal content, respectively. No significant direct effect of the Staphylococcus addition was observed, however, there was a clear correspondence between high initial amount of MbFe(II)NO in the different sausages and the colour stability during storage. Autofluorescence data correlated well with hexanal content, and may be used as predictive tools. Overall, nitrite- and nitrate-reductase activities of Staphylococcus strains in nitrite-cured sausages were of limited importance regarding colour development, while in nitrate-cured sausages strains with higher nitrate reductase activity were crucial for ensuring optimal colour formation during initial fermentation stages.

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

Directory of Open Access Journals (Sweden)

Józef Buczek

2014-01-01

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.

A study on superoxide dismutase activity of some model compounds.

Science.gov (United States)

Liao, Z; Liu, W; Liu, J; Jiang, Y; Shi, J; Liu, C

1994-08-15

The synthesis and characteristics of a binuclear ligand N,N,N',N'-tetrakis (2'-benzimidazolyl methyl)-1,4-diethylene amino glycol ether (EGTB) and its series of coordination compounds containing copper(II), iron(III), and manganese(II) with and without exogenous bridging ligand which was imidazolate ion (Im-), bipyridine (bpy), or 1,10-phenanthroline (phen) are reported. Depending on the redox potentials by cyclic voltammetry, the coordination compounds can act as catalysts for the dismutation of superoxide radicals (O2-). The detection of the rate constant of the reaction of superoxide ion with nitroblue tetrazolium (NBT) which is inhibited by superoxide dismutase (SOD) and its model compounds of the EGTB system has been performed by a modified illumination method. The rate constants kQ of the catalytic dismutation have been obtained.

Molecular Diagnosis of 5α-Reductase Type II Deficiency in Brazilian Siblings with 46,XY Disorder of Sex Development

Directory of Open Access Journals (Sweden)

Maricilda Palandi de Mello

2011-12-01

Full Text Available The steroid 5α-reductase type II enzyme catalyzes the conversion of testosterone (T to dihydrotestosterone (DHT, and its deficiency leads to undervirilization in 46,XY individuals, due to an impairment of this conversion in genital tissues. Molecular analysis in the steroid 5α-reductase type II gene (SRD5A2 was performed in two 46,XY female siblings. SRD5A2 gene sequencing revealed that the patients were homozygous for p.Gln126Arg missense mutation, which results from the CGA > CAA nucleotide substitution. The molecular result confirmed clinical diagnosis of 46,XY disorder of sex development (DSD for the older sister and directed the investigation to other family members. Studies on SRD5A2 protein structure showed severe changes at NADPH binding region indicating that structural modeling analysis can be useful to evaluate the deleterious role of a mutation as causing 5α-reductase type II enzyme deficiency.

Zymogram profiling of superoxide dismutase and catalase activities allows Saccharomyces and non-Saccharomyces species differentiation and correlates to their fermentation performance.

Science.gov (United States)

Gamero-Sandemetrio, Esther; Gómez-Pastor, Rocío; Matallana, Emilia

2013-05-01

Aerobic organisms have devised several enzymatic and non-enzymatic antioxidant defenses to deal with reactive oxygen species (ROS) produced by cellular metabolism. To combat such stress, cells induce ROS scavenging enzymes such as catalase, peroxidase, superoxide dismutase (SOD) and glutathione reductase. In the present research, we have used a double staining technique of SOD and catalase enzymes in the same polyacrylamide gel to analyze the different antioxidant enzymatic activities and protein isoforms present in Saccharomyces and non-Saccharomyces yeast species. Moreover, we used a technique to differentially detect Sod1p and Sod2p on gel by immersion in NaCN, which specifically inhibits the Sod1p isoform. We observed unique SOD and catalase zymogram profiles for all the analyzed yeasts and we propose this technique as a new approach for Saccharomyces and non-Saccharomyces yeast strains differentiation. In addition, we observed functional correlations between SOD and catalase enzyme activities, accumulation of essential metabolites, such as glutathione and trehalose, and the fermentative performance of different yeasts strains with industrial relevance.

Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

Energy Technology Data Exchange (ETDEWEB)

Rosenthal, Cindy [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Mueller, Uwe [Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung mbH, Albert-Einstein-Strasse 15, D-12489 Berlin (Germany); Panjikar, Santosh [European Molecular Biology Laboratory Hamburg, Outstation Deutsches Elektronen-Synchrotron, Notkestrasse 85, D-22603 Hamburg (Germany); Sun, Lianli [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China); Ruppert, Martin [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Zhao, Yu [Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China); Stöckigt, Joachim [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China)

2006-12-01

Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution. Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222{sub 1} and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å.

Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

International Nuclear Information System (INIS)

Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

2006-01-01

Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution. Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222 1 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å

Endogenous superoxide dismutase and catalase activities and radiation resistance in mouse cell lines

International Nuclear Information System (INIS)

Davy, C.A.; Tesfay, Z.; Jones, J.; Rosenberg, R.C.; McCarthy, C.; Ostrand-Rosenberg, S.

1988-01-01

The relationship between the endogenous cytoplasmic levels of the enzymes superoxide dismutase and catalase and the inhibition of cell proliferation by γ-radiation has been studied in 11 mouse cell lines. The resistance of these mouse cell lines to radiation was found to vary by over 25-fold. No correlation was found between the cytoplasmic level of CuZn-superoxide dismutase or catalase and the resistance to radiation as measured by extrapolation number (EN), quasi-threshold dose (Dsub(q)), or Dsub(o). None of the cell lines had detectable cytoplasmic Mn-superoxide dismutase. The apparent Ksub(i) of potassium cyanide for mouse CuZn-superoxide dismutase was determined (Ksub(i) = 6.5 μmol dm -3 ). (author)

Effect of oral administration of green tea extract in various dosage schemes on oxidative stress status of mice in vivo

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Bártíková Hana

2015-03-01

Full Text Available Green tea is a favorite beverage and its extracts are popular components of dietary supplements. The aim of the present in vivo study was to obtain detailed information about the effect of a standard green tea extract (Polyphenon, P, at different doses, on antioxidant enzymes and oxidative stress markers in murine blood, liver, small and large intestine. In all doses, P improved the oxidative stress status via an increased content of plasmatic SH-groups (by 21-67 %. Regarding antioxidant enzymes in tissues, the low dose of P had the best positive effect as it elevated the activity of NADPH quinone reductase in liver and small intestine, thioredoxin reductase in small intestine and hepatic superoxide dismutase. Based on these facts, consumption of green tea seems to be safe and beneficial, while consumption of dietary supplements containing high doses of catechins may disturb oxidative balance by lowering the activity of thioredoxin reductase, glutathione S-transferase, glutathione reductase and superoxide dismutase

Identification of the 7-Hydroxymethyl Chlorophyll a Reductase of the Chlorophyll Cycle in Arabidopsis[W

Science.gov (United States)

Meguro, Miki; Ito, Hisashi; Takabayashi, Atsushi; Tanaka, Ryouichi; Tanaka, Ayumi

2011-01-01

The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation. PMID:21934147

Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.

Science.gov (United States)

Wang, Xinning; Liang, Zhenzhen; Hou, Jin; Bao, Xiaoming; Shen, Yu

2016-04-01

Vanillin, a type of phenolic released during the pre-treatment of lignocellulosic materials, is toxic to microorganisms and therefore its presence inhibits the fermentation. The vanillin can be reduced to vanillyl alcohol, which is much less toxic, by the ethanol producer Saccharomyces cerevisiae. The reducing capacity of S. cerevisiae and its vanillin resistance are strongly correlated. However, the specific enzymes and their contribution to the vanillin reduction are not extensively studied. In our previous work, an evolved vanillin-resistant strain showed an increased vanillin reduction capacity compared with its parent strain. The transcriptome analysis suggested the reductases and dehydrogenases of this vanillin resistant strain were up-regulated. Using this as a starting point, 11 significantly regulated reductases and dehydrogenases were selected in the present work for further study. The roles of these reductases and dehydrogenases in the vanillin tolerance and detoxification abilities of S. cerevisiae are described. Among the candidate genes, the overexpression of the alcohol dehydrogenase gene ADH6, acetaldehyde dehydrogenase gene ALD6, glucose-6-phosphate 1-dehydrogenase gene ZWF1, NADH-dependent aldehyde reductase gene YNL134C, and aldo-keto reductase gene YJR096W increased 177, 25, 6, 15, and 18 % of the strain μmax in the medium containing 1 g L(-1) vanillin. The in vitro detected vanillin reductase activities of strain overexpressing ADH6, YNL134C and YJR096W were notably higher than control. The vanillin specific reduction rate increased by 8 times in ADH6 overexpressed strain but not in YNL134C and YJR096W overexpressed strain. This suggested that the enzymes encoded by YNL134C and YJR096W might prefer other substrate and/or could not show their effects on vanillin on the high background of Adh6p in vivo. Overexpressing ALD6 and ZWF1 mainly increased the [NADPH]/[NADP(+)] and [GSH]/[GSSG] ratios but not the vanillin reductase activities. Their

Reduction of azo dyes by flavin reductase from Citrobacter freundii A1

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Mohd Firdaus Abdul-Wahab

2012-12-01

Full Text Available Citrobacter freundii A1 isolated from a sewage treatment facility was demonstrated to be able to effectively decolorize azo dyes as pure and mixed culture. This study reports on the investigation on the enzymatic systems involved. An assay performed suggested the possible involvement of flavin reductase (Fre as an azo reductase. A heterologouslyexpressed recombinant Fre from C. freundii A1 was used to investigate its involvement in the azo reduction process. Three model dyes were used, namely Acid Red 27 (AR27, Direct Blue 15 (DB15 and Reactive Black 5 (RB5. AR27 was found to be reduced the fastest by Fre, followed by RB5, and lastly DB15. Redox mediators nicotinamide adenine dinucleotide (NADH and riboflavin enhance the reduction, suggesting the redox activity of the enzyme. The rate and extent of reduction of the model dyes correlate well with the reduction potentials (Ep. The data presented here strongly suggest that Fre is one of the enzymes responsible for azo reduction in C. freundii A1, acting via an oxidation-reduction reaction.

Genome sequence analysis of predicted polyprenol reductase gene from mangrove plant kandelia obovata

Science.gov (United States)

Basyuni, M.; Sagami, H.; Baba, S.; Oku, H.

2018-03-01

It has been previously reported that dolichols but not polyprenols were predominated in mangrove leaves and roots. Therefore, the occurrence of larger amounts of dolichol in leaves of mangrove plants implies that polyprenol reductase is responsible for the conversion of polyprenol to dolichol may be active in mangrove leaves. Here we report the early assessment of probably polyprenol reductase gene from genome sequence of mangrove plant Kandelia obovata. The functional assignment of the gene was based on a homology search of the sequences against the non-redundant (nr) peptide database of NCBI using Blastx. The degree of sequence identity between DNA sequence and known polyprenol reductase was confirmed using the Blastx probability E-value, total score, and identity. The genome sequence data resulted in three partial sequences, termed c23157 (700 bp), c23901 (960 bp), and c24171 (531 bp). The c23157 gene showed the highest similarity (61%) to predicted polyprenol reductase 2- like from Gossypium raimondii with E-value 2e-100. The second gene was c23901 to exhibit high similarity (78%) to the steroid 5-alpha-reductase Det2 from J. curcas with E-value 2e-140. Furthermore, the c24171 gene depicted highest similarity (79%) to the polyprenol reductase 2 isoform X1 from Jatropha curcas with E- value 7e-21.The present study suggested that the c23157, c23901, and c24171, genes may encode predicted polyprenol reductase. The c23157, c23901, c24171 are therefore the new type of predicted polyprenol reductase from K. obovata.

Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription

DEFF Research Database (Denmark)

Cheng, Chi-Lien; Acedo, Gregoria N; Kristensen, Michael

1992-01-01

Nitrate reductase, the first enzyme in nitrate assimilation, is located at the crossroad of two energy-consuming pathways: nitrate assimilation and carbon fixation. Light, which regulates the expression of many higher-plant carbon fixation genes, also regulates nitrate reductase gene expression. ...

Role of nitric oxide and superoxide in Giardia lamblia killing

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P.D. Fernandes

1997-01-01

Full Text Available Giardia lamblia trophozoites were incubated for 2 h with activated murine macrophages, nitric oxide (NO donors or a superoxide anion generator (20 mU/ml xanthine oxidase plus 1 mM xanthine. Activated macrophages were cytotoxic to Giardia trophozoites (~60% dead trophozoites. This effect was inhibited (>90% by an NO synthase inhibitor (200 µM and unaffected by superoxide dismutase (SOD, 300 U/ml. Giardia trophozoites were killed by the NO donors, S-nitroso-acetyl-penicillamine (SNAP and sodium nitroprusside (SNP in a dose-dependent manner (LD50 300 and 50 µM, respectively. A dual NO-superoxide anion donor, 3-morpholino-sydnonimine hydrochloride (SIN-1, did not have a killing effect in concentrations up to 1 mM. However, when SOD (300 U/ml was added simultaneously with SIN-1 to Giardia, a significant trophozoite-killing effect was observed (~35% dead trophozoites at 1 mM. The mixture of SNAP or SNP with superoxide anion, which yields peroxynitrite, abolished the trophozoite killing induced by NO donors. Authentic peroxynitrite only killed trophozoites at very high concentrations (3 mM. These results indicate that NO accounts for Giardia trophozoite killing and this effect is not mediated by peroxynitrite

Mn(II) oxidation by an ascomycete fungus is linked to superoxide production during asexual reproduction.

Science.gov (United States)

Hansel, Colleen M; Zeiner, Carolyn A; Santelli, Cara M; Webb, Samuel M

2012-07-31

Manganese (Mn) oxides are among the most reactive minerals within the environment, where they control the bioavailability of carbon, nutrients, and numerous metals. Although the ability of microorganisms to oxidize Mn(II) to Mn(III/IV) oxides is scattered throughout the bacterial and fungal domains of life, the mechanism and physiological basis for Mn(II) oxidation remains an enigma. Here, we use a combination of compound-specific chemical assays, microspectroscopy, and electron microscopy to show that a common Ascomycete filamentous fungus, Stilbella aciculosa, oxidizes Mn(II) to Mn oxides by producing extracellular superoxide during cell differentiation. The reactive Mn oxide phase birnessite and the reactive oxygen species superoxide and hydrogen peroxide are colocalized at the base of asexual reproductive structures. Mn oxide formation is not observed in the presence of superoxide scavengers (e.g., Cu) and inhibitors of NADPH oxidases (e.g., diphenylene iodonium chloride), enzymes responsible for superoxide production and cell differentiation in fungi. Considering the recent identification of Mn(II) oxidation by NADH oxidase-based superoxide production by a common marine bacterium (Roseobacter sp.), these results introduce a surprising homology between some prokaryotic and eukaryotic organisms in the mechanisms responsible for Mn(II) oxidation, where oxidation appears to be a side reaction of extracellular superoxide production. Given the versatility of superoxide as a redox reactant and the widespread ability of fungi to produce superoxide, this microbial extracellular superoxide production may play a central role in the cycling and bioavailability of metals (e.g., Hg, Fe, Mn) and carbon in natural systems.

Bioinformatics analysis of the predicted polyprenol reductase genes in higher plants

Science.gov (United States)

Basyuni, M.; Wati, R.

2018-03-01

The present study evaluates the bioinformatics methods to analyze twenty-four predicted polyprenol reductase genes from higher plants on GenBank as well as predicted the structure, composition, similarity, subcellular localization, and phylogenetic. The physicochemical properties of plant polyprenol showed diversity among the observed genes. The percentage of the secondary structure of plant polyprenol genes followed the ratio order of α helix > random coil > extended chain structure. The values of chloroplast but not signal peptide were too low, indicated that few chloroplast transit peptide in plant polyprenol reductase genes. The possibility of the potential transit peptide showed variation among the plant polyprenol reductase, suggested the importance of understanding the variety of peptide components of plant polyprenol genes. To clarify this finding, a phylogenetic tree was drawn. The phylogenetic tree shows several branches in the tree, suggested that plant polyprenol reductase genes grouped into divergent clusters in the tree.

Trypsin Binding with Copper Ions Scavenges Superoxide: Molecular Dynamics-Based Mechanism Investigation

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Xin Li

2018-01-01

Full Text Available Trypsin is a serine protease, which has been proved to be a novel superoxide scavenger. The burst of superoxide induced by polychlorinated biphenyls can be impeded by trypsin in both wild type and sod knockout mutants of Escherichia coli. The experimental results demonstrated that the activities of superoxide scavenging of trypsin were significantly accelerated by Cu ions. Also, with the addition of Cu ions, a new β-sheet (β7 transited from a random coil in the Cu(II-trypsin (TP system, which was favorable for the formation of more contacts with other sheets of trypsin. Residue–residue network analysis and the porcupine plots proved that the Cu ion in trypsin strengthened some native interactions among residues, which ultimately resulted in much greater stability of the Cu(II-TP system. Moreover, compact and stable trypsin structures with Cu ions might be responsible for significantly provoking the activity of superoxide scavenging.

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

International Nuclear Information System (INIS)

Hou, Feng; Miyakawa, Takuya; Kataoka, Michihiko; Takeshita, Daijiro; Kumashiro, Shoko; Uzura, Atsuko; Urano, Nobuyuki; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2014-01-01

Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

Energy Technology Data Exchange (ETDEWEB)

Hou, Feng; Miyakawa, Takuya [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kataoka, Michihiko [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Takeshita, Daijiro [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kumashiro, Shoko [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Uzura, Atsuko [Research and Development Center, Nagase and Co., Ltd., 2-2-3 Muratani, Nishi-ku, Kobe 651-2241 (Japan); Urano, Nobuyuki [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Nagata, Koji [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Shimizu, Sakayu [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Faculty of Bioenvironmental Science, Kyoto Gakuen University, Sogabe-cho, Kameoka 621-8555 (Japan); Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

2014-04-18

Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.

Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance

International Nuclear Information System (INIS)

Beckman, J.S.; Minor, R.L. Jr.; White, C.W.; Repine, J.E.; Rosen, G.M.; Freeman, B.A.

1988-01-01

Covalent conjugation of superoxide dismutase and catalase with polyethylene glycol (PEG) increases the circulatory half-lives of these enzymes from 125 I-PEG-catalase or 125 I-PEG-superoxide dismutase produced a linear, concentration-dependent increase in cellular enzyme activity and radioactivity. Fluorescently labeled PEG-superoxide dismutase incubated with endothelial cells showed a vesicular localization. Mechanical injury to cell monolayers, which is known to stimulate endocytosis, further increased the uptake of fluorescent PEG-superoxide dismutase. Addition of PEG and PEG-conjugated enzymes perturbed the spin-label binding environment, indicative of producing an increase in plasma membrane fluidity. Thus, PEG conjugation to superoxide dismutase and catalase enhances cell association of these enzymes in a manner which increases cellular enzyme activities and provides prolonged protection from partially reduced oxygen species

Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis.

Science.gov (United States)

Kataoka, M; Delacruz-Hidalgo, A-R G; Akond, M A; Sakuradani, E; Kita, K; Shimizu, S

2004-04-01

The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to D-pantoyl lactone.

Identification of 5α-reductase isoenzymes in canine skin.

Science.gov (United States)

Bernardi de Souza, Lucilene; Paradis, Manon; Zamberlam, Gustavo; Benoit-Biancamano, Marie-Odile; Price, Christopher

2015-10-01

Alopecia X in dogs is a noninflammatory alopecia that may be caused by a hormonal dysfunction. It may be similar to androgenic alopecia in men that is caused by the effect of dihydrotestosterone (DHT). The 5α-reductase isoenzymes, 5αR1 and 5αR2, and a recently described 5αR3, are responsible for the conversion of testosterone into DHT. However, which 5α-reductases are present in canine skin has not yet been described. The main objective of this study was to determine the pattern of expression of 5α-reductase genes in canine skin. Skin biopsies were obtained from healthy, intact young-mature beagles (three males, four females) at three anatomical sites normally affected by alopecia X (dorsal neck, back of thighs and base of tail) and two sites generally unaffected (dorsal head and ventral thorax). Prostate samples (n = 3) were collected as positive controls for 5α-reductase mRNA abundance measurement by real-time PCR. We detected mRNA encoding 5αR1 and 5αR3 but not 5αR2. There were no significant differences in 5αR1 and 5αR3 mRNA levels between the different anatomical sites, irrespective of gender (P > 0.05). Moreover, the mean mRNA abundance in each anatomical site did not differ between males and females (P > 0.05). To the best of the authors' knowledge, this is the first study demonstrating the expression of 5α-reductases in canine skin and the expression of 5αR3 in this tissue. These results may help to elucidate the pathogenesis of alopecia X and to determine more appropriate treatments for this disorder. © 2015 ESVD and ACVD.

Ultraviolet-B- and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana

International Nuclear Information System (INIS)

Rao, M.V.; Paliyath, G.; Ormrod, D.P.

1996-01-01

Earlier studies with Arabidopsis thaliana exposed to ultraviolet B (UV-B) and ozone (O 3 ) have indicated the differential responses of superoxide dismutase and glutathione reductase. In this study, we have investigated whether A. thaliana genotype Landsberg erecta and its flavonoid-deficient mutant transparent testa (tt5) is capable of metabolizing UV-B- and O 3 -induced activated oxygen species by invoking similar antioxidant enzymes. UV-B exposure preferentially enhanced guaiacol-peroxidases, ascorbate peroxidase, and peroxidases specific to coniferyl alcohol and modified the substrate affinity of ascorbate peroxidase. O 3 exposure enhanced superoxide dismutase, peroxidases, glutathione reductase, and ascorbate peroxidase to a similar degree and modified the substrate affinity of both glutathione reductase and ascorbate peroxidase. Both UV-B and O 3 exposure enhanced similar Cu,Zn-superoxide dismutase isoforms. New isoforms of peroxidases and ascorbate peroxidase were synthesized in tt5 plants irradiated with UV-B. UV-B radiation, in contrast to O 3 , enhanced the activation oxygen species by increasing membrane-localized NADPH-oxidase activity and decreasing catalase activities. These results collectively suggest that (a) UV-B exposure preferentially induces peroxidase-related enzymes, whereas O 3 exposure invokes the enzymes of superoxide dismutase/ascorbate-glutathione cycle, and (b) in contrast to O 3 , UV-B exposure generated activated oxygen species by increasing NADPH-oxidase activity. 10 figs., 4 tabs

Gamma-irradiation activates biochemical systems: induction of nitrate reductase activity in plant callus.

OpenAIRE

Pandey, K N; Sabharwal, P S

1982-01-01

Gamma-irradiation induced high levels of nitrate reductase activity (NADH:nitrate oxidoreductase, EC 1.6.6.1) in callus of Haworthia mirabilis Haworth. Subcultures of gamma-irradiated tissues showed autonomous growth on minimal medium. We were able to mimic the effects of gamma-irradiation by inducing nitrate reductase activity in unirradiated callus with exogenous auxin and kinetin. These results revealed that induction of nitrate reductase activity by gamma-irradiation is mediated through i...

Role of a novel dual flavin reductase (NR1) and an associated histidine triad protein (DCS-1) in menadione-induced cytotoxicity

International Nuclear Information System (INIS)

Kwasnicka-Crawford, Dorota A.; Vincent, Steven R.

2005-01-01

Microsomal cytochrome P450 reductase catalyzes the one-electron transfer from NADPH via FAD and FMN to various electron acceptors, such as cytochrome P450s or to some anti-cancer quinone drugs. This results in generation of free radicals and toxic oxygen metabolites, which can contribute to the cytotoxicity of these compounds. Recently, a cytosolic NADPH-dependent flavin reductase, NR1, has been described which is highly homologous to the microsomal cytochrome P450 reductase. In this study, we show that over-expression of NR1 in human embryonic kidney cells enhances the cytotoxic action of the model quinone, menadione. Furthermore, we show that a novel human histidine triad protein DCS-1, which is expressed together with NR1 in many tissues, can significantly reduce menadione-induced cytotoxicity in these cells. We also show that DCS-1 binds NF1 and directly modulates its activity. These results suggest that NR1 may play a role in carcinogenicity and cell death associated with one-electron reductions

Catalase and Superoxide Dismutase of Root-Colonizing Saprophytic Fluorescent Pseudomonads †

OpenAIRE

Katsuwon, Jirasak; Anderson, Anne J.

1990-01-01

Root-colonizing, saprophytic fluorescent pseudomonads of the Pseudomonas putida-P. fluorescens group express similar levels of catalase and superoxide dismutase activities during growth on a sucrose- and amino acid-rich medium. Increased specific activities of catalase but not superoxide dismutase were observed during growth of these bacteria on components washed from root surfaces. The specific activities of both enzymes were also regulated during contact of these bacteria with intact bean r...

Superoxide dismutases: Dual roles in controlling ROS damage and regulating ROS signaling.

Science.gov (United States)

Wang, Ying; Branicky, Robyn; Noë, Alycia; Hekimi, Siegfried

2018-04-18

Superoxide dismutases (SODs) are universal enzymes of organisms that live in the presence of oxygen. They catalyze the conversion of superoxide into oxygen and hydrogen peroxide. Superoxide anions are the intended product of dedicated signaling enzymes as well as the byproduct of several metabolic processes including mitochondrial respiration. Through their activity, SOD enzymes control the levels of a variety of reactive oxygen species (ROS) and reactive nitrogen species, thus both limiting the potential toxicity of these molecules and controlling broad aspects of cellular life that are regulated by their signaling functions. All aerobic organisms have multiple SOD proteins targeted to different cellular and subcellular locations, reflecting the slow diffusion and multiple sources of their substrate superoxide. This compartmentalization also points to the need for fine local control of ROS signaling and to the possibility for ROS to signal between compartments. In this review, we discuss studies in model organisms and humans, which reveal the dual roles of SOD enzymes in controlling damage and regulating signaling. © 2018 Wang et al.

Enzymatic Activity Enhancement of Non-Covalent Modified Superoxide Dismutase and Molecular Docking Analysis

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Fa-Jun Song

2012-03-01

Full Text Available The enzyme activity of superoxide dismutase was improved in the pyrogallol autoxidation system by about 27%, after interaction between hydroxypropyl-β-cyclo- dextrin and superoxide dismutase. Fluorescence spectrometry was used to study the interaction between hydroxypropyl-β-cyclodextrin and superoxide dismutase at different temperatures. By doing this, it can be found that these interactions increase fluorescence sensitivity. In the meantime, the synchronous fluorescence intensity revealed the interaction sites to be close to the tryptophan (Trp and tyrosine (Tyr residues of superoxide dismutase. Furthermore, molecular docking was applied to explore the binding mode between the ligands and the receptor. This suggested that HP-β-CD interacted with the B ring, G ring and the O ring and revealed that the lysine (Lys residues enter the nanocavity. It was concluded that the HP-β-CD caused specific conformational changes in SOD by non-covalent modification.

Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase

DEFF Research Database (Denmark)

Huppke, Peter; Brendel, Cornelia; Korenke, Georg Christoph

2012-01-01

chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution...... of a highly conserved arginine residue at position 163, with tryptophan in domain II of CCS, which interacts directly with superoxide dismutase 1 (SOD1). Biochemical analyses of the patient's fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation...... support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result, this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal Cu homeostasis. CCS-Arg163Trp represents the primary example of a human mutation...

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

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Yeon Bok Kim

2014-01-01

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.

Superoxide dismutase 1 is positively selected to minimize protein aggregation in great apes

DEFF Research Database (Denmark)

Dasmeh, Pouria; Kepp, Kasper Planeta

2017-01-01

Positive (adaptive) selection has recently been implied in human superoxide dismutase 1 (SOD1), a highly abundant antioxidant protein with energy signaling and antiaging functions, one of very few examples of direct selection on a human protein product (exon); the molecular drivers...... and SOD1 aggregates and triggered by aging. Our study thus marks an example of direct selection for a particular chemical phenotype (high net charge and stability) in a single human protein with possible implications for the evolution of aging....... of this selection are unknown. We mapped 30 extant SOD1 sequences to the recently established mammalian species tree and inferred ancestors, key substitutions, and signatures of selection during the protein's evolution. We detected elevated substitution rates leading to great apes (Hominidae) at ~1 per 2 million...

Comparative modelling and molecular docking of nitrate reductase from Bacillus weihenstephanensis (DS45

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R. Seenivasagan

2016-07-01

Full Text Available Nitrate reductase catalyses the oxidation of NAD(PH and the reduction of nitrate to nitrite. NR serves as a central point for the integration of metabolic pathways by governing the flux of reduced nitrogen through several regulatory mechanisms in plants, algae and fungi. Bacteria express nitrate reductases that convert nitrate to nitrite, but mammals lack these specific enzymes. The microbial nitrate reductase reduces toxic compounds to nontoxic compounds with the help of NAD(PH. In the present study, our results revealed that Bacillus weihenstephanensis expresses a nitrate reductase enzyme, which was made to generate the 3D structure of the enzyme. Six different modelling servers, namely Phyre2, RaptorX, M4T Server, HHpred, SWISS MODEL and Mod Web, were used for comparative modelling of the structure. The model was validated with standard parameters (PROCHECK and Verify 3D. This study will be useful in the functional characterization of the nitrate reductase enzyme and its docking with nitrate molecules, as well as for use with autodocking.

Superoxide dismutase from Trichuris ovis, inhibiton by benzimidazoles and pyrimidine derivatives

OpenAIRE

Sanchez-Moreno, M.; Garcia-Rejon, L.; Salas, I.; Osuna, A.; Monteoliva, M.

1992-01-01

Three superoxide dismutase isoenzymes of different cellular location were detected in an homogenate of Thrichuris ovis. Each of these molecular forms was purified by differential centrifugation and precipitation with ammonium sulphate, followed by chromatography on DEAE-cellulose and Sephadex G-75 columns. The activity levels of the two molecular forms detected in the mitochondrial (one cyanide sensitive Cu-Zn-SOD and the other cyanide intensitive Mn-Sod were higher than that of the superoxid...

Designing Superoxide-Generating Quantum Dots for Selective Light-Activated Nanotherapy

Science.gov (United States)

Goodman, Samuel M.; Levy, Max; Li, Fei-Fei; Ding, Yuchen; Courtney, Colleen M.; Chowdhury, Partha P.; Erbse, Annette; Chatterjee, Anushree; Nagpal, Prashant

2018-03-01

The rapid emergence of superbugs or multi-drug resistant (MDR) organisms has prompted a search for novel antibiotics, beyond traditional small-molecule therapies. Nanotherapeutics are being investigated as alternatives, and recently superoxide-generating quantum dots (QDs) have been shown as important candidates for selective light-activated therapy and potentiating existing antibiotics against MDR superbugs. Their therapeutic action is selective, can be tailored by simply changing their quantum-confined conduction-valence bands and their alignment with different redox half-reactions, and hence their ability to generate specific radical species in biological media. Here, we show the design of superoxide-generating QDs using optimal QD material and size well matched to superoxide redox potential, charged ligands to modulate their uptake in cells and selective redox interventions, and core/shell structures to improve their stability for therapeutic action. We show that cadmium telluride (CdTe) QDs with conduction band position at -0.5V with respect to Normal Hydrogen Electron (NHE) and visible 2.4 eV bandgap generate a large flux of selective superoxide radicals, thereby demonstrating the most effective light-activated therapy. Although the positively charged QDs demonstrate large cellular uptake, they bind indiscriminately to cell surfaces and cause non-selective cell death, while negatively charged and zwitterionic QD ligands reduce the uptake and allow selective therapeutic action via interaction with redox species. The stability of designed QDs in biologically-relevant media increases with the formation of core-shell QD structures, but an appropriate design of core-shell structures is needed to minimize any reduction in charge injection efficiency to adsorbed oxygen molecules (to form superoxide) and maintain similar quantitative generation of tailored redox species, as measured using electron paramagnetic resonance (EPR) spectroscopy and electrochemical

Designing Superoxide-Generating Quantum Dots for Selective Light-Activated Nanotherapy.

Science.gov (United States)

Goodman, Samuel M; Levy, Max; Li, Fei-Fei; Ding, Yuchen; Courtney, Colleen M; Chowdhury, Partha P; Erbse, Annette; Chatterjee, Anushree; Nagpal, Prashant

2018-01-01

The rapid emergence of superbugs, or multi-drug resistant (MDR) organisms, has prompted a search for novel antibiotics, beyond traditional small-molecule therapies. Nanotherapeutics are being investigated as alternatives, and recently superoxide-generating quantum dots (QDs) have been shown as important candidates for selective light-activated therapy, while also potentiating existing antibiotics against MDR superbugs. Their therapeutic action is selective, can be tailored by simply changing their quantum-confined conduction-valence band (CB-VB) positions and alignment with different redox half-reactions-and hence their ability to generate specific radical species in biological media. Here, we show the design of superoxide-generating QDs using optimal QD material and size well-matched to superoxide redox potential, charged ligands to modulate their uptake in cells and selective redox interventions, and core/shell structures to improve their stability for therapeutic action. We show that cadmium telluride (CdTe) QDs with conduction band (CB) position at -0.5 V with respect to Normal Hydrogen Electron (NHE) and visible 2.4 eV bandgap generate a large flux of selective superoxide radicals, thereby demonstrating the effective light-activated therapy. Although the positively charged QDs demonstrate large cellular uptake, they bind indiscriminately to cell surfaces and cause non-selective cell death, while negatively charged and zwitterionic QD ligands reduce the uptake and allow selective therapeutic action via interaction with redox species. The stability of designed QDs in biologically-relevant media increases with the formation of core-shell QD structures, but an appropriate design of core-shell structures is needed to minimize any reduction in charge injection efficiency to adsorbed oxygen molecules (to form superoxide) and maintain similar quantitative generation of tailored redox species, as measured using electron paramagnetic resonance (EPR) spectroscopy and

Glutathione reductase: solvent equilibrium and kinetic isotope effects

International Nuclear Information System (INIS)

Wong, K.K.; Vanoni, M.A.; Blanchard, J.S.

1988-01-01

Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D 2 O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 microM and 13 microM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456

Increased expression of native cytosolic Cu/Zn superoxide dismutase and ascorbate peroxidase improves tolerance to oxidative and chilling stresses in cassava (Manihot esculenta Crantz).

Science.gov (United States)

Xu, Jia; Yang, Jun; Duan, Xiaoguang; Jiang, Yueming; Zhang, Peng

2014-08-05

Cassava (Manihot esculenta Crantz) is a tropical root crop, and is therefore, extremely sensitive to low temperature; its antioxidative response is pivotal for its survival under stress. Timely turnover of reactive oxygen species (ROS) in plant cells generated by chilling-induced oxidative damages, and scavenging can be achieved by non-enzymatic and enzymatic reactions in order to maintain ROS homeostasis. Transgenic cassava plants that co-express cytosolic superoxide dismutase (SOD), MeCu/ZnSOD, and ascorbate peroxidase (APX), MeAPX2, were produced and tested for tolerance against oxidative and chilling stresses. The up-regulation of MeCu/ZnSOD and MeAPX2 expression was confirmed by the quantitative reverse transcriptase-polymerase chain reaction, and enzymatic activity analyses in the leaves of transgenic cassava plant lines with a single-transgene integration site. Upon exposure to ROS-generating agents, 100 μM ROS-generating reagent methyl viologen and 0.5 M H₂O₂, higher levels of enzymatic activities of SOD and APX were detected in transgenic plants than the wild type. Consequently, the oxidative stress parameters, such as lipid peroxidation, chlorophyll degradation and H₂O₂ synthesis, were lower in the transgenic lines than the wild type. Tolerance to chilling stress at 4°C for 2 d was greater in transgenic cassava, as observed by the higher levels of SOD, catalase, and ascorbate-glutathione cycle enzymes (e.g., APX, monodehydroascorbate reductase, dehydroascorbate reducatase and glutathione reductase) and lower levels of malondialdehyde content. These results suggest that the expression of native cytosolic SOD and APX simultaneously activated the antioxidative defense mechanisms via cyclic ROS scavenging, thereby improving its tolerance to cold stress.

Neutrophil superoxide-anion generating capacity in chronic smoking ...

Indian Academy of Sciences (India)

Unknown

To this purpose, we randomly assigned 128 male chronic smokers (37 ± 21 pack years of smoking) ... Secondly, in non-smoking human subjects we have recently shown ... therapy in chronic smoking affects superoxide generating capacity of ...

The Cholesterol-Lowering Effect of Alisol Acetates Based on HMG-CoA Reductase and Its Molecular Mechanism

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Fei Xu

2016-01-01

Full Text Available This study measured the impact of alisol B 23-acetate and alisol A 24-acetate, the main active ingredients of the traditional Chinese medicine Alismatis rhizoma, on total cholesterol (TC, triglyceride (TG, high density lipoprotein-cholesterol (HDL-C, and low density lipoprotein-cholesterol (LDL-C levels of hyperlipidemic mice. The binding of alisol B 23-acetate and alisol A 24-acetate to the key enzyme involved in the metabolism of TC, 3-hydroxy-3-methylglutary-coenzyme A (HMG-CoA reductase, was studied using the reagent kit method and the western blotting technique combined with a molecular simulation technique. According to the results, alisol acetates significantly lower the TC, TG, and LDL-C concentrations of hyperlipidemic mice, while raising HDL-C concentrations. Alisol acetates lower HMG-CoA reductase activity in a dose-dependent fashion, both in vivo and in vitro. Neither of these alisol acetates significantly lower the protein expression of HMG-CoA. This suggests that alisol acetates lower the TC level via inhibiting the activity of HMG-CoA reductase by its prototype drug, which may exhibit an inhibition effect via directly and competitively binding to HMG-CoA. The side chain of the alisol acetate was the steering group via molecular simulation.

Substrate and cofactor binding to nitrile reductase : A mass spectrometry based study

NARCIS (Netherlands)

Gjonaj, L.; Pinkse, M.W.H.; Fernandez Fueyo, E.; Hollmann, F.; Hanefeld, U.

2016-01-01

Nitrile reductases catalyse a two-step reduction of nitriles to amines. This requires the binding of two NADPH molecules during one catalytic cycle. For the nitrile reductase from E. coli (EcoNR) mass spectrometry studies of the catalytic mechanism were performed. EcoNR is dimeric and has no Rossman

[Cell surface peroxidase--generator of superoxide anion in wheat root cells under wound stress].

Science.gov (United States)

Chasov, A V; Gordon, L Kh; Kolesnikov, O P; Minibaeva, F V

2002-01-01

Development of wound stress in excised wheat roots is known to be accompanied with an increase in reactive oxygen species (ROS) production, fall of membrane potential, release of K+ from cells, alkalization of extracellular solution, changes in respiration and metabolism of structural lipids. Dynamics of superoxide release correlates with changes in other physiological parameters, indicating the cross-reaction of these processes. Activity of peroxidase in extracellular solution after a 1 h incubation and removal of roots was shown to be stimulated by the range of organic acids, detergents, metals, and to be inhibited by cyanide. Superoxide production was sensitive to the addition of Mn2+ and H2O2. Increase in superoxide production correlates with the enhancement of peroxidase activity at the application of organic acids and detergents. The results obtained indicate that cell surface peroxidase is one of the main generators of superoxide in wounded wheat root cells. Different ways of stimulation of the ROS producing activity in root cells is supposed. By controlling superoxide and hydrogen peroxide formation, the cell surface peroxidase can control the adaptation processes in stressed plant cells.

Human Manganese Superoxide Dismutase Tyrosine 34 Contribution to Structure and Catalysis

Science.gov (United States)

Perry, J. Jefferson P.; Hearn, Amy S.; Cabelli, Diane E.; Nick, Harry S.; Tainer, John A.; Silverman, David N.

2009-01-01

Superoxide dismutase (SOD) enzymes are critical in controlling levels of reactive oxygen species (ROS) that are linked to aging, cancer and neurodegenerative disease. Superoxide (O2 •−) produced during respiration is removed by the product of the SOD2 gene, the homotetrameric manganese superoxide dismutase (MnSOD). Here, we examine the structural and catalytic roles of the highly conserved active-site residue Tyr34, based upon structure-function studies of MnSOD enzymes with mutations at this site. Substitution of Tyr34 with five different amino acids retained the active site protein structure and assembly, but causes a substantial decrease in the catalytic rate constant for the reduction of superoxide. The rate constant for formation of product inhibition complex also decreases but to a much lesser extent, resulting in a net increase in the product inhibition form of the mutant enzymes. Comparisons of crystal structures and catalytic rates also suggest that one mutation, Y34V, interrupts the hydrogen-bonded network, which is associated with a rapid dissociation of the product-inhibited complex. Notably, with three of the Tyr34 mutants we also observe an intermediate in catalysis, which has not been reported previously. Thus, these mutants establish a means to trap a catalytic intermediate that promises to help elucidate the mechanism of catalysis. PMID:19265433

Phagocytosis of mast cell granules results in decreased macrophage superoxide production

Directory of Open Access Journals (Sweden)

Bobby A. Shah

1995-01-01

Full Text Available The mechanism by which phagocytosed mast cell granules (MCGs inhibit macrophage superoxide production has not been defined. In this study, rat peritoneal macrophages were co-incubated with either isolated intact MCGs or MCG-sonicate, and their respiratory burst capacity and morphology were studied. Co-incubation of macrophages with either intact MCGs or MCG-sonicate resulted in a dose-dependent inhibition of superoxide- mediated cytochrome c reduction. This inhibitory effect was evident within 5 min of incubation and with MCG-sonicate was completely reversed when macrophages were washed prior to activation with PMA. In the case of intact MCGs, the inhibitory effect was only partially reversed by washing after a prolonged co-incubation time. Electron microscopic analyses revealed that MCGs were rapidly phagocytosed by macrophages and were subsequently disintegrated within the phagolysosomes. Assay of MCGs for superoxide dismutase (SOD revealed the presence of significant activity of this enzyme. A comparison of normal macrophages and those containing phagocytosed MCGs did not reveal a significant difference in total SOD activity. It is speculated that, although there was no significant increase in total SOD activity in macrophages containing phagocytosed MCGs, the phagocytosed MCGs might cause a transient increase in SOD activity within the phagolysosomes. This transient rise in SOD results in scavenging of the newly generated superoxide. Alternatively, MCG inhibition of NADPH oxidase would explain the reported observations.

Extraction of Erythrocyte Enzymes for the Preparation of Polyhemoglobin-catalase-superoxide Dismutase

OpenAIRE

Gu, Jingsong; Chang, Thomas Ming Swi

2009-01-01

In sustained severe ischemia, reperfusion with oxygen carriers may result in ischemia-reperfusion injuries because of the release of damaging oxygen radicals. A nanobiotechnology-based polyhemogloin-calatase-superoxide dismutase can prevent this because the oxygen carrier, polyhemoglobin, is linked to antioxidant enzymes, catalase and superoxide dismutase. However, these antioxidant enzymes come from nonhuman sources and recombinant human enzymes are expensive. This paper describes our study ...

The activity of catalase and superoxide dismutase in isogenous bacteria strains with different radioresistance

International Nuclear Information System (INIS)

Vasil'eva, E.I.; Goncharenko, E.N.; Yudz, T.I.; Samojlenko, I.I.

1984-01-01

The catalase and superoxide dismutase activity in isogenous bacterial strains with various radiosensitivity is investigated. In micrococcus radiodurans mutants with defects in the DNA repair systems the superoxide dismutase activity is lower than in the wild type cells. In investigated Escherichia coli strains differing in radiosensitivity, no alteration in catalase and superoxide dismutase activity is found. The conclusion is drawn that viability of bacteria subjected to the effect of ionizing radiations is determined by the efficiency of DNA repair systems whose functional reliability is sometimes connected with the catalase and suferoxide dismutase activity

Deficiency of superoxide dismutase promotes cerebral vascular hypertrophy and vascular dysfunction in hyperhomocysteinemia.

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Sanjana Dayal

Full Text Available There is an emerging consensus that hyperhomocysteinemia is an independent risk factor for cerebral vascular disease and that homocysteine-lowering therapy protects from ischemic stroke. However, the mechanisms by which hyperhomocysteinemia produces abnormalities of cerebral vascular structure and function remain largely undefined. Our objective in this study was to define the mechanistic role of superoxide in hyperhomocysteinemia-induced cerebral vascular dysfunction and hypertrophy. Unlike previous studies, our experimental design included a genetic approach to alter superoxide levels by using superoxide dismutase 1 (SOD1-deficient mice fed a high methionine/low folate diet to produce hyperhomocysteinemia. In wild-type mice, the hyperhomocysteinemic diet caused elevated superoxide levels and impaired responses to endothelium-dependent vasodilators in cerebral arterioles, and SOD1 deficiency compounded the severity of these effects. The cross-sectional area of the pial arteriolar wall was markedly increased in mice with SOD1 deficiency, and the hyperhomocysteinemic diet sensitized SOD1-deficient mice to this hypertrophic effect. Analysis of individual components of the vascular wall demonstrated a significant increase in the content of smooth muscle and elastin. We conclude that superoxide is a key driver of both cerebral vascular hypertrophy and vasomotor dysfunction in this model of dietary hyperhomocysteinemia. These findings provide insight into the mechanisms by which hyperhomocysteinemia promotes cerebral vascular disease and ischemic stroke.

N-terminus determines activity and specificity of styrene monooxygenase reductases.

Science.gov (United States)

Heine, Thomas; Scholtissek, Anika; Westphal, Adrie H; van Berkel, Willem J H; Tischler, Dirk

2017-12-01

Styrene monooxygenases (SMOs) are two-enzyme systems that catalyze the enantioselective epoxidation of styrene to (S)-styrene oxide. The FADH 2 co-substrate of the epoxidase component (StyA) is supplied by an NADH-dependent flavin reductase (StyB). The genome of Rhodococcus opacus 1CP encodes two SMO systems. One system, which we define as E1-type, displays homology to the SMO from Pseudomonas taiwanensis VLB120. The other system, originally reported as a fused system (RoStyA2B), is defined as E2-type. Here we found that E1-type RoStyB is inhibited by FMN, while RoStyA2B is known to be active with FMN. To rationalize the observed specificity of RoStyB for FAD, we generated an artificial reductase, designated as RoStyBart, in which the first 22 amino acid residues of RoStyB were joined to the reductase part of RoStyA2B, while the oxygenase part (A2) was removed. RoStyBart mainly purified as apo-protein and mimicked RoStyB in being inhibited by FMN. Pre-incubation with FAD yielded a turnover number at 30°C of 133.9±3.5s -1 , one of the highest rates observed for StyB reductases. RoStyBart holo-enzyme switches to a ping-pong mechanism and fluorescence analysis indicated for unproductive binding of FMN to the second (co-substrate) binding site. In summary, it is shown for the first time that optimization of the N-termini of StyB reductases allows the evolution of their activity and specificity. Copyright © 2017 Elsevier B.V. All rights reserved.

Physiological Levels of Nitric Oxide Diminish Mitochondrial Superoxide. Potential Role of Mitochondrial Dinitrosyl Iron Complexes and Nitrosothiols

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Sergey I. Dikalov

2017-11-01

Full Text Available Mitochondria are the major source of superoxide radicals and superoxide overproduction contributes to cardiovascular diseases and metabolic disorders. Endothelial dysfunction and diminished nitric oxide levels are early steps in the development of these pathological conditions. It is known that physiological production of nitric oxide reduces oxidative stress and inflammation, however, the precise mechanism of “antioxidant” effect of nitric oxide is not clear. In this work we tested the hypothesis that physiological levels of nitric oxide diminish mitochondrial superoxide production without inhibition of mitochondrial respiration. In order to test this hypothesis we analyzed effect of low physiological fluxes of nitric oxide (20 nM/min on superoxide and hydrogen peroxide production by ESR spin probes and Amplex Red in isolated rat brain mitochondria. Indeed, low levels of nitric oxide substantially attenuated both basal and antimycin A-stimulated production of reactive oxygen species in the presence of succinate or glutamate/malate as mitochondrial substrates. Furthermore, slow releasing NO donor DPTA-NONOate (100 μM did not change oxygen consumption in State 4 and State 3. However, the NO-donor strongly inhibited oxygen consumption in the presence of uncoupling agent CCCP, which is likely associated with inhibition of the over-reduced complex IV in uncoupled mitochondria. We have examined accumulation of dinitrosyl iron complexes and nitrosothiols in mitochondria treated with fast-releasing NO donor MAHMA NONOate (10 μM for 30 min until complete release of NO. Following treatment with NO donor, mitochondria were frozen for direct detection of dinitrosyl iron complexes using Electron Spin Resonance (ESR while accumulation of nitrosothiols was measured by ferrous-N-Methyl-D-glucamine dithiocarbamate complex, Fe(MGD2, in lysed mitochondria. Treatment of mitochondria with NO-donor gave rise to ESR signal of dinitrosyl iron complexes while ESR

Models of Superoxide Dismutases

Energy Technology Data Exchange (ETDEWEB)

Cabelli, Diane E.; Riley, Dennis; Rodriguez, Jorge A.; Valentine, Joan Selverstone; Zhu, Haining

1998-05-20

In this review we have focused much of our discussion on the mechanistic details of how the native enzymes function and how mechanistic developments/insights with synthetic small molecule complexes possessing SOD activity have influenced our understanding of the electron transfer processes involved with the natural enzymes. A few overriding themes have emerged. Clearly, the SOD enzymes operate at near diffusion controlled rates and to achieve such catalytic turnover activity, several important physical principles must be operative. Such fast electron transfer processes requires a role for protons; i.e., proton-coupled electron transfer (''H-atom transfer'') solves the dilemma of charge separation developing in the transition state for the electron transfer step. Additionally, outer-sphere electron transfer is likely a most important pathway for manganese and iron dismutases. This situation arises because the ligand exchange rates on these two ions in water never exceed {approx}10{sup +7} s{sup -1}; consequently, 10{sup +9} catalytic rates require more subtle mechanistic insights. In contrast, copper complexes can achieve diffusion controlled (>10{sup +9}) exchange rates in water; thus inner-sphere electron transfer processes are more likely to be operative in the Cu/Zn enzymes. Recent studies have continued to expand our understanding of the mechanism of action of this most important class of redox active enzymes, the superoxide dismutases, which have been critical in the successful adaptation of life on this planet to an oxygen-based metabolism. The design of SOD mimic drugs, synthetic models compounds that incorporate this superoxide dismutase catalytic activity and are capable of functioning in vivo, offers clear potential benefits in the control of diseases, ranging from the control of neurodegenerative conditions, such as Parkinson's or Alzheimer's disease, to cancer.

Methemoglobin reductase activity in intact fish red blood cells

DEFF Research Database (Denmark)

Jensen, Frank B; Nielsen, Karsten

2018-01-01

RBCs in physiological saline at normal Pco2 and pH. After initial loading of oxygenated RBCs with nitrite (partly oxidizing Hb to metHb), the nitrite is removed by three washes of the RBCs in nitrite-free physiological saline to enable the detection of RBC metHb reductase activity in the absence......Hb reductase activity in fish offsets their higher Hb autoxidation and higher likelihood of encountering elevated nitrite. Deoxygenation significantly raised the rates of RBC metHb reduction, and more so in rainbow trout than in carp. The temperature sensitivity of metHb reduction in rainbow trout RBCs...

Molecular mechanisms of drug resistance and tumor promotion involving mammalian ribonucleotide reductase

Energy Technology Data Exchange (ETDEWEB)

Choy, B.B.K.

1991-01-01

Mammalian ribonucleotide reductase is a highly regulated, rate-limiting activity responsible for converting ribonucleoside diphosphates to the deoxyribonucleotide precursors of DNA. The enzyme consists of two nonidentical proteins called M1 and M2, both of which are required for activity. Hydroxyurea is an antitumor agent which inhibits ribonucleotide reductase by interacting with the M2 component specifically at a unique tyrosyl free radical. Studies were conducted on a series of drug resistant mouse cell lines, selected by a step-wise procedure for increasing levels of resistance to the cytotoxic effects of hydroxyurea. Each successive drug selection step leading to the isolation of highly resistant cells was accompanied by stable elevations in cellular resistance and ribonucleotide reductase activity. The drug resistant cell lines exhibited gene amplification of the M2 gene, elevated M2 mRNA, and M2 protein. In addition to M2 gene amplification, posttranscriptional modulation also occurred during the drug selection. Studies of the biosynthesis rates with exogenously added iron suggest a role for iron in regulating the level of M2 protein when cells are cultured in the presence of hydroxyurea. The hydroxyurea-inactivated ribonucleotide reductase protein M2 has a destabilized iron centre, which readily releases iron. Altered expression of ferritin appears to be required for the development of hydroxyurea resistance in nammalian cells. The results show an interesting relationship between the expressions of ribonucleotide reductase and ferritin. The phorbol ester tumor promoter, TPA, is also able to alter the expression of M2. TPA was able to induce M2 mRNA levels transiently up to 18-fold within 1/2 hour. This rapid and large elevation of ribonucleotide reductase suggests that the enzyme may play a role in tumor promotion. Studies of the M2 promoter region were undertaken to better understand the mechanism of TPA induction of M2.

Identification of Multiple Soluble Fe(III Reductases in Gram-Positive Thermophilic Bacterium Thermoanaerobacter indiensis BSB-33

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Subrata Pal

2014-01-01

Full Text Available Thermoanaerobacter indiensis BSB-33 has been earlier shown to reduce Fe(III and Cr(VI anaerobically at 60°C optimally. Further, the Gram-positive thermophilic bacterium contains Cr(VI reduction activity in both the membrane and cytoplasm. The soluble fraction prepared from T. indiensis cells grown at 60°C was found to contain the majority of Fe(III reduction activity of the microorganism and produced four distinct bands in nondenaturing Fe(III reductase activity gel. Proteins from each of these bands were partially purified by chromatography and identified by mass spectrometry (MS with the help of T. indiensis proteome sequences. Two paralogous dihydrolipoamide dehydrogenases (LPDs, thioredoxin reductase (Trx, NADP(H-nitrite reductase (Ntr, and thioredoxin disulfide reductase (Tdr were determined to be responsible for Fe(III reductase activity. Amino acid sequence and three-dimensional (3D structural similarity analyses of the T. indiensis Fe(III reductases were carried out with Cr(VI reducing proteins from other bacteria. The two LPDs and Tdr showed very significant sequence and structural identity, respectively, with Cr(VI reducing dihydrolipoamide dehydrogenase from Thermus scotoductus and thioredoxin disulfide reductase from Desulfovibrio desulfuricans. It appears that in addition to their iron reducing activity T. indiensis LPDs and Tdr are possibly involved in Cr(VI reduction as well.

Cell death in response to antimetabolites directed at ribonucleotide reductase and thymidylate synthase

Science.gov (United States)

Asuncion Valenzuela, Malyn M; Castro, Imilce; Gonda, Amber; Diaz Osterman, Carlos J; Jutzy, Jessica M; Aspe, Jonathan R; Khan, Salma; Neidigh, Jonathan W; Wall, Nathan R

2015-01-01

New agent development, mechanistic understanding, and combinatorial partnerships with known and novel modalities continue to be important in the study of pancreatic cancer and its improved treatment. In this study, known antimetabolite drugs such as gemcitabine (ribonucleotide reductase inhibitor) and 5-fluorouracil (thymidylate synthase inhibitor) were compared with novel members of these two drug families in the treatment of a chemoresistant pancreatic cancer cell line PANC-1. Cellular survival data, along with protein and messenger ribonucleic acid expression for survivin, XIAP, cIAP1, and cIAP2, were compared from both the cell cytoplasm and from exosomes after single modality treatment. While all antimetabolite drugs killed PANC-1 cells in a time- and dose-dependent manner, neither family significantly altered the cytosolic protein level of the four inhibitors of apoptosis (IAPs) investigated. Survivin, XIAP, cIAP1, and cIAP2 were found localized to exosomes where no significant difference in expression was recorded. This inability for significant and long-lasting expression may be a reason why pancreatic cancer lacks responsiveness to these and other cancer-killing agents. Continued investigation is required to determine the responsibilities of these IAPs in their role in chemoresistance in pancreatic adenocarcinoma. PMID:25767396

Isolation and characterization of Cu/Zn-superoxide dismutase in Fasciola gigantica.

Science.gov (United States)

Lalrinkima, H; Raina, O K; Chandra, Dinesh; Jacob, Siju Susan; Bauri, R K; Chandra, Subhash; Yadav, H S; Singh, M N; Rialch, A; Varghese, A; Banerjee, P S; Kaur, Navneet; Sharma, Arvind

2015-01-01

A full-length complementary DNA (cDNA) encoding Cu/Zn-superoxide dismutase was isolated from Fasciola gigantica that on nucleotide sequencing showed a close homology (98.9%) with Cu/Zn-superoxide dismutase (SOD) of the temperate liver fluke, F. hepatica. Expression of the gene was found in all the three developmental stages of the parasite viz. adult, newly excysted juvenile and metacercaria at transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. F. gigantica Cu/Zn-SOD cDNA was cloned and expressed in Escherichia coli. Enzyme activity of the recombinant protein was determined by nitroblue tetrazolium (NBT)-polyacrylamide gel electrophoresis (PAGE) and this activity was inactivated by hydrogen peroxide but not by sodium azide, indicating that the recombinant protein is Cu/Zn-SOD. The enzyme activity was relatively stable at a broad pH range of pH 4.0-10.0. Native Cu/Zn-superoxide dismutase protein was detected in the somatic extract and excretory-secretory products of the adult F. gigantica by Western blotting. NBT-PAGE showed a single Cu/Zn-SOD present in the somatic extract while three SODs are released ex vivo by the adult parasite. The recombinant superoxide dismutase did not react with the serum from buffaloes infected with F. gigantica. The role of this enzyme in defense by the parasite against the host reactive oxygen species is discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

In silico docking studies of aldose reductase inhibitory activity of commercially available flavonoids

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Arumugam Madeswaran

2012-12-01

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.

Crystallization and preliminary X-ray diffraction analysis of maize aldose reductase

Energy Technology Data Exchange (ETDEWEB)

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: aparicio@iqm.unicamp.br [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil)

2007-11-01

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.

Crystallization and preliminary X-ray diffraction analysis of maize aldose reductase

International Nuclear Information System (INIS)

Kiyota, Eduardo; Sousa, Sylvia Morais de; Santos, Marcelo Leite dos; Costa Lima, Aline da; Menossi, Marcelo; Yunes, José Andrés; Aparicio, Ricardo

2007-01-01

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 1 2 1 2 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

Prolonged exposure of resveratrol induces reactive superoxide species-independent apoptosis in murine prostate cells.

Science.gov (United States)

Kumar, Sanjay; Stokes, James; Singh, Udai P; Scissum-Gunn, Karyn; Singh, Rajesh; Manne, Upender; Mishra, Manoj K

2017-10-01

Nitric oxide, a signaling molecule, inhibits mitochondrial respiration by binding with cytochrome c oxidase, resulting in elevated production of reactive superoxide species (reactive oxygen and nitrogen) in the mitochondria and increased susceptibility to cell death. Generation of mitochondrial superoxide species can be suppressed by natural compounds such as resveratrol, a dietary polyphenol found in the skin of red fruits. In various cancer cells, resveratrol shows anti-oxidant and cancer preventive properties. Since, the effect of resveratrol on reactive superoxide species-independent apoptosis in prostate cancer cells is not well illustrated; therefore, we investigated this phenomenon in TRAMP murine prostate cancer cells. To accomplish this, TRAMP cells were incubated with resveratrol, resveratrol + DETA-NONOate, DETA-NONOate (nitric oxide donor), resveratrol + L-NMMA, or L-NMMA (nitric oxide inhibitor) for 48 h, and reactive superoxide species in the mitochondria and culture supernatant were measured. In addition, the mitochondrial membrane potential, cell viability, expression of apoptotic markers (Bax and Bcl2), γ-H2A.x, p53, and caspase-3 was determined. We found that resveratrol suppressed reactive superoxide species such as reactive oxygen species in the mitochondria and nitric oxide in culture supernatant when compared to the DETA-NONOate treatment and disrupted the mitochondrial membrane potential. Resveratrol also reduced cell viability, altered the expression of apoptotic markers (Bax and Bcl2), and increased expression of γ-H2A.x (indicative marker of DNA fragmentation) and p53 (a critical DNA damage response protein). However, there was no appreciable modulation of the caspase-3. Therefore, our data suggest that resveratrol induces superoxide species-independent apoptosis and may act as a therapeutic agent against prostate cancer.

Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana plumbaginifolia.

Science.gov (United States)

Kaye, C; Crawford, N M; Malmberg, R L

1997-04-01

We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.

Chemistry of superoxide radical in seawater: CDOM associated sink of superoxide in coastal waters

Energy Technology Data Exchange (ETDEWEB)

Goldstone, J.V.; Voelker, B.M.

2000-03-15

Colored dissolved organic matter (CDOM) and humic substances contain a nonmetallic redox-cycling component capable of catalyzing superoxide (O{sub 2}{sup {minus}}) dismutation. First-order rate coefficients (k{sub pseudo}) measured for this O{sub 2}{sup {minus}} sink in a number of coastal and Chesapeake Bay water samples range up to 1.4s{sup {minus}1}, comparable in magnitude to catalyzed dismutation by Cu species. A significant (r{sup 2}=0.73) correlation is observed between k{sub pseudo} and the optical absorption and salinity of individual coastal water samples, suggesting an association with non-marine-derived CDOM. The activity of this sink is not changed by acidification or boiling of samples but is removed by photooxidation, indicating that it is an organic compound, but that it is neither enzymatic nor likely to consist of tightly bound metals. The stoichiometry of hydrogen peroxide formation from O{sub 2}{sup {minus}} decay indicates that this sink is capable of a redox cycle catalyzing the dismutation of O{sub 2}{sup {minus}}. This CDOM sink combined with the organic copper sink previously described will produce a steady-state superoxide concentration in coastal waters that is 100--1000-fold lower than that predicted from bimolecular dismutation alone. Catalyzed O{sub 2}{sup {minus}} decay was also observed in a variety of humic and fulvic acid samples, possibly occurring through quinone functionalities. Although the presence of quinone moieties in humic and fulvic acids has been demonstrated, there do not appear to be good correlations between several measures of quinone content and the O{sub 2}{sup {minus}} dismutation rates of these samples.

Radiation-induced damage in E. coli B: The effect of superoxide radicals and molecular oxygen. Progress report, December 1, 1978--November 30, 1979

International Nuclear Information System (INIS)

Samuni, A.; Czapski, G.

The roles of superoxide radicals and of molecular oxygen in the radiodamage of E. coli B suspended in dilute phosphate buffer were studied. The presence of high concentrations of polyethylene glycol in the γ-irradiated cell suspensions, had no effect on bacterial radiosensitivity. This indicates that the damage was primarily endogenous, i.e. originated intracellularly. Saturation of the cell suspensions with N 2 O doubled the radiosensitivity, thus indicating that OH radicals are responsible for the majority of the damage (indirect radiation effect). The presence of oxygen either in the absence or presence of N 2 O brought about roughly a three-fold increase in the radiosensitivity. Since in the presence of N 2 O all e - /sub aq/ are scavenged by the nitrous oxide rather than by oxygen, this shows that superoxide radicals play no role in the bacterial radiodamage. Our results substantiate the attribution of the oxygen effect to a direct interaction of O 2 with the hydroxyl-radical-damaged sites on vital biomolecules, and exclude any significant contribution of e - /sub aq/ and superoxide radicals to the cellular radiodamage

The effects of the sulfonylurea glyburide on glutathione peroxidase, superoxide dismutase and catalase activities in the heart tissue of streptozotocin-induced diabetic rat.

Science.gov (United States)

Bukan, N; Sancak, B; Bilgihan, A; Kosova, F; Buğdayci, G; Altan, N

2004-09-01

Oxygen free radicals have been suggested to be a contributory factor in diabetes complications. The aim of this study was to examine the effects of glyburide on the antioxidant enzyme activities in the heart tissue of diabetic rats. We investigated the activities of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) in the hearts of both control and streptozotocin-induced diabetic rats. In the heart of diabetic rats, the activity of total superoxide dismutase decreased significantly (p < 0.005), whereas the activity of catalase and glutathione peroxidase increased to a large extent (p < 0.0001 and p = 0.05, respectively) at the end of the fourth week compared with the control group. Glyburide treatment of diabetic rats for 4 weeks corrected the changes observed in diabetic heart. In addition, blood glucose levels of untreated diabetic rats decreased following the glyburide treatment. These results demonstrate that the sulfonylurea glyburide is capable of exerting direct insulin-like effect on heart superoxide dismutase, catalase and glutathione peroxidase activities of diabetic rats in vivo.

Homology modeling of dissimilatory APS reductases (AprBA of sulfur-oxidizing and sulfate-reducing prokaryotes.

Directory of Open Access Journals (Sweden)

Birte Meyer

Full Text Available BACKGROUND: The dissimilatory adenosine-5'-phosphosulfate (APS reductase (cofactors flavin adenine dinucleotide, FAD, and two [4Fe-4S] centers catalyzes the transformation of APS to sulfite and AMP in sulfate-reducing prokaryotes (SRP; in sulfur-oxidizing bacteria (SOB it has been suggested to operate in the reverse direction. Recently, the three-dimensional structure of the Archaeoglobus fulgidus enzyme has been determined in different catalytically relevant states providing insights into its reaction cycle. METHODOLOGY/PRINCIPAL FINDINGS: Full-length AprBA sequences from 20 phylogenetically distinct SRP and SOB species were used for homology modeling. In general, the average accuracy of the calculated models was sufficiently good to allow a structural and functional comparison between the beta- and alpha-subunit structures (78.8-99.3% and 89.5-96.8% of the AprB and AprA main chain atoms, respectively, had root mean square deviations below 1 A with respect to the template structures. Besides their overall conformity, the SRP- and SOB-derived models revealed the existence of individual adaptations at the electron-transferring AprB protein surface presumably resulting from docking to different electron donor/acceptor proteins. These structural alterations correlated with the protein phylogeny (three major phylogenetic lineages: (1 SRP including LGT-affected Archaeoglobi and SOB of Apr lineage II, (2 crenarchaeal SRP Caldivirga and Pyrobaculum, and (3 SOB of the distinct Apr lineage I and the presence of potential APS reductase-interacting redox complexes. The almost identical protein matrices surrounding both [4Fe-4S] clusters, the FAD cofactor, the active site channel and center within the AprB/A models of SRP and SOB point to a highly similar catalytic process of APS reduction/sulfite oxidation independent of the metabolism type the APS reductase is involved in and the species it has been originated from. CONCLUSIONS: Based on the comparative

The French Paradox: Determining the Superoxide-Scavenging Capacity of Red Wine and Other Beverages

Science.gov (United States)

Logan, Barry A.; Hammond, Matthew P.; Stormo, Benjamin M.

2008-01-01

Plant-derived phenolic compounds such as those found in red wine, tea, and certain fruit juices may protect against cardiovascular disease by detoxifying (scavenging) superoxide and other unstable reactive oxygen species. We present a laboratory exercise that can be used to assess the superoxide-scavenging capacity of beverages. Among the…

Copper, Zinc Superoxide Dismutase is Primarily a Cytosolic Protein in Human Cells

Science.gov (United States)

Crapo, James D.; Oury, Tim; Rabouille, Catherine; Slot, Jan W.; Chang, Ling-Yi

1992-11-01

The intracellular localization of human copper, zinc superoxide dismutase (Cu,Zn-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) was evaluated by using EM immunocytochemistry and both isolated human cell lines and human tissues. Eight monoclonal antibodies raised against either native or recombinant human Cu,Zn-SOD and two polyclonal antibodies raised against either native or recombinant human Cu,Zn-SOD were used. Fixation with 2% paraformaldehyde/0.2% glutaraldehyde was found necessary to preserve normal distribution of the protein. Monoclonal antibodies were less effective than polyclonal antibodies in recognizing the antigen after adequate fixation of tissue. Cu,Zn-SOD was found widely distributed in the cell cytosol and in the cell nucleus, consistent with it being a soluble cytosolic protein. Mitochondria and secretory compartments did not label for this protein. In human cells, peroxisomes showed a labeling density slightly less than that of cytoplasm.

The structure of Lactococcus lactis thioredoxin reductase reveals molecular features of photo-oxidative damage

DEFF Research Database (Denmark)

Skjoldager, Nicklas; Bang, Maria Blanner; Rykær, Martin

2017-01-01

The NADPH-dependent homodimeric flavoenzyme thioredoxin reductase (TrxR) provides reducing equivalents to thioredoxin, a key regulator of various cellular redox processes. Crystal structures of photo-inactivated thioredoxin reductase (TrxR) from the Gram-positive bacterium Lactococcus lactis have...

Treating infected diabetic wounds with superoxidized water as anti-septic agent: a preliminary experience

International Nuclear Information System (INIS)

Hadi, S.F.; Khaliq, T.; Zubair, M.; Saaiq, M.; Sikandar, I.

2007-01-01

To evaluate the effectiveness of superoxidized water (MicrocynTM) in diabetic patients with different wounds. One hundred known diabetic patients were enrolled. Half were randomized to the intervention group (those whose wounds were managed with superoxidized water) and half to the control group (whose wounds were treated with normal saline) using a table of random numbers. The two groups were matched for age, gender, duration of diabetes and category of wound. All patients received appropriate surgical treatment for their wounds as required. Local wound treatment was carried out daily using superoxidized water soaked gauzes on twice daily basis in the intervention group and normal saline in the control group. The treatment was continued until wound healing. The main outcome measures were duration of hospital stay, downgrading of the wound category, wound healing time and need for interventions such as amputation. Statistically significant differences were found in favour of the superoxidized water group with respect to duration of hospital stay, downgrading of the wound category and wound healing time. Although the initial results of employing superoxidized water for the management of infected diabetic wounds are encouraging, further multicentre clinical trials are warranted before this antiseptic is recommended for general use. It may offer an economical alternative to other expensive antiseptics with positive impact on the prevailing infection rates, patient outcomes and patient satisfaction. (author)

Caveolin-1 sensitizes cisplatin-induced lung cancer cell apoptosis via superoxide anion-dependent mechanism.

Science.gov (United States)

Pongjit, Kanittha; Chanvorachote, Pithi

2011-12-01

Caveolin-1 (Cav-1) expression frequently found in lung cancer was linked with disease prognosis and progression. This study reveals for the first time that Cav-1 sensitizes cisplatin-induced lung carcinoma cell death by the mechanism involving oxidative stress modulation. We established stable Cav-1 overexpressed (H460/Cav-1) cells and investigated their cisplatin susceptibility in comparison with control-transfected cells and found that Cav-1 expression significantly enhanced cisplatin-mediated cell death. Results indicated that the different response to cisplatin between these cells was resulted from different level of superoxide anion induced by cisplatin. Inhibitory study revealed that superoxide anion inhibitor MnTBAP could inhibit cisplatin-mediated toxicity only in H460/Cav-1 cells while had no effect on H460 cells. Further, superoxide anion detected by DHE probe indicated that H460/Cav-1 cells generated significantly higher superoxide anion level in response to cisplatin than that of control cells. The role of Cav-1 in regulating cisplatin sensitivity was confirmed in shRNA-mediated Cav-1 down-regulated (H460/shCav-1) cells and the cells exhibited decreased cisplatin susceptibility and superoxide generation. In summary, these findings reveal novel aspects regarding role of Cav-1 in modulating oxidative stress induced by cisplatin, possibly providing new insights for cancer biology and cisplatin-based chemotherapy.

Attenuation of 6-hydroxydopamine-induced dopaminergic nigrostriatal lesions in superoxide dismutase transgenic mice

International Nuclear Information System (INIS)

Cadet, J.L.; Hirata, H.; Asanuma, M.

1998-01-01

6-Hydroxydopamine is a neurotoxin that produces degeneration of the nigrostriatal dopaminergic pathway in rodents. Its toxicity is thought to involve the generation of superoxide anion secondary to its autoxidation. To examine the effects of the overexpression of Cu,Zn-superoxide dismutase activity on 6-hydroxydopamine-induced dopaminergic neuronal damage, we have measured the effects of 6-hydroxydopamine on striatal and nigral dopamine transporters and nigral tyrosine hydroxylase-immunoreactive neurons in Cu,Zn-superoxide dismutase transgenic mice. Intracerebroventricular injection of 6-hydroxydopamine (50 μg) in non-transgenic mice produced reductions in the size of striatal area and an enlargement of the cerebral ventricle on both sides of the brains of mice killed two weeks after the injection. In addition, 6-hydroxydopamine caused marked decreases in striatal and nigral [ 125 I]RTI-121-labelled dopamine transporters not only on the injected side but also on the non-injected side of non-transgenic mice; this was associated with decreased cell number and size of tyrosine hydroxylase-immunoreactive dopamine neurons in the substantia nigra pars compacta on both sides in these mice. In contrast, superoxide dismutase transgenic mice were protected against these neurotoxic effects of 6-hydroxydopamine, with the homozygous transgenic mice showing almost complete protection.These results provide further support for a role of superoxide anion in the toxic effects of 6-hydroxydopamine. They also provide further evidence that reactive oxygen species may be the main determining factors in the neurodegenerative effects of catecholamines. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase

DEFF Research Database (Denmark)

Montalvetti, A; Pena Diaz, Javier; Hurtado, R

2000-01-01

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 L...

Neutrophil superoxide-anion generating capacity in chronic smoking ...

Indian Academy of Sciences (India)

We investigated whether long-term -tocopherol therapy in chronic smoking affects superoxide generating capacity of neutrophils ex vivo. To this purpose, we randomly assigned 128 male chronic smokers (37 ± 21 pack years of smoking) to treatment with placebo ( = 64) or -tocopherol (400 IU dL--tocopherol daily, ...

Methylenetetrahydrofolate reductase gene polymorphism in type 1 ...

African Journals Online (AJOL)

In patients with type-I diabetes mellitus folate deficiency is associated with endothelial dysfunction. So, polymorphism in genes involved in folate metabolism may have a role in vascular disease. This study was designed to evaluate the relationship between methylenetetrahydrofolate reductase (MTHFR) gene polymorphism ...

Characterization of human warfarin reductase

OpenAIRE

Sokolová, Simona

2016-01-01

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Simona Sokolová Supervisor: PharmDr. Petra Malátková, Ph.D. Title of diploma thesis: Characterization of human warfarin reductase Warfarin is widely used anticoagulant drug. Considering the narrow therapeutic window of warfarin, it is important to fully understand its metabolism in human body. Oxidative, reductive and conjugation reactions are involved in warfarin metabolism. Howev...

Superoxide radicals can act synergistically with hypochlorite to induce damage to proteins

DEFF Research Database (Denmark)

Hawkins, Clare Louise; Rees, Martin D; Davies, Michael Jonathan

2002-01-01

Activated phagocytes generate both superoxide radicals via a respiratory burst, and HOCl via the concurrent release of the haem enzyme myeloperoxidase. Amine and amide functions on proteins and carbohydrates are major targets for HOCl, generating chloramines (RNHCl) and chloramides (RC(O)NClR'), ......Activated phagocytes generate both superoxide radicals via a respiratory burst, and HOCl via the concurrent release of the haem enzyme myeloperoxidase. Amine and amide functions on proteins and carbohydrates are major targets for HOCl, generating chloramines (RNHCl) and chloramides (RC...

X-ray effects of lens DNA-implications of superoxide (O2.-)

International Nuclear Information System (INIS)

Srivastava, V.K.; Richards, R.D.; Varma, S.D.

1983-01-01

The photocemical generation of superoxide (O 2 .-) during in vitro exposure of bovine lenses induced damage in the structure of lens DNA as indicated by hyperchromicity and Tm measurements. The damage in lens DNA was significantly protected by the inclusion of superoxide dismutase (SOD), glutathione (GSH) and ascorbate in the incubation medium before X-ray exposure. The protection by SOD, GSH and ascorbate occurred due to their interaction with O 2 .- radicals. These results thus indicate the deleterious effect of O 2 .- in lens physiology and the protective role of such compounds against radiation damage. (author)

Induction of Apoptosis by Superoxide Anion and the Protective Effects of Selenium and Vitamin E

Institute of Scientific and Technical Information of China (English)

无

2001-01-01

Objective The purpose of this study is to investigate the effect of superoxide anion on the apoptosis of cultured fibroblasts and the protective role of selenium and Vitamin E. Methods Cultured fibroblasts (NIH3T3), with or without selenium or vitamin E in the medium, were treated by superoxide anion produced by xanthine/xanthine oxidase reaction system and changes in cell structure and DNA were observed microscopically and electrophoretically. Results Apoptosis was observed when superoxide anion at a concentration of 5 nmol/L or 10 nmol/L had acted on the fibroblasts for 5-10 h. Selenium and Vitamin E in the medium inhibited the apoptosis significantly when their concentrations reached 1.15 mol/L and 2.3 mol/L respectively. Conclusion　Selenium and vitamin E have protective effect against the apoptosis induced by superoxide anion. The effect of selenium is more remarkable than that of vitamin E.

Immobilization of Superoxide Dismutase on Polyelectrolyte-Functionalized Titania Nanosheets.

Science.gov (United States)

Rouster, Paul; Pavlovic, Marko; Szilagyi, Istvan

2018-02-16

The superoxide dismutase (SOD) enzyme was successfully immobilized on titania nanosheets (TNS) functionalized with the poly(diallyldimethylammonium chloride) (PDADMAC) polyelectrolyte. The TNS-PDADMAC solid support was prepared by hydrothermal synthesis followed by self-assembled polyelectrolyte layer formation. It was found that SOD strongly adsorbed onto oppositely charged TNS-PDADMAC through electrostatic and hydrophobic interactions. The TNS-PDADMAC-SOD material was characterized by light scattering and microscopy techniques. Colloidal stability studies revealed that the obtained nanocomposites possessed good resistance against salt-induced aggregation in aqueous suspensions. The enzyme kept its functional integrity upon immobilization; therefore, TNS-PDADMAC-SOD showed excellent superoxide radical anion scavenging activity. The developed system is a promising candidate for applications in which suspensions of antioxidant activity are required in the manufacturing processes. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metabolic regulation of manganese superoxide dismutase expression via essential amino acid deprivation.

Science.gov (United States)

Aiken, Kimberly J; Bickford, Justin S; Kilberg, Michael S; Nick, Harry S

2008-04-18

Organisms respond to available nutrient levels by rapidly adjusting metabolic flux, in part through changes in gene expression. A consequence of adaptations in metabolic rate is the production of mitochondria-derived reactive oxygen species. Therefore, we hypothesized that nutrient sensing could regulate the synthesis of the primary defense of the cell against superoxide radicals, manganese superoxide dismutase. Our data establish a novel nutrient-sensing pathway for manganese superoxide dismutase expression mediated through essential amino acid depletion concurrent with an increase in cellular viability. Most relevantly, our results are divergent from current mechanisms governing amino acid-dependent gene regulation. This pathway requires the presence of glutamine, signaling via the tricarboxylic acid cycle/electron transport chain, an intact mitochondrial membrane potential, and the activity of both the MEK/ERK and mammalian target of rapamycin kinases. Our results provide evidence for convergence of metabolic cues with nutrient control of antioxidant gene regulation, revealing a potential signaling strategy that impacts free radical-mediated mutations with implications in cancer and aging.

Formation and scavenging of superoxide in chloroplasts, with relation to injury by sulfur dioxide

Energy Technology Data Exchange (ETDEWEB)

Asada, K

1980-01-01

Injury of plant leaf cells by sulfur dioxide-exposure is greater in day time than in night. A hypothesis is proposed that the free radical chain oxidation of sulfite is initiated by the superoxide radicals (O/sub 2//sup -/) produced in illuminated chloroplasts, and that the resulting amplified production of O/sub 2//sup -/, the hydroxyl radicals and the bisulfite radicals causes the injury of leaf tissues. In this review, the production of O/sub 2//sup -/ in illuminated chloroplasts and scavenging of O/sub 2//sup -/ by superoxide dismutase and their relation to oxidation of sulfite in chloroplasts are discussed. Superoxide dismutase in chloroplasts plays an important role in protecting leaf cells from injury by sulfur dioxide.

Cadmium phytotoxicity: Quantitative sensitivity relationships between classical endpoints and antioxidative enzyme biomarkers

International Nuclear Information System (INIS)

Rosa Correa, Albertina Xavier da; Roerig, Leonardo Rubi; Verdinelli, Miguel A.; Cotelle, Sylvie; Ferard, Jean-Francois; Radetski, Claudemir Marcos

2006-01-01

In this work, cadmium phytotoxicity and quantitative sensitivity relationships between different hierarchical endpoints in plants cultivated in a contaminated soil were studied. Thus, germination rate, biomass growth and antioxidative enzyme activity (i.e. superoxide dismutase, peroxidase, catalase and glutathione reductase) in three terrestrial plants (Avena sativa L., Brassica campestris L. cv. Chinensis, Lactuca sativa L. cv. hanson) were analyzed. Plant growth tests were carried out according to an International Standard Organization method and the results were analyzed by ANOVA followed by Williams' test. The concentration of Cd 2+ that had the smallest observed significant negative effect (LOEC) on plant biomass was 6.25, 12.5 and 50 mg Cd/kg dry soil for lettuce, oat and Chinese cabbage, respectively. Activity of all enzymes studied increased significantly compared to enzyme activity in plant controls. For lettuce, LOEC values (mg Cd/kg dry soil) for enzymic activity ranged from 0.05 (glutathione reductase) to 0.39 (catalase). For oat, LOEC values (mg Cd/kg dry soil) ranged from 0.19 (for superoxide dismutase and glutathione reductase) to 0.39 (for catalase and peroxidase). For Chinese cabbage, LOEC values (mg Cd/kg dry soil) ranged from 0.19 (peroxidase, catalase and glutathione reductase) to 0.39 (superoxide dismutase). Classical (i.e. germination and biomass) and biochemical (i.e. enzyme activity) endpoints were compared to establish a sensitivity ranking, which was: enzyme activity > biomass > germination rate. For cadmium-soil contamination, the determination of quantitative sensitivity relationships (QSR) between classical and antioxidative enzyme biomarkers showed that the most sensitive plant species have, generally, the lowest QSR values

Inhibitory effect of rhetsinine isolated from Evodia rutaecarpa on aldose reductase activity.

Science.gov (United States)

Kato, A; Yasuko, H; Goto, H; Hollinshead, J; Nash, R J; Adachi, I

2009-03-01

Aldose reductase inhibitors have considerable potential for the treatment of diabetic complications, without increased risk of hypoglycemia. Search for components inhibiting aldose reductase led to the discovery of active compounds contained in Evodia rutaecarpa Bentham (Rutaceae), which is the one of the component of Kampo-herbal medicine. The hot water extract from the E. rutaecarpa was subjected to distribution or gel filtration chromatography to give an active compound, N2-(2-methylaminobenzoyl)tetrahydro-1H-pyrido[3,4-b]indol-1-one (rhetsinine). It inhibited aldose reductase with IC(50) values of 24.1 microM. Furthermore, rhetsinine inhibited sorbitol accumulation by 79.3% at 100 microM. These results suggested that the E. rutaecarpa derived component, rhetsinine, would be potentially useful in the treatment of diabetic complications.

Extraction of erythrocyte enzymes for the preparation of polyhemoglobin-catalase-superoxide dismutase.

Science.gov (United States)

Gu, Jingsong; Chang, Thomas Ming Swi

2009-01-01

In sustained severe ischemia, reperfusion with oxygen carriers may result in ischemia-reperfusion injuries because of the release of damaging oxygen radicals. A nanobiotechnology-based polyhemogloin-calatase-superoxide dismutase can prevent this because the oxygen carrier, polyhemoglobin, is linked to antioxidant enzymes, catalase and superoxide dismutase. However, these antioxidant enzymes come from nonhuman sources and recombinant human enzymes are expensive. This paper describes our study on extracting these enzymes from red blood cells and analyzing the amount of enzymes needed for adequate protection from ischemia-reperfusion.

21 CFR 864.7375 - Glutathione reductase assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

A lithium-oxygen battery based on lithium superoxide.

Science.gov (United States)

Lu, Jun; Lee, Yun Jung; Luo, Xiangyi; Lau, Kah Chun; Asadi, Mohammad; Wang, Hsien-Hau; Brombosz, Scott; Wen, Jianguo; Zhai, Dengyun; Chen, Zonghai; Miller, Dean J; Jeong, Yo Sub; Park, Jin-Bum; Fang, Zhigang Zak; Kumar, Bijandra; Salehi-Khojin, Amin; Sun, Yang-Kook; Curtiss, Larry A; Amine, Khalil

2016-01-21

Batteries based on sodium superoxide and on potassium superoxide have recently been reported. However, there have been no reports of a battery based on lithium superoxide (LiO2), despite much research into the lithium-oxygen (Li-O2) battery because of its potential high energy density. Several studies of Li-O2 batteries have found evidence of LiO2 being formed as one component of the discharge product along with lithium peroxide (Li2O2). In addition, theoretical calculations have indicated that some forms of LiO2 may have a long lifetime. These studies also suggest that it might be possible to form LiO2 alone for use in a battery. However, solid LiO2 has been difficult to synthesize in pure form because it is thermodynamically unstable with respect to disproportionation, giving Li2O2 (refs 19, 20). Here we show that crystalline LiO2 can be stabilized in a Li-O2 battery by using a suitable graphene-based cathode. Various characterization techniques reveal no evidence for the presence of Li2O2. A novel templating growth mechanism involving the use of iridium nanoparticles on the cathode surface may be responsible for the growth of crystalline LiO2. Our results demonstrate that the LiO2 formed in the Li-O2 battery is stable enough for the battery to be repeatedly charged and discharged with a very low charge potential (about 3.2 volts). We anticipate that this discovery will lead to methods of synthesizing and stabilizing LiO2, which could open the way to high-energy-density batteries based on LiO2 as well as to other possible uses of this compound, such as oxygen storage.

Glutathione oxidation in response to intracellular H2O2: Key but overlapping roles for dehydroascorbate reductases.

Science.gov (United States)

Rahantaniaina, Marie-Sylviane; Li, Shengchun; Chatel-Innocenti, Gilles; Tuzet, Andrée; Mhamdi, Amna; Vanacker, Hélène; Noctor, Graham

2017-08-03

Glutathione is a pivotal molecule in oxidative stress, during which it is potentially oxidized by several pathways linked to H 2 O 2 detoxification. We have investigated the response and functional importance of 3 potential routes for glutathione oxidation pathways mediated by glutathione S-transferases (GST), glutaredoxin-dependent peroxiredoxins (PRXII), and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Loss-of-function gstU8, gstU24, gstF8, prxIIE and prxIIF mutants as well as double gstU8 gstU24, gstU8 gstF8, gstU24 gstF8, prxIIE prxIIF mutants were obtained. No mutant lines showed marked changes in their phenotype and glutathione profiles in comparison to the wild-type plants in either optimal conditions or oxidative stress triggered by catalase inhibition. By contrast, multiple loss of DHAR functions markedly decreased glutathione oxidation triggered by catalase deficiency. To assess whether this effect was mediated directly by loss of DHAR enzyme activity, or more indirectly by upregulation of other enzymes involved in glutathione and ascorbate recycling, we measured expression of glutathione reductase (GR) and expression and activity of monodehydroascorbate reductases (MDHAR). No evidence was obtained that either GRs or MDHARs were upregulated in plants lacking DHAR function. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H 2 O 2 metabolism.

Characterisation of a desmosterol reductase involved in phytosterol dealkylation in the silkworm, Bombyx mori.

Directory of Open Access Journals (Sweden)

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.

Characterisation of a Desmosterol Reductase Involved in Phytosterol Dealkylation in the Silkworm, Bombyx mori

Science.gov (United States)

Ciufo, Leonora F.; Murray, Patricia A.; Thompson, Anu; Rigden, Daniel J.; Rees, Huw H.

2011-01-01

Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). 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 250kDa. 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. PMID:21738635

Inhibition of human anthracycline reductases by emodin — A possible remedy for anthracycline resistance

International Nuclear Information System (INIS)

Hintzpeter, Jan; Seliger, Jan Moritz; Hofman, Jakub; Martin, Hans-Joerg; Wsol, Vladimir; Maser, Edmund

2016-01-01

The clinical application of anthracyclines, like daunorubicin and doxorubicin, is limited by two factors: dose-related cardiotoxicity and drug resistance. Both have been linked to reductive metabolism of the parent drug to their metabolites daunorubicinol and doxorubicinol, respectively. These metabolites show significantly less anti-neoplastic properties as their parent drugs and accumulate in cardiac tissue leading to chronic cardiotoxicity. Therefore, we aimed to identify novel and potent natural inhibitors for anthracycline reductases, which enhance the anticancer effect of anthracyclines by preventing the development of anthracycline resistance. Human enzymes responsible for the reductive metabolism of daunorubicin were tested for their sensitivity towards anthrachinones, in particular emodin and anthraflavic acid. Intense inhibition kinetic data for the most effective daunorubicin reductases, including IC 50 - and K i -values, the mode of inhibition, as well as molecular docking, were compiled. Subsequently, a cytotoxicity profile and the ability of emodin to reverse daunorubicin resistance were determined using multiresistant A549 lung cancer and HepG2 liver cancer cells. Emodin potently inhibited the four main human daunorubicin reductases in vitro. Further, we could demonstrate that emodin is able to synergistically sensitize human cancer cells towards daunorubicin at clinically relevant concentrations. Therefore, emodin may yield the potential to enhance the therapeutic effectiveness of anthracyclines by preventing anthracycline resistance via inhibition of the anthracycline reductases. In symphony with its known pharmacological properties, emodin might be a compound of particular interest in the management of anthracycline chemotherapy efficacy and their adverse effects. - Highlights: • Natural and synthetic compounds were identified as inhibitors for human daunorubicin reductases. • Emodin is a potent inhibitor for human daunorubicin reductases. �

Sulfite-oxido-reductase is involved in the oxidation of sulfite in Desulfocapsa sulfoexigens during disproportionation of thiosulfate and elemental sulfur.

Science.gov (United States)

Frederiksen, Trine-Maria; Finster, Kai

2003-06-01

The enzymatic pathways of elemental sulfur and thiosulfate disproportionation were investigated using cell-free extract of Desulfocapsa sulfoexigens. Sulfite was observed to be an intermediate in the metabolism of both compounds. Two distinct pathways for the oxidation of sulfite have been identified. One pathway involves APS reductase and ATP sulfurylase and can be described as the reversion of the initial steps of the dissimilatory sulfate reduction pathway. The second pathway is the direct oxidation of sulfite to sulfate by sulfite oxidoreductase. This enzyme has not been reported from sulfate reducers before. Thiosulfate reductase, which cleaves thiosulfate into sulfite and sulfide, was only present in cell-free extract from thiosulfate disproportionating cultures. We propose that this enzyme catalyzes the first step in thiosulfate disproportionation. The initial step in sulfur disproportionation was not identified. Dissimilatory sulfite reductase was present in sulfur and thiosulfate disproportionating cultures. The metabolic function of this enzyme in relation to elemental sulfur or thiosulfate disproportionation was not identified. The presence of the uncouplers HQNO and CCCP in growing cultures had negative effects on both thiosulfate and sulfur disproportionation. CCCP totally inhibited sulfur disproportionation and reduced thiosulfate disproportionation by 80% compared to an unamended control. HQNO reduced thiosulfate disproportionation by 80% and sulfur disproportionation by 90%.

Colour formation in fermented sausages by meat-associated staphylococci with different nitrite- and nitrate-reductase activities

DEFF Research Database (Denmark)

Gøtterup, Jacob; Olsen, Karsten; Knøchel, Susanne

2008-01-01

nitrate depended on the specific Staphylococcus strain. Strains with high nitrate-reductase activity showed a significantly faster rate of pigment formation, but other factors were of influence as well. Product stability for the sliced, packaged sausage was evaluated as surface colour and oxidation......Three Staphylococcus strains, S. carnosus, S. simulans and S. saprophyticus, selected due to their varying nitrite and/or nitrate-reductase activities, were used to initiate colour formation during sausage fermentation. During fermentation of sausages with either nitrite or nitrate added, colour...... with hexanal content, and may be used as predictive tools. Overall, nitrite- and nitrate-reductase activities of Staphylococcus strains in nitrite-cured sausages were of limited importance regarding colour development, while in nitrate-cured sausages strains with higher nitrate reductase activity were crucial...

Water stress induces overexpression of superoxide dismutases that ...

African Journals Online (AJOL)

Water stress is known to induce active oxygen species in plants. The accumulation of these harmful species must be prevented by plants as rapidly as possible to maintain growth and productivity. The aim of this study was to determine the effect of water stress on superoxide dismutase isozymes (SOD, EC 1.15.1.1.) in two ...

Biochemical markers of oxidative stress in Perna viridis exposed to mercury and temperature

Digital Repository Service at National Institute of Oceanography (India)

Verlecar, X.N.; Jena, K.B.; Chainy, G.B.N.

peroxidation (LPX). Increased activities of antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-S-transferase (GST) both in gills and digestive glands under long...

Aldose Reductase Inhibitory and Antiglycation Activities of Four ...

African Journals Online (AJOL)

Aldose Reductase Inhibitory and Antiglycation Activities of Four Medicinal Plant Standardized Extracts and Their Main Constituents for the Prevention of ... levels in galactosemic condition by using reverse phase high pressure liquid chromatography (RP-HPLC) and gas liquid chromatography (GLC) was determined.

Xylose reductase from the thermophilic fungus Talaromyces emersonii

Indian Academy of Sciences (India)

Prakash

Xylose reductase is involved in the first step of the fungal pentose catabolic pathway. The gene .... proteins with reversed coenzyme preference from NADPH to NADH ..... 399–404. Hasper A A, Visser J and de Graaff L H 2000 The Aspergillus.

Regulation of schistosome egg production by HMG CoA reductase

International Nuclear Information System (INIS)

VandeWaa, E.A.; Bennett, J.L.

1986-01-01

Hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase) catalyzes the conversion of HMG CoA to mevalonate in the synthesis of steroids, isoprenoids and terpenes. Mevinolin, an inhibitor of this enzyme, decreased egg production in Schistosoma mansoni during in vitro incubations. This was associated with a reduction in the incorporation of 14 C-acetate into polyisoprenoids and a reduction in the formation of a lipid-linked oligosaccharide. In vivo, mevinolin in daily doses of 50 mg/kg (p.o., from days 30-48 post-infection) caused no change in gross liver pathology in S. mansoni infected mice. However, when parasites exposed to mevinolin or its vehicle in vivo were cultured in vitro, worms from mevinolin-treated mice produced six times more eggs than control parasites. When infected mice were dosed with 250 mg/kg mevinolin daily (p.o., from days 35-45 post-infection), liver pathology was reduced in comparison to control mice. Thus, during in vivo exposure to a high dose of the drug egg production is decreased, while at a lower dose it appears unaffected until the parasites are cultured in a drug-free in vitro system wherein egg production is stimulated to extraordinarily high levels. It may be that at low doses mevinolin, by inhibiting the enzyme, is blocking the formation of a product (such as an isoprenoid) which normally acts to down-regulate enzyme synthesis, resulting in enzyme induction. Induction of HMG CoA reductase is then expressed as increased egg production when the worms are removed from the drug. These data suggest that HMG CoA reductase plays a role in schistosome egg production

Inhibition of aldose reductase activity by Cannabis sativa chemotypes extracts with high content of cannabidiol or cannabigerol.

Science.gov (United States)

Smeriglio, Antonella; Giofrè, Salvatore V; Galati, Enza M; Monforte, Maria T; Cicero, Nicola; D'Angelo, Valeria; Grassi, Gianpaolo; Circosta, Clara

2018-02-07

Aldose reductase (ALR2) is a key enzyme involved in diabetic complications and the search for new aldose reductase inhibitors (ARIs) is currently very important. The synthetic ARIs are often associated with deleterious side effects and medicinal and edible plants, containing compounds with aldose reductase inhibitory activity, could be useful for prevention and therapy of diabetic complications. Non-psychotropic phytocannabinoids exert multiple pharmacological effects with therapeutic potential in many diseases such as inflammation, cancer, diabetes. Here, we have investigated the inhibitory effects of extracts and their fractions from two Cannabis sativa L. chemotypes with high content of cannabidiol (CBD)/cannabidiolic acid (CBDA) and cannabigerol (CBG)/cannabigerolic acid (CBGA), respectively, on human recombinant and pig kidney aldose reductase activity in vitro. A molecular docking study was performed to evaluate the interaction of these cannabinoids with the active site of ALR2 compared to known ARIs. The extracts showed significant dose-dependent aldose reductase inhibitory activity (>70%) and higher than fractions. The inhibitory activity of the fractions was greater for acidic cannabinoid-rich fractions. Comparative molecular docking results have shown a higher stability of the ALR2-cannabinoid acids complex than the other inhibitors. The extracts of Cannabis with high content of non-psychotropic cannabinoids CBD/CBDA or CBG/CBGA significantly inhibit aldose reductase activity. These results may have some relevance for the possible use of C. sativa chemotypes based preparations as aldose reductase inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

Metabolic Regulation of Manganese Superoxide Dismutase Expression via Essential Amino Acid Deprivation*

Science.gov (United States)

Aiken, Kimberly J.; Bickford, Justin S.; Kilberg, Michael S.; Nick, Harry S.

2008-01-01

Organisms respond to available nutrient levels by rapidly adjusting metabolic flux, in part through changes in gene expression. A consequence of adaptations in metabolic rate is the production of mitochondria-derived reactive oxygen species. Therefore, we hypothesized that nutrient sensing could regulate the synthesis of the primary defense of the cell against superoxide radicals, manganese superoxide dismutase. Our data establish a novel nutrient-sensing pathway for manganese superoxide dismutase expression mediated through essential amino acid depletion concurrent with an increase in cellular viability. Most relevantly, our results are divergent from current mechanisms governing amino acid-dependent gene regulation. This pathway requires the presence of glutamine, signaling via the tricarboxylic acid cycle/electron transport chain, an intact mitochondrial membrane potential, and the activity of both the MEK/ERK and mammalian target of rapamycin kinases. Our results provide evidence for convergence of metabolic cues with nutrient control of antioxidant gene regulation, revealing a potential signaling strategy that impacts free radical-mediated mutations with implications in cancer and aging. PMID:18187411

JS-K, a Nitric Oxide Prodrug, Has Enhanced Cytotoxicity in Colon Cancer Cells with Knockdown of Thioredoxin Reductase 1

Science.gov (United States)

Edes, Kornelia; Cassidy, Pamela; Shami, Paul J.; Moos, Philip J.

2010-01-01

Background The selenoenzyme thioredoxin reductase 1 has a complex role relating to cell growth. It is induced as a component of the cellular response to potentially mutagenic oxidants, but also appears to provide growth advantages to transformed cells by inhibiting apoptosis. In addition, selenocysteine-deficient or alkylated forms of thioredoxin reductase 1 have also demonstrated oxidative, pro-apoptotic activity. Therefore, a greater understanding of the role of thioredoxin reductase in redox initiated apoptotic processes is warranted. Methodology The role of thioredoxin reductase 1 in RKO cells was evaluated by attenuating endogenous thioredoxin reductase 1 expression with siRNA and then either inducing a selenium-deficient thioredoxin reductase or treatment with distinct redox challenges including, hydrogen peroxide, an oxidized lipid, 4-hydroxy-2-nonenol, and a nitric oxide donating prodrug. Thioredoxin redox status, cellular viability, and effector caspase activity were measured. Conclusions/Significance In cells with attenuated endogenous thioredoxin reductase 1, a stably integrated selenocysteine-deficient form of the enzyme was induced but did not alter either the thioredoxin redox status or the cellular growth kinetics. The oxidized lipid and the nitric oxide donor demonstrated enhanced cytotoxicity when thioredoxin reductase 1 was knocked-down; however, the effect was more pronounced with the nitric oxide prodrug. These results are consistent with the hypothesis that attenuation of the thioredoxin-system can promote apoptosis in a nitric oxide-dependent manner. PMID:20098717

Inorganic Polyphosphates Regulate Hexokinase Activity and Reactive Oxygen Species Generation in Mitochondria of Rhipicephalus (Boophilus) microplus Embryo

Science.gov (United States)

Fraga, Amanda; Moraes, Jorge; da Silva, José Roberto; Costa, Evenilton P.; Menezes, Jackson; da Silva Vaz Jr, Itabajara; Logullo, Carlos; da Fonseca, Rodrigo Nunes; Campos, Eldo

2013-01-01

The physiological roles of polyphosphates (poly P) recently found in arthropod mitochondria remain obscure. Here, the possible involvement of poly P with reactive oxygen species generation in mitochondria of Rhipicephalus microplus embryos was investigated. Mitochondrial hexokinase and scavenger antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione reductase were assayed during embryogenesis of R. microplus. The influence of poly P3 and poly P15 were analyzed during the period of higher enzymatic activity during embryogenesis. Both poly Ps inhibited hexokinase activity by up to 90% and, interestingly, the mitochondrial membrane exopolyphosphatase activity was stimulated by the hexokinase reaction product, glucose-6-phosphate. Poly P increased hydrogen peroxide generation in mitochondria in a situation where mitochondrial hexokinase is also active. The superoxide dismutase, catalase and glutathione reductase activities were higher during embryo cellularization, at the end of embryogenesis and during embryo segmentation, respectively. All of the enzymes were stimulated by poly P3. However, superoxide dismutase was not affected by poly P15, catalase activity was stimulated only at high concentrations and glutathione reductase was the only enzyme that was stimulated in the same way by both poly Ps. Altogether, our results indicate that inorganic polyphosphate and mitochondrial membrane exopolyphosphatase regulation can be correlated with the generation of reactive oxygen species in the mitochondria of R. microplus embryos. PMID:23983617

Superoxide dismutase of human platelets

International Nuclear Information System (INIS)

Kimura, Akiro; Fujimura, Kingo; Kuramoto, Atsushi

1979-01-01

Superoxide dismutase (S.O.D.) is the enzyme to protect from destructive effect of superoxide (O 2 -) produced in many metabolic pathways related to oxygen. The purpose of this study was to investigate the possibility that S.O.D. may play an important role in the platelet function. The cytoplasmic and mitochondrial S.O.D. has been investigated spectrophotometrically and gel electrophoretically in human platelets from eleven patients of chronic myelogenous leukemia (CML) and three patients of primary thrombocythemia (P.Th.). Neither deficiency nor abnormality of cytoplasmic and mitochondrial S.O.D. has been found electrophoretically in any case compared to normal platelets. However, the total activity from three of the CML patients and one of the P.Th. patients were above 3 unit/mg platelet protein (normal subject: 2.11 - 2.70 unit/mg protein), suggesting the possibility either that more O 2 -production occurs in the platelets or that rather little O 2 -production due to much O 2 -deprivation by the increased S.O.D. The S.O.D. activity of human platelets has been also investigated in several conditions, where much O 2 -generation might occur in platelets. Sodium fluoride (2 mM), which increases platelet O 2 -production about 3 fold, had no effect on platelet S.O.D. The aggregated platelets induced by ADP (10 -5 M), epinephrin (50 μg/ml), ristocetin (1.5 mg/ml) or collagen (1 - 20 μg/ml) had no increase of S.O.D. activity compared to that from non aggregated platelets. X-ray irradiation (1,000 - 20,000R) had not induced its activity increase or decrease. These findings indicated the induction of platelet S.O.D. was not brought about under these conditions. (author)

Seasonal variation of oxidative biomarkers in gills and digestive gland of green-lipped mussel Perna viridis from Arabian Sea

Digital Repository Service at National Institute of Oceanography (India)

Verlecar, X.N.; Jena, K.B.; Chainy, G.B.N.

peroxidation (LPX), hydrogen peroxide (H sub(2) O sub(2)), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione transferase (GST), glutathione reductase (GR), reduced glutathione (GSH) and ascorbic acid (ASA) were measured...

Modulation of antioxidant and biotransformation enzymes in the gills of Perna viridis (L.) exposed to water accomodate fraction of diesel

Digital Repository Service at National Institute of Oceanography (India)

Jena, K.B.; Verlecar, X.N.; Chainy, G.B.N.

tested for cell damage and antioxidant responses in P. viridis for over 15 day period. The parameters measured included lipid peroxidation (LPX), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR...

Measurement of Antioxidant Activity Towards Superoxide in Natural Waters.

Directory of Open Access Journals (Sweden)

D. Whitney King

2016-11-01

Full Text Available Antioxidants are a class of molecules that provide a protective function against reactive oxygen species (ROS in biological systems by out competing physiologically important molecules for ROS oxidation. In natural waters, the reactivity of antioxidants gives an estimate of oxidative stress and may determine the reactivity and distribution of reactive oxidants. We present an analytical method to measure antioxidant activity in natural waters through the competition between ascorbic acid, an antioxidant, and MCLA, a chemiluminescent probe for superoxide. A numerical kinetic model of the analytical method has been developed to optimize analytical performance. Measurements of antioxidant concentrations in pure and seawater are possible with detection limits below 0.1 nM. Surface seawater samples collected at solar noon contained over 0.4 nM of antioxidants and exhibited first-order decay with a half-life of 3-7 minutes, consistent with a reactive species capable of scavenging photochemically produced superoxide.

ACTIVITY OF SUPEROXIDE DISMUTASE ENZYME IN YEAST SACCHAROMYCES CEREVISIAE

Directory of Open Access Journals (Sweden)

Blažena Lavová

2014-02-01

Full Text Available Reactive oxygen species (ROS with reactive nitrogen species (RNS are known to play dual role in biological systems, they can be harmful or beneficial to living systems. ROS can be important mediators of damage to cell structures, including proteins, lipids and nucleic acids termed as oxidative stress. The antioxidant enzymes protect the organism against the oxidative damage caused by active oxygen forms. The role of superoxide dismutase (SOD is to accelerate the dismutation of the toxic superoxide radical, produced during oxidative energy processes, to hydrogen peroxide and molecular oxygen. In this study, SOD activity of three yeast strains Saccharomyces cerevisiae was determined. It was found that SOD activity was the highest (23.7 U.mg-1 protein in strain 612 after 28 hours of cultivation. The lowest SOD activity from all tested strains was found after 56 hours of cultivation of strain Gyöng (0.7 U.mg-1 protein.

Superoxide activates a GDP-sensitive proton conductance in skeletal muscle mitochondria from king penguin (Aptenodytes patagonicus).

Science.gov (United States)

Talbot, Darren A; Hanuise, Nicolas; Rey, Benjamin; Rouanet, Jean-Louis; Duchamp, Claude; Brand, Martin D

2003-12-26

We present the partial nucleotide sequence of the avian uncoupling protein (avUCP) gene from king penguin (Aptenodytes patagonicus), showing that the protein is 88-92% identical to chicken (Gallus gallus), turkey (Meleagris gallopavo), and hummingbird (Eupetomena macroura). We show that superoxide activates the proton conductance of mitochondria isolated from king penguin skeletal muscle. GDP abolishes the superoxide-activated proton conductance, indicating that it is mediated via avUCP. In the absence of superoxide there is no GDP-sensitive component of the proton conductance from penguin muscle mitochondria demonstrating that avUCP plays no role in the basal proton leak.

Inhibition of human anthracycline reductases by emodin — A possible remedy for anthracycline resistance

Energy Technology Data Exchange (ETDEWEB)

Hintzpeter, Jan, E-mail: hintzpeter@toxi.uni-kiel.de [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Seliger, Jan Moritz [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Hofman, Jakub [Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove (Czech Republic); Martin, Hans-Joerg [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Wsol, Vladimir [Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove (Czech Republic); Maser, Edmund [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany)

2016-02-15

The clinical application of anthracyclines, like daunorubicin and doxorubicin, is limited by two factors: dose-related cardiotoxicity and drug resistance. Both have been linked to reductive metabolism of the parent drug to their metabolites daunorubicinol and doxorubicinol, respectively. These metabolites show significantly less anti-neoplastic properties as their parent drugs and accumulate in cardiac tissue leading to chronic cardiotoxicity. Therefore, we aimed to identify novel and potent natural inhibitors for anthracycline reductases, which enhance the anticancer effect of anthracyclines by preventing the development of anthracycline resistance. Human enzymes responsible for the reductive metabolism of daunorubicin were tested for their sensitivity towards anthrachinones, in particular emodin and anthraflavic acid. Intense inhibition kinetic data for the most effective daunorubicin reductases, including IC{sub 50}- and K{sub i}-values, the mode of inhibition, as well as molecular docking, were compiled. Subsequently, a cytotoxicity profile and the ability of emodin to reverse daunorubicin resistance were determined using multiresistant A549 lung cancer and HepG2 liver cancer cells. Emodin potently inhibited the four main human daunorubicin reductases in vitro. Further, we could demonstrate that emodin is able to synergistically sensitize human cancer cells towards daunorubicin at clinically relevant concentrations. Therefore, emodin may yield the potential to enhance the therapeutic effectiveness of anthracyclines by preventing anthracycline resistance via inhibition of the anthracycline reductases. In symphony with its known pharmacological properties, emodin might be a compound of particular interest in the management of anthracycline chemotherapy efficacy and their adverse effects. - Highlights: • Natural and synthetic compounds were identified as inhibitors for human daunorubicin reductases. • Emodin is a potent inhibitor for human daunorubicin

Structural and biochemical properties of cloned and expressed human and rat steroid 5α-reductases

International Nuclear Information System (INIS)

Andersson, S.; Russell, D.W.

1990-01-01

The microsomal enzyme steroid 5α-reductase is responsible for the conversion of testosterone into the more potent androgen dihydrotestosterone. In man, this steroid acts on a variety of androgen-responsive target tissues to mediate such diverse endocrine processes as male sexual differentiation in the fetus and prostatic growth in men. Here we describe the isolation, structure, and expression of a cDNA encoding the human steroid 5α-reductase. A rat cDNA was used as a hybridization probe to screen a human prostate cDNA library. A 2.1-kilobase cDNA was identified and DNA sequence analysis indicated that the human steroid 5α-reductase was a hydrophobic protein of 259 amino acids with a predicted molecular weight of 29,462. A comparison of the human and rat protein sequences revealed a 60% identity. Transfection of expression vectors containing the human and rat cDNAs into simian COS cells resulted in the synthesis of high levels of steroid 5α-reductase enzyme activity. Both enzymes expressed in COS cells showed similar substrate specificities for naturally occurring steroid hormones. However, synthetic 4-azasteroids demonstrated marked differences in their abilities to inhibit the human and rat steroid 5α-reductases

Overview of Catalytic Properties of Fungal Xylose Reductases and Molecular Engineering Approaches for Improved Xylose Utilisation in Yeast

Directory of Open Access Journals (Sweden)

Sk Amir Hossain

2018-03-01

Full Text Available Background and Objective: Xylose reductases belong to the aldo-keto reductase family of enzymes, which catalyse the conversion of xylose to xylitol. Yeast xylose reductases have been intensively studied in the last two decades due to their significance in biotechnological production of ethanol and xylitol from xylose. Due to its GRAS status and pronounced tolerance to harsh conditions, Saccharomyces cerevisiae is the ideal organism for industrial production of both xylitol and ethanol. However, Saccharomyces cerevisiae is unable to use xylose as the sole carbon source due to the lack of xylose specific transporters and insufficient activity of metabolic pathways for xylose utilisation. The aim of this paper is to give an overview of attempts in increasing biotechnological potential of xylose reductases and to highlight the prospective of this application. Results and Conclusion: In order to create strains with improved xylose utilization, different approaches were attempted including simultaneous overexpression of xylitol dehydrogenase, xylose reductase and pentose phosphate pathway enzymes, heterologous expression of putative xylose transporters or heterologous expression of genes coding for enzymes included in the xylose metabolism, respectively. Furthermore, number of attempts to genetically modify different xylose reductases is increasing. This review presents current knowledge about yeast xylose reductases and the different approaches applied in order to improve xylose metabolism in yeast.Conflict of interest: The authors declare no conflict of interest.

Aldose Reductase Inhibitory Activity of Compounds from  Zea mays L.

Science.gov (United States)

Kim, Tae Hyeon; Kim, Jin Kyu; Kang, Young-Hee; Lee, Jae-Yong; Kang, Il Jun; Lim, Soon Sung

2013-01-01

Aldose reductase (AR) inhibitors have a considerable therapeutic potential against diabetes complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of an EtOH extract of the kernel from purple corn (Zea mays L.), 7 nonanthocyanin phenolic compounds (compound 1–7) and 5 anthocyanins (compound 8–12) were isolated. These compounds were investigated by rat lens aldose reductase (RLAR) inhibitory assays. Kinetic analyses of recombinant human aldose reductase (rhAR) were performed, and intracellular galactitol levels were measured. Hirsutrin, one of 12 isolated compounds, showed the most potent RLAR inhibitory activity (IC50, 4.78 μM). In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate concentration, hirsutrin showed competitive inhibition against rhAR. Furthermore, hirsutrin inhibited galactitol formation in rat lens and erythrocytes sample incubated with a high concentration of galactose; this finding indicates that hirsutrin may effectively prevent osmotic stress in hyperglycemia. Therefore, hirsutrin derived from Zea mays L. may be a potential therapeutic agent against diabetes complications. PMID:23586057

Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency.

Science.gov (United States)

Waddell, D; Ullman, B

1983-04-10

From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.

Do Superoxide Dismutase (SOD) and Catalase (CAT) protect Cells from DNA Damage Induced by Active Arsenicals?

Science.gov (United States)

Superoxide dismutase (SOD) catalyzes the conversion of superoxide to hydrogen peroxide, which can be converted to water and oxygen through the action of catalase. Heterozygous mice of strain B6: 129S7-SodltmlLeb/J were obtained from Jackson Laboratories and bred to produce offspr...

Plasmid-encoded diacetyl (acetoin) reductase in Leuconostoc pseudomesenteroides

DEFF Research Database (Denmark)

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

2003-01-01

A plasmid-borne diacetyl (acetoin) reductase (butA) from Leuconostoc pseudomesenteroides CHCC2114 was sequenced and cloned. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin...

Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct hydride transfer

International Nuclear Information System (INIS)

Kaspera, Rüdiger; Sahele, Tariku; Lakatos, Kyle; Totah, Rheem A.

2012-01-01

Highlights: ► Cytochrome P450BM-3 reduced aldehydes to alcohols efficiently (k cat ∼ 25 min −1 ). ► Reduction is a direct hydride transfer from R-NADP 2 H to the carbonyl moiety. ► P450 domain variants enhance reduction through potential allosteric/redox interactions. ► Novel reaction will have implications for metabolism of xenobiotics. -- Abstract: Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k cat of ∼25 min −1 was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. The reduction was caused by a direct hydride transfer from preferentially R-NADP 2 H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP 2 H but not D 2 O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.

Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis.

Science.gov (United States)

Castro-Cerritos, Karla Viridiana; Yasbin, Ronald E; Robleto, Eduardo A; Pedraza-Reyes, Mario

2017-02-15

The Gram-positive microorganism Bacillus subtilis relies on a single class Ib ribonucleotide reductase (RNR) to generate 2'-deoxyribonucleotides (dNDPs) for DNA replication and repair. In this work, we investigated the influence of RNR levels on B. subtilis stationary-phase-associated mutagenesis (SPM). Since RNR is essential in this bacterium, we engineered a conditional mutant of strain B. subtilis YB955 (hisC952 metB5 leu427) in which expression of the nrdEF operon was modulated by isopropyl-β-d-thiogalactopyranoside (IPTG). Moreover, genetic inactivation of ytcG, predicted to encode a repressor (NrdR) of nrdEF in this strain, dramatically increased the expression levels of a transcriptional nrdE-lacZ fusion. The frequencies of mutations conferring amino acid prototrophy in three genes were measured in cultures under conditions that repressed or induced RNR-encoding genes. The results revealed that RNR was necessary for SPM and overexpression of nrdEF promoted growth-dependent mutagenesis and SPM. We also found that nrdEF expression was induced by H 2 O 2 and such induction was dependent on the master regulator PerR. These observations strongly suggest that the metabolic conditions operating in starved B. subtilis cells increase the levels of RNR, which have a direct impact on SPM. Results presented in this study support the concept that the adverse metabolic conditions prevailing in nutritionally stressed bacteria activate an oxidative stress response that disturbs ribonucleotide reductase (RNR) levels. Such an alteration of RNR levels promotes mutagenic events that allow Bacillus subtilis to escape from growth-limited conditions. Copyright © 2017 American Society for Microbiology.

Cadmium phytotoxicity: Quantitative sensitivity relationships between classical endpoints and antioxidative enzyme biomarkers

Energy Technology Data Exchange (ETDEWEB)

Rosa Correa, Albertina Xavier da [Centro de Ciencias Tecnologicas da Terra e do Mar, Universidade do Vale do Itajai, Rua Uruguai, 458, 88302-202 Itajai SC (Brazil); Roerig, Leonardo Rubi [Centro de Ciencias Tecnologicas da Terra e do Mar, Universidade do Vale do Itajai, Rua Uruguai, 458, 88302-202 Itajai SC (Brazil); Verdinelli, Miguel A. [Centro de Ciencias Tecnologicas da Terra e do Mar, Universidade do Vale do Itajai, Rua Uruguai, 458, 88302-202 Itajai SC (Brazil); Cotelle, Sylvie [Centre des Sciences de l' Environnement, Universite de Metz, 57000 Metz (France); Ferard, Jean-Francois [Centre des Sciences de l' Environnement, Universite de Metz, 57000 Metz (France); Radetski, Claudemir Marcos [Centro de Ciencias Tecnologicas da Terra e do Mar, Universidade do Vale do Itajai, Rua Uruguai, 458, 88302-202 Itajai SC (Brazil)]. E-mail: radetski@univali.br

2006-03-15

In this work, cadmium phytotoxicity and quantitative sensitivity relationships between different hierarchical endpoints in plants cultivated in a contaminated soil were studied. Thus, germination rate, biomass growth and antioxidative enzyme activity (i.e. superoxide dismutase, peroxidase, catalase and glutathione reductase) in three terrestrial plants (Avena sativa L., Brassica campestris L. cv. Chinensis, Lactuca sativa L. cv. hanson) were analyzed. Plant growth tests were carried out according to an International Standard Organization method and the results were analyzed by ANOVA followed by Williams' test. The concentration of Cd{sup 2+} that had the smallest observed significant negative effect (LOEC) on plant biomass was 6.25, 12.5 and 50 mg Cd/kg dry soil for lettuce, oat and Chinese cabbage, respectively. Activity of all enzymes studied increased significantly compared to enzyme activity in plant controls. For lettuce, LOEC values (mg Cd/kg dry soil) for enzymic activity ranged from 0.05 (glutathione reductase) to 0.39 (catalase). For oat, LOEC values (mg Cd/kg dry soil) ranged from 0.19 (for superoxide dismutase and glutathione reductase) to 0.39 (for catalase and peroxidase). For Chinese cabbage, LOEC values (mg Cd/kg dry soil) ranged from 0.19 (peroxidase, catalase and glutathione reductase) to 0.39 (superoxide dismutase). Classical (i.e. germination and biomass) and biochemical (i.e. enzyme activity) endpoints were compared to establish a sensitivity ranking, which was: enzyme activity > biomass > germination rate. For cadmium-soil contamination, the determination of quantitative sensitivity relationships (QSR) between classical and antioxidative enzyme biomarkers showed that the most sensitive plant species have, generally, the lowest QSR values.

X-Ray crystal structure of GarR—tartronate semialdehyde reductase from Salmonella typhimurium

OpenAIRE

Osipiuk, J.; Zhou, M.; Moy, S.; Collart, F.; Joachimiak, A.

2009-01-01

Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determi...

Cell lysis and superoxide dismutase activities of highly radioresistant bacteria

Energy Technology Data Exchange (ETDEWEB)

Yoshinaka, T; Yano, K; Yamaguchi, H [Tokyo Univ. (Japan). Faculty of Agriculture

1976-01-01

The highly radioresistant bacterium, Arthrobacter radiotolerans, has been isolated from the radioactive hot spring of Misasa, and it does not sporulate, it is Gram-positive, and its color is pink to red. This bacterium shows the highest resistance to gamma-ray among Gram-positive resistants, but the lytic enzyme capable of lysing the cells of strong radioresistants and the surface structure of the cells are little known except those about Micrococcus radiodurans. The cells of the M. radiodurans can be lysed by Achramobacter lyticus enzyme, and electron microscopic observation and chemical analysis revealed the mutilayered surface structure of the cells consisting of an inner membrane, a mucopeptide wall layer and a very outer layer. The superoxide dismutase (SOD) activity of aerobic and anaerobic bacteria was studied, and the relatively high SOD activity of the M. radiodurans was found. The SOD function acts against the threat posed by the reactive superoxide radical being generated biologically, photochemically and radiochemically in the presence of molecular oxygen. In this paper, it is reported that the lytic enzyme No.2 obtained from Cytophaga sp., containing N-acetyl-muramyl-L-alanine amidase, peptidase and endopeptidase, and showing broad lytic spectra, was able to lyse the cells of A. radiotolerans and four radioresistant micrococci, and the radioresistant bacteria showedrelatively high SOD activity except M. sp. 248. It is well known that superoxide anions are generated by aerobic irradiation, and are toxic to microbial cells.

Cell lysis and superoxide dismutase activities of highly radioresistant bacteria

International Nuclear Information System (INIS)

Yoshinaka, Taeko; Yano, Keiji; Yamaguchi, Hikoyuki

1976-01-01

The highly radioresistant bacterium, Arthrobacter radiotolerans, has been isolated from the radioactive hot spring of Misasa, and it does not sporulate, it is Gram-positive, and its color is pink to red. This bacterium shows the highest resistance to gamma-ray among Gram-positive resistants, but the lytic enzyme capable of lysing the cells of strong radioresistants and the surface structure of the cells are little known except those about Micrococcus radiodurans. The cells of the M. radiodurans can be lysed by Achramobacter lyticus enzyme, and electron microscopic observation and chemical analysis revealed the mutilayered surface structure of the cells consisting of an inner membrane, a mucopeptide wall layer and a very outer layer. The superoxide dismutase (SOD) activity of aerobic and anaerobic bacteria was studied, and the relatively high SOD activity of the M. radiodurans was found. The SOD function acts against the threat posed by the reactive superoxide radical being generated biologically, photochemically and radiochemically in the presence of molecular oxygen. In this paper, it is reported that the lytic enzyme No.2 obtained from Cytophaga sp., containing N-acetyl-muramyl-L-alanine amidase, peptidase and endopeptidase, and showing broad lytic spectra, was able to lyse the cells of A. radiotolerans and four radioresistant micrococci, and the radioresistant bacteria showed relatively high SOD activity except M. sp. 248. It is well known that superoxide anions are generated by aerobic irradiation, and are toxic to microbial cells. (Kako, I.)

Electrolyte Chemistry for Simultaneous Stabilization of Potassium Metal and Superoxide in K-O₂ Batteries.

Science.gov (United States)

Xiao, Neng; Gourdin, Gerald; Wu, Yiying

2018-05-22

In the superoxide batteries based on O2/O2- redox chemistry, identifying an electrolyte to stabilize both alkali metal and superoxide remains challenging due to their reactivity towards electrolyte components. Bis(fluorosulfonyl)imide (FSI-) has been recognized as a "magical anion" for passivating alkali metals. Herein, we illustrate the chemical reactions between FSI- and superoxide, and the resultant dilemma when considering an anode-compatible electrolyte vs. a cathode-compatible one in K-O2 batteries. On one side, the KFSI-dimethoxyethane (DME) electrolyte passivates the potassium metal anode via the cleavage of S-F bond and formation of a KF-rich solid electrolyte interface (SEI). Nevertheless, the KFSI salt is chemically unstable due to the nucleophilic attack by superoxide and/or hydroxide species. On the other hand, potassium bis(trifluorosulfonyl)imide (KTFSI) is stable for KO2, but results in mossy deposition and irreversible plating and stripping. In order to circumvent this dilemma, we develop an artificial SEI for K metal anode to achieve long cycle-life K-O2 batteries. This work contributes to the understanding of electrolyte chemistry and guides the development of stable electrolytes and artificial SEI in metal-O2 batteries. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reduced bone mass and muscle strength in male 5α-reductase type 1 inactivated mice.

Directory of Open Access Journals (Sweden)

Sara H Windahl

Full Text Available Androgens are important regulators of bone mass but the relative importance of testosterone (T versus dihydrotestosterone (DHT for the activation of the androgen receptor (AR in bone is unknown. 5α-reductase is responsible for the irreversible conversion of T to the more potent AR activator DHT. There are two well established isoenzymes of 5α-reductase (type 1 and type 2, encoded by separate genes (Srd5a1 and Srd5a2. 5α-reductase type 2 is predominantly expressed in male reproductive tissues whereas 5α-reductase type 1 is highly expressed in liver and moderately expressed in several other tissues including bone. The aim of the present study was to investigate the role of 5α-reductase type 1 for bone mass using Srd5a1⁻/⁻ mice. Four-month-old male Srd5a1⁻/⁻ mice had reduced trabecular bone mineral density (-36%, p<0.05 and cortical bone mineral content (-15%, p<0.05 but unchanged serum androgen levels compared with wild type (WT mice. The cortical bone dimensions were reduced in the male Srd5a1⁻/⁻ mice as a result of a reduced cortical periosteal circumference compared with WT mice. T treatment increased the cortical periosteal circumference (p<0.05 in orchidectomized WT mice but not in orchidectomized Srd5a1⁻/⁻ mice. Male Srd5a1⁻/⁻ mice demonstrated a reduced forelimb muscle grip strength compared with WT mice (p<0.05. Female Srd5a1⁻/⁻ mice had slightly increased cortical bone mass associated with elevated circulating levels of androgens. In conclusion, 5α-reductase type 1 inactivated male mice have reduced bone mass and forelimb muscle grip strength and we propose that these effects are due to lack of 5α-reductase type 1 expression in bone and muscle. In contrast, the increased cortical bone mass in female Srd5a1⁻/⁻ mice, is an indirect effect mediated by elevated circulating androgen levels.

Effect of cystamine on rat tissue GSH level and glutathione reductase activity

International Nuclear Information System (INIS)

Kovarova, H.; Pulpanova, J.

1979-01-01

Reduced glutathione (GSH) level and glutathione reductase activity were determined by means of the spectrophotometric method in various rat tissues after i.p. administration of cystamine (50 mg/kg and 20 mg/kg). GSH amount dropped in the spleen and kidney at 10 and 20 min; following this interval, an increase of GSH level was observed in the liver at 20-30 min, in the spleen and kidney at 60 min after the treatment with a radioprotective cystamine dose (50 mg/kg). The changes in GSH level induced by a non-radioprotective cystamine dose (20 mg/kg) had an opposite tendency. The activity of glutathione reductase was decreased in all tissues studied. As to the mechanism of the radioprotective action, both the inactivation of glutathione reductase activity and the changes in GSH level seem to be the factors contributing to the radioprotective effect of cystamine by strengthening the cellular radioresistance. (orig.) 891 MG/orig. 892 RKD [de

Expression, purification, crystallization and preliminary X-ray diffraction analysis of carbonyl reductase from Candida parapsilosis ATCC 7330

International Nuclear Information System (INIS)

Aggarwal, Nidhi; Mandal, P. K.; Gautham, Namasivayam; Chadha, Anju

2013-01-01

The expression, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies on C. parapsilosis carbonyl reductase are reported. The NAD(P)H-dependent carbonyl reductase from Candida parapsilosis ATCC 7330 catalyses the asymmetric reduction of ethyl 4-phenyl-2-oxobutanoate to ethyl (R)-4-phenyl-2-hydroxybutanoate, a precursor of angiotensin-converting enzyme inhibitors such as Cilazapril and Benazepril. The carbonyl reductase was expressed in Escherichia coli and purified by GST-affinity and size-exclusion chromatography. Crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to 1.86 Å resolution. The asymmetric unit contained two molecules of carbonyl reductase, with a solvent content of 48%. The structure was solved by molecular replacement using cinnamyl alcohol dehydrogenase from Saccharomyces cerevisiae as a search model

Proanthocyanidin synthesis in Theobroma cacao: genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase.

Science.gov (United States)

Liu, Yi; Shi, Zi; Maximova, Siela; Payne, Mark J; Guiltinan, Mark J

2013-12-05

The proanthocyanidins (PAs), a subgroup of flavonoids, accumulate to levels of approximately 10% total dry weight of cacao seeds. PAs have been associated with human health benefits and also play important roles in pest and disease defense throughout the plant. To dissect the genetic basis of PA biosynthetic pathway in cacao (Theobroma cacao), we have isolated three genes encoding key PA synthesis enzymes, anthocyanidin synthase (ANS), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). We measured the expression levels of TcANR, TcANS and TcLAR and PA content in cacao leaves, flowers, pod exocarp and seeds. In all tissues examined, all three genes were abundantly expressed and well correlated with PA accumulation levels, suggesting their active roles in PA synthesis. Overexpression of TcANR in an Arabidopsis ban mutant complemented the PA deficient phenotype in seeds and resulted in reduced anthocyanidin levels in hypocotyls. Overexpression of TcANS in tobacco resulted in increased content of both anthocyanidins and PAs in flower petals. Overexpression of TcANS in an Arabidopsis ldox mutant complemented its PA deficient phenotype in seeds. Recombinant TcLAR protein converted leucoanthocyanidin to catechin in vitro. Transgenic tobacco overexpressing TcLAR had decreased amounts of anthocyanidins and increased PAs. Overexpressing TcLAR in Arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin. Our results confirm the in vivo function of cacao ANS and ANR predicted based on sequence homology to previously characterized enzymes from other species. In addition, our results provide a clear functional analysis of a LAR gene in vivo.

Oxidative stress and superoxide dismutase activity in brain of rats ...

African Journals Online (AJOL)

JTEkanem

effect of superoxide dismutase (SOD) activity in brain homogenates of Wistar rats. Oxidative stress measured as ..... on the brain and nervous system of humans as handlers and ... environment may be at higher health risk in that their internal ...

Photoaffinity labeling of steroid 5 alpha-reductase of rat liver and prostate microsomes

International Nuclear Information System (INIS)

Liang, T.; Cheung, A.H.; Reynolds, G.F.; Rasmusson, G.H.

1985-01-01

21-Diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione (Diazo-MAPD) inhibits steroid 5 alpha-reductase in liver microsomes of female rats with a K/sub i/ value of 8.7 +/- 1.7 nM, and the inhibition is competitive with testosterone. It also inhibits the binding of a 5 alpha-reductase inhibitor, [ 3 H] 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([ 3 H]4-MA), to the enzyme in liver microsomes. The inhibition of 5 alpha-reductase activity and of inhibitor binding activity by diazo-MAPD becomes irreversible upon UV irradiation. [1,2- 3 H]Diazo-MAPD binds to a single high affinity site in liver microsomes of female rats, and this binding requires NADPH. Without UV irradiation, this binding is reversible, and it becomes irreversible upon UV irradiation. Both the initial reversible binding and the subsequent irreversible conjugation after UV irradiation are inhibited by inhibitors (diazo-MAPD and 4-MA) and substrates (progesterone and testosterone) of 5 alpha-reductase, but they are not inhibited by 5 alpha-reduced steroids. Photoaffinity labeled liver microsomes of female rats were solubilized and fractionated by high performance gel filtration. The radioactive conjugate eluted in one major peak at Mr 50,000

Superoxide dismutating molecules rescue the toxic effects of PINK1 and parkin loss.

Science.gov (United States)

Biosa, Alice; Sanchez-Martinez, Alvaro; Filograna, Roberta; Terriente-Felix, Ana; Alam, Sarah M; Beltramini, Mariano; Bubacco, Luigi; Bisaglia, Marco; Whitworth, Alexander J

2018-05-01

Reactive oxygen species exert important functions in regulating several cellular signalling pathways. However, an excessive accumulation of reactive oxygen species can perturb the redox homeostasis leading to oxidative stress, a condition which has been associated to many neurodegenerative disorders. Accordingly, alterations in the redox state of cells and mitochondrial homeostasis are established hallmarks in both familial and sporadic Parkinson's disease cases. PINK1 and Parkin are two genes which account for a large fraction of autosomal recessive early-onset forms of Parkinson's disease and are now firmly associated to both mitochondria and redox homeostasis. In this study we explored the hypothesis that superoxide anions participate in the generation of the Parkin and PINK1 associated phenotypic effect by testing the capacity of endogenous and exogenous superoxide dismutating molecules to rescue the toxic effects induced by loss of PINK1 or Parkin, in both cellular and fly models. Our results demonstrate the positive effect of an increased level of superoxide dismutase proteins on the pathological phenotypes, both in vitro and in vivo. A more pronounced effectiveness for mitochondrial SOD2 activity points to the superoxide radicals generated in the mitochondrial matrix as the prime suspect in the definition of the observed phenotypes. Moreover, we also demonstrate the efficacy of a SOD-mimetic compound, M40403, to partially ameliorate PINK1/Parkin phenotypes in vitro and in vivo. These results support the further exploration of SOD-mimetic compounds as a therapeutic strategy against Parkinson's disease.

Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase.

Science.gov (United States)

Liew, Li Phing; Lim, Zun Yi; Cohen, Matan; Kong, Ziqing; Marjavaara, Lisette; Chabes, Andrei; Bell, Stephen D

2016-11-01

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Nitrate reductase gene involvement in hexachlorobiphenyl dechlorination by Phanerochaete chrysosporium

International Nuclear Information System (INIS)

De, Supriyo; Perkins, Michael; Dutta, Sisir K.

2006-01-01

Polychlorobiphenyl (PCB) degradation usually occurs through reductive dechlorination under anaerobic conditions and phenolic ring cleavage under aerobic conditions. In this paper, we provide evidence of nitrate reductase (NaR) mediated dechlorination of hexachlorobiphenyl (PCB-153) in Phanerochaete chrysosporium under non-ligninolytic condition and the gene involved. The NaR enzyme and its cofactor, molybdenum (Mo), were found to mediate reductive dechlorination of PCBs even in aerobic condition. Tungsten (W), a competitive inhibitor of this enzyme, was found to suppress this dechlorination. Chlorine release assay provided further evidence of this nitrate reductase mediated dechlorination. Commercially available pure NaR enzyme from Aspergillus was used to confirm these results. Through homology search using TBLASTN program, NaR gene was identified, primers were designed and the RT-PCR product was sequenced. The NaR gene was then annotated in the P. chrysosporium genome (GenBank accession no. AY700576). This is the first report regarding the presence of nitrate reductase gene in this fungus with the explanation why this fungus can dechlorinate PCBs even in aerobic condition. These fungal inoculums are used commercially as pellets in sawdust for enhanced bioremediation of PCBs at the risk of depleting soil nitrates. Hence, the addition of nitrates to the pellets will reduce this risk as well as enhance its activity

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

Directory of Open Access Journals (Sweden)

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.

Preliminary neutron diffraction analysis of challenging human manganese superoxide dismutase crystals.

Science.gov (United States)

Azadmanesh, Jahaun; Trickel, Scott R; Weiss, Kevin L; Coates, Leighton; Borgstahl, Gloria E O

2017-04-01

Superoxide dismutases (SODs) are enzymes that protect against oxidative stress by dismutation of superoxide into oxygen and hydrogen peroxide through cyclic reduction and oxidation of the active-site metal. The complete enzymatic mechanisms of SODs are unknown since data on the positions of hydrogen are limited. Here, methods are presented for large crystal growth and neutron data collection of human manganese SOD (MnSOD) using perdeuteration and the MaNDi beamline at Oak Ridge National Laboratory. The crystal from which the human MnSOD data set was obtained is the crystal with the largest unit-cell edge (240 Å) from which data have been collected via neutron diffraction to sufficient resolution (2.30 Å) where hydrogen positions can be observed.

Isolation and primary structural analysis of two conjugated polyketone reductases from Candida parapsilosis.

Science.gov (United States)

Hidalgo, A R; Akond, M A; Kita, K; Kataoka, M; Shimizu, S

2001-12-01

Two conjugated polyketone reductases (CPRs) were isolated from Candida parapsilosis IFO 0708. The primary structures of CPRs (C1 and C2) were analyzed by amino acid sequencing. The amino acid sequences of both enzymes had high similarity to those of several proteins of the aldo-keto-reductase (AKR) superfamily. However, several amino acid residues in the putative active sites of AKRs were not conserved in CPRs-C1 and -C2.

The role of extended Fe4S4 cluster ligands in mediating sulfite reductase hemoprotein activity.

Science.gov (United States)

Cepeda, Marisa R; McGarry, Lauren; Pennington, Joseph M; Krzystek, J; Elizabeth Stroupe, M

2018-05-28

The siroheme-containing subunit from the multimeric hemoflavoprotein NADPH-dependent sulfite reductase (SiR/SiRHP) catalyzes the six electron-reduction of SO 3 2- to S 2- . Siroheme is an iron-containing isobacteriochlorin that is found in sulfite and homologous siroheme-containing nitrite reductases. Siroheme does not work alone but is covalently coupled to a Fe 4 S 4 cluster through one of the cluster's ligands. One long-standing hypothesis predicted from this observation is that the environment of one iron-containing cofactor influences the properties of the other. We tested this hypothesis by identifying three amino acids (F437, M444, and T477) that interact with the Fe 4 S 4 cluster and probing the effect of altering them to alanine on the function and structure of the resulting enzymes by use of activity assays, X-ray crystallographic analysis, and EPR spectroscopy. We showed that F437 and M444 gate access for electron transfer to the siroheme-cluster assembly and the direct hydrogen bond between T477 and one of the cluster sulfides is important for determining the geometry of the siroheme active site. Copyright © 2018. Published by Elsevier B.V.

Progress in Understanding Algal Bloom-Mediated Fish Kills: The Role of Superoxide Radicals, Phycotoxins and Fatty Acids.

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Juan José Dorantes-Aranda

Full Text Available Quantification of the role of reactive oxygen species, phycotoxins and fatty acids in fish toxicity by harmful marine microalgae remains inconclusive. An in vitro fish gill (from rainbow trout Oncorhynchus mykiss assay was used to simultaneously assess the effect in superoxide dismutase, catalase and lactate dehydrogenase enzymatic activities caused by seven species of ichthyotoxic microalgae (Chattonella marina, Fibrocapsa japonica, Heterosigma akashiwo, Karenia mikimotoi, Alexandrium catenella, Karlodinium veneficum, Prymnesium parvum. Quantification of superoxide production by these algae was also performed. The effect of purified phycotoxins and crude extracts was compared, and the effect of fatty acids is discussed. The raphidophyte Chattonella was the most ichthyotoxic (gill cell viability down to 35% and also the major producer of superoxide radicals (14 pmol cell-1 hr-1 especially after cell lysis. The raphidophyte Heterosigma and dinoflagellate Alexandrium were the least toxic and had low superoxide production, except when A. catenella was lysed (5.6 pmol cell-1 hr-1. Catalase showed no changes in activity in all the treatments. Superoxide dismutase (SOD and lactate dehydrogenase exhibited significant activity increases of ≤23% and 51.2% TCC (total cellular content, respectively, after exposure to C. marina, but SOD showed insignificant changes with remaining algal species. A strong relationship between gill cell viability and superoxide production or superoxide dismutase was not observed. Purified brevetoxins PbTx-2 and -3 (from Karenia brevis, LC50 of 22.1 versus 35.2 μg mL-1 and karlotoxin KmTx-2 (from Karlodinium; LC50 = 380 ng mL-1 could almost entirely account for the fish killing activity by those two dinoflagellates. However, the paralytic shellfish toxins (PST GTX1&4, C1&C2, and STX did not account for Alexandrium ichthyotoxicity. Only aqueous extracts of Alexandrium were cytotoxic (≤65% decrease of viability, whereas

Molecular Cloning and Expression of Bacterial Mercuric Reductase ...

African Journals Online (AJOL)

In order to characterize the bacterial mercuric reductase (merA) gene, mercury resistant (Hgr) Escherichia coli strains have been isolated from various mercury contaminated sites of India. Their minimum inhibitory concentration (MIC) for Hg and zone of inhibition for different antibiotics were measured, and finally mer operon ...

Molecular Cloning and Expression of Bacterial Mercuric Reductase ...

African Journals Online (AJOL)

USER

2010-06-21

Jun 21, 2010 ... In order to characterize the bacterial mercuric reductase (merA) gene, mercury resistant (Hgr). Escherichia coli strains have been isolated from various mercury contaminated sites of India. Their minimum inhibitory concentration (MIC) for Hg and zone of inhibition for different antibiotics were measured, and ...

Potency of a novel saw palmetto ethanol extract, SPET-085, for inhibition of 5alpha-reductase II.

Science.gov (United States)

Pais, Pilar

2010-08-01

The nicotinamide adenine dinucleotide phosphate (NADPH)-dependent membrane protein 5alpha-reductase irreversibly catalyses the conversion of testosterone to the most potent androgen, 5alpha-dihydrotestosterone (DHT). In humans, two 5alpha-reductase isoenyzmes are expressed: type I and type II. Type II is found primarily in prostate tissue. Saw palmetto extract (SPE) has been widely used for the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH). The mechanisms of the pharmacological effects of SPE include the inhibition of 5alpha-reductase, among other actions. Clinical studies of SPE have been equivocal, with some showing significant results and others not. These inconsistent results may be due, in part, to varying bioactivities of the SPE used in the studies. The aim of the present study was to determine the in vitro potency of a novel saw palmetto ethanol extract (SPET-085), an inhibitor of the 5alpha-reductase isoenzyme type II, in a cell-free test system. On the basis of the enzymatic conversion of the substrate androstenedione to the 5alpha-reduced product 5alpha-androstanedione, the inhibitory potency was measured and compared to those of finasteride, an approved 5alpha-reductase inhibitor. SPET-085 concentration-dependently inhibited 5alpha-reductase type II in vitro (IC(50)=2.88+/-0.45 microg/mL). The approved 5alpha-reductase inhibitor, finasteride, tested as positive control, led to 61% inhibition of 5alpha-reductase type II. SPET-085 effectively inhibits the enzyme that has been linked to BPH, and the amount of extract required for activity is very low compared to data reported for other extracts. It can be concluded from data in the literature that SPET-085 is as effective as a hexane extract of saw palmetto that exhibited the highest levels of bioactivity, and is more effective than other SPEs tested. This study confirmed that SPET-085 has prostate health-promoting bioactivity that also corresponds favorably to

Intraethnic variation in steroid-5-alpha-reductase polymorphisms in ...

Indian Academy of Sciences (India)

2015-06-01

Jun 1, 2015 ... in prostate cancer patients: a potential factor implicated ... reductase alpha polypeptides 1 and 2 in a set of 601 prostate cancer patients from four ..... tion in the key androgen-regulating genes androgen receptor, cytochrome ...

Molecular and phenotypic characterization of transgenic soybean expressing the Arabidopsis ferric chelate reductase gene, FRO2.

Science.gov (United States)

Vasconcelos, Marta; Eckert, Helene; Arahana, Venancio; Graef, George; Grusak, Michael A; Clemente, Tom

2006-10-01

Soybean (Glycine max Merr.) production is reduced under iron-limiting calcareous soils throughout the upper Midwest regions of the US. Like other dicotyledonous plants, soybean responds to iron-limiting environments by induction of an active proton pump, a ferric iron reductase and an iron transporter. Here we demonstrate that heterologous expression of the Arabidopsis thaliana ferric chelate reductase gene, FRO2, in transgenic soybean significantly enhances Fe(+3) reduction in roots and leaves. Root ferric reductase activity was up to tenfold higher in transgenic plants and was not subjected to post-transcriptional regulation. In leaves, reductase activity was threefold higher in the transgenic plants when compared to control. The enhanced ferric reductase activity led to reduced chlorosis, increased chlorophyll concentration and a lessening in biomass loss in the transgenic events between Fe treatments as compared to control plants grown under hydroponics that mimicked Fe-sufficient and Fe-deficient soil environments. However, the data indicate that constitutive FRO2 expression under non-iron stress conditions may lead to a decrease in plant productivity as reflected by reduced biomass accumulation in the transgenic events under non-iron stress conditions. When grown at Fe(III)-EDDHA levels greater than 10 microM, iron concentration in the shoots of transgenic plants was significantly higher than control. The same observation was found in the roots in plants grown at iron levels higher than 32 microM Fe(III)-EDDHA. These results suggest that heterologous expression of an iron chelate reductase in soybean can provide a route to alleviate iron deficiency chlorosis.

Stereoselective hydrogenation of olefins using rhodium-substituted carbonic anhydrase--a new reductase.

Science.gov (United States)

Jing, Qing; Okrasa, Krzysztof; Kazlauskas, Romas J

2009-01-01

One useful synthetic reaction missing from nature's toolbox is the direct hydrogenation of substrates using hydrogen. Instead nature uses cofactors like NADH to reduce organic substrates, which adds complexity and cost to these reductions. To create an enzyme that can directly reduce organic substrates with hydrogen, researchers have combined metal hydrogenation catalysts with proteins. One approach is an indirect link where a ligand is linked to a protein and the metal binds to the ligand. Another approach is direct linking of the metal to protein, but nonspecific binding of the metal limits this approach. Herein, we report a direct hydrogenation of olefins catalyzed by rhodium(I) bound to carbonic anhydrase (CA-[Rh]). We minimized nonspecific binding of rhodium by replacing histidine residues on the protein surface using site-directed mutagenesis or by chemically modifying the histidine residues. Hydrogenation catalyzed by CA-[Rh] is slightly slower than for uncomplexed rhodium(I), but the protein environment induces stereoselectivity favoring cis- over trans-stilbene by about 20:1. This enzyme is the first cofactor-independent reductase that reduces organic molecules using hydrogen. This catalyst is a good starting point to create variants with tailored reactivity and selectivity. This strategy to insert transition metals in the active site of metalloenzymes opens opportunities to a wider range of enzyme-catalyzed reactions.

Research progress on the roles of aldose reductase in diabetic retinopathy

Directory of Open Access Journals (Sweden)

Hong-Zhe Li

2015-07-01

Full Text Available Aldose reductase(ARbelonging to nicotinamide-adenine dinucleotide phosphate(NADPH-dependent aldehyde-keto reductase superfamily, is the key rate-limiting enzyme in the polyol pathway which plays an important role in the body's high-sugar metabolism. AR is widely present in the kidneys, blood vessels, lens, retina, heart, skeletal muscle and other tissues and organs, converts glucose to sorbitol which easy permeability of cell membranes, cause cell swelling, degeneration, necrosis, and have a close relationship with the development of chronic complications of diabetes mellitus. Diabetic retinopathy(DRis a multifactorial disease, the exact cause is currently unknown, but polyol pathway has been demonstrated to play an important role in the pathogenesis of DR. Clinical risk factors such as blood sugar control, blood pressure and other treatments for DR only play a part effect of remission or invalid, if we can find out DR genes associated with the disease, this will contribute to a better understanding of the pathological mechanisms and contribute to the development of new treatments and drugs. The current research progress of AR, AR gene polymorphism, Aldose reductase inhibitors to DR was reviewed in this article.

Hydrogen peroxide induce modifications of human extracellular superoxide dismutase that results in enzyme inhibition

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Randi H. Gottfredsen

2013-01-01

Full Text Available Superoxide dismutase (EC-SOD controls the level of superoxide in the extracellular space by catalyzing the dismutation of superoxide into hydrogen peroxide and molecular oxygen. In addition, the enzyme reacts with hydrogen peroxide in a peroxidase reaction which is known to disrupt enzymatic activity. Here, we show that the peroxidase reaction supports a site-specific bond cleavage. Analyses by peptide mapping and mass spectrometry shows that oxidation of Pro112 supports the cleavage of the Pro112–His113 peptide bond. Substitution of Ala for Pro112 did not inhibit fragmentation, indicating that the oxidative fragmentation at this position is dictated by spatial organization and not by side-chain specificity. The major part of EC-SOD inhibited by the peroxidase reaction was not fragmented but found to encompass oxidations of histidine residues involved in the coordination of copper (His98 and His163. These oxidations are likely to support the dissociation of copper from the active site and thus loss of enzymatic activity. Homologous modifications have also been described for the intracellular isozyme, Cu/Zn-SOD, reflecting the almost identical structures of the active site within these enzymes. We speculate that the inactivation of EC-SOD by peroxidase activity plays a role in regulating SOD activity in vivo, as even low levels of superoxide will allow for the peroxidase reaction to occur.

Inhibition of steroid 5 alpha-reductase by specific aliphatic unsaturated fatty acids.

Science.gov (United States)

Liang, T; Liao, S

1992-01-01

Human or rat microsomal 5 alpha-reductase activity, as measured by enzymic conversion of testosterone into 5 alpha-dihydrotestosterone or by binding of a competitive inhibitor, [3H]17 beta-NN-diethulcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA) to the reductase, is inhibited by low concentrations (less than 10 microM) of certain polyunsaturated fatty acids. The relative inhibitory potencies of unsaturated fatty acids are, in decreasing order: gamma-linolenic acid greater than cis-4,7,10,13,16,19-docosahexaenoic acid = cis-6,9,12,15-octatetraenoic acid = arachidonic acid = alpha-linolenic acid greater than linoleic acid greater than palmitoleic acid greater than oleic acid greater than myristoleic acid. Other unsaturated fatty acids such as undecylenic acid, erucic acid and nervonic acid, are inactive. The methyl esters and alcohol analogues of these compounds, glycerols, phospholipids, saturated fatty acids, retinoids and carotenes were inactive even at 0.2 mM. The results of the binding assay and the enzymic assay correlated well except for elaidic acid and linolelaidic acid, the trans isomers of oleic acid and linoleic acid respectively, which were much less active than their cis isomers in the binding assay but were as potent in the enzymic assay. gamma-Linolenic acid had no effect on the activities of two other rat liver microsomal enzymes: NADH:menadione reductase and glucuronosyl transferase. gamma-Linolenic acid, the most potent inhibitor tested, decreased the Vmax. and increased Km values of substrates, NADPH and testosterone, and promoted dissociation of [3H]4-MA from the microsomal reductase. gamma-Linolenic acid, but not the corresponding saturated fatty acid (stearic acid), inhibited the 5 alpha-reductase activity, but not the 17 beta-dehydrogenase activity, of human prostate cancer cells in culture. These results suggest that unsaturated fatty acids may play an important role in regulating androgen action in target cells. PMID:1637346

Mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in colon and liver of Big Blue Rats: role of DNA adducts, strand breaks, DNA repair and oxidative stress

DEFF Research Database (Denmark)

Møller, Peter; Wallin, Håkan; Vogel, Ulla

2002-01-01

, indicating a higher rate of protein oxidation in the liver following IQ administration. In plasma and erythrocytes there were unaltered levels of oxidized protein, malondialdehyde, and antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase, catalase, glutathione reductase) indicating...

Selenium supplementation restores the antioxidative capacity and prevents cell damage in bone marrow stromal cells in vitro

DEFF Research Database (Denmark)

Ebert, Regina; Ulmer, Matthias; Zeck, Sabine

2006-01-01

signaling, cumulative cell damage, senescence, and tumor development. Selenium-dependent (glutathione peroxidases [GPxs] and thioredoxin reductases [TrxRs]) and selenium-independent (superoxide dismutases [SODs] and catalase [CAT]) enzyme systems regulate cellular ROS steady state levels. SODs process...

Mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in colon and liver of Big Blue rats: role of DNA adducts, strand breaks, DNA repair and oxidative stress

DEFF Research Database (Denmark)

Moller, P.; Wallin, H.; Vogel, U.

2002-01-01

, indicating a higher rate of protein oxidation in the liver following IQ administration. In plasma and erythrocytes there were unaltered levels of oxidized protein, malondialdehyde, and antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase, catalase, glutathione reductase) indicating...

Myocardial capillary permeability after regional ischemia and reperfusion in the in vivo canine heart. Effect of superoxide dismutase

DEFF Research Database (Denmark)

Svendsen, J H; Bjerrum, P J; Haunsø, S

1991-01-01

This study assesses the effect of the superoxide anion scavenger superoxide dismutase on myocardial capillary permeability-surface area (PS) products for small hydrophilic molecules after ischemia and reperfusion. Open-chest dogs underwent a 20-minute occlusion of the left anterior descending...... the start of reperfusion. In 13 dogs, no scavenger treatment was given (nonprotected control group), whereas eight dogs were treated systemically with 15,000 units/kg superoxide dismutase during 1 hour, starting 20 minutes before ischemia. In the control group, three dogs developed reperfusion ventricular...

Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

Science.gov (United States)

Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

2006-01-01

Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C2221 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å. PMID:17142919

Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct hydride transfer

Energy Technology Data Exchange (ETDEWEB)

Kaspera, Ruediger; Sahele, Tariku; Lakatos, Kyle [Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195-7610 (United States); Totah, Rheem A., E-mail: rtotah@u.washington.edu [Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195-7610 (United States)

2012-02-17

Highlights: Black-Right-Pointing-Pointer Cytochrome P450BM-3 reduced aldehydes to alcohols efficiently (k{sub cat} {approx} 25 min{sup -1}). Black-Right-Pointing-Pointer Reduction is a direct hydride transfer from R-NADP{sup 2}H to the carbonyl moiety. Black-Right-Pointing-Pointer P450 domain variants enhance reduction through potential allosteric/redox interactions. Black-Right-Pointing-Pointer Novel reaction will have implications for metabolism of xenobiotics. -- Abstract: Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k{sub cat} of {approx}25 min{sup -1} was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. The reduction was caused by a direct hydride transfer from preferentially R-NADP{sup 2}H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP{sup 2}H but not D{sub 2}O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.

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

Science.gov (United States)

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

2010-09-01

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. Copyright © 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

International Nuclear Information System (INIS)

Nascimento, Alessandro S.; Ferrarezi, Thiago; Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A.; Polikarpov, Igor

2006-01-01

Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP + reductase. Ferredoxin-NADP + 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

Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

Energy Technology Data Exchange (ETDEWEB)

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: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil)

2006-07-01

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.

Oxidative stress and superoxide dismutase activity in brain of rats ...

African Journals Online (AJOL)

The present study was envisaged to investigate the possible role of oxidative stress in permethrin neurotoxicity and to evaluate the protective effect of superoxide dismutase (SOD) activity in brain homogenates of Wistar rats. Oxidative stress measured as thiobarbituric acid reacting substances (TBARS) was found to ...

The Drosophila carbonyl reductase sniffer is an efficient 4-oxonon-2-enal (4ONE) reductase.

Science.gov (United States)

Martin, Hans-Jörg; Ziemba, Marta; Kisiela, Michael; Botella, José A; Schneuwly, Stephan; Maser, Edmund

2011-05-30

Studies with the fruit-fly Drosophila melanogaster demonstrated that the enzyme sniffer prevented oxidative stress-induced neurodegeneration. Mutant flies overexpressing sniffer had significantly extended life spans in a 99.5% oxygen atmosphere compared to wild-type flies. However, the molecular mechanism of this protection remained unclear. Sequence analysis and database searches identified sniffer as a member of the short-chain dehydrogenase/reductase superfamily with a 27.4% identity to the human enzyme carbonyl reductase type I (CBR1). As CBR1 catalyzes the reduction of the lipid peroxidation products 4HNE and 4ONE, we tested whether sniffer is able to metabolize these lipid derived aldehydes by carbonyl reduction. To produce recombinant enzyme, the coding sequence of sniffer was amplified from a cDNA-library, cloned into a bacterial expression vector and the His-tagged protein was purified by Ni-chelate chromatography. We found that sniffer catalyzed the NADPH-dependent carbonyl reduction of 4ONE (K(m)=24±2 μM, k(cat)=500±10 min(-1), k(cat)/K(m)=350 s(-1) mM(-1)) but not that of 4HNE. The reaction product of 4ONE reduction by sniffer was mainly 4HNE as shown by HPLC- and GC/MS analysis. Since 4HNE, though still a potent electrophile, is less neurotoxic and protein reactive than 4ONE, one mechanism by which sniffer exerts its neuroprotective effects in Drosophila after oxidative stress may be enzymatic reduction of 4ONE. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

[Comparison of Physico-chemical Aspects between E. coli and Human Dihydrofolate Reductase: an Equilibrium Unfolding Study].

Science.gov (United States)

Thapliyal, Charu; Jain, Neha; Chaudhuri, Pratima

2015-01-01

A protein, differing in origin, may exhibit variable physicochemical behaviour, difference in sequence homology, fold and function. Thus studying structure-function relationship of proteins from altered sources is meaningful in the sense that it may give rise to comparative aspects of their sequence-structure-function relationship. Dihydrofolate reductase is an enzyme involved in cell cycle regulation. It is a significant enzyme as.a target for developing anticancer drugs. Hence, detailed understanding of structure-function relationships of wide variants of the enzyme dihydrofolate reductase would be important for developing an inhibitor or an antagonist against the enzyme involved in the cellular developmental processes. In this communication, we have reported the comparative structure-function relationship between E. coli and human dihydrofolate reductase. The differences in the unfolding behaviour of these two proteins have been investigated to understand various properties of these two proteins like relative' stability differences and variation in conformational changes under identical denaturing conditions. The equilibrium unfolding mechanism of dihydrofolate reductase proteins using guanidine hydrochloride as a denaturant in the presence of various types of osmolytes has been monitored using loss in enzymatic activity, intrinsic tryptophan fluorescence and an extrinsic fluorophore 8-anilino-1-naphthalene-sulfonic acid as probes. It has been observed that osmolytes, such as 1M sucrose, and 30% glycerol, provided enhanced stability to both variants of dihydrofolate reductase. Their level of stabilisation has been observed to be dependent on intrinsic protein stability. It was observed that 100 mM proline does not show any 'significant stabilisation to either of dihydrofolate reductases. In the present study, it has been observed that the human protein is relatively less stable than the E.coli counterpart.

Crystallization of purple nitrous oxide reductase from Pseudomonas stutzeri

International Nuclear Information System (INIS)

Pomowski, Anja; Zumft, Walter G.; Kroneck, Peter M. H.; Einsle, Oliver

2010-01-01

The physiologically active form of nitrous oxide reductase was isolated and crystallized under strict exclusion of dioxygen and diffraction data were collected from crystals belonging to two different space groups. Nitrous oxide reductase (N 2 OR) from Pseudomonas stutzeri catalyzes the final step in denitrification: the two-electron reduction of nitrous oxide to molecular dinitrogen. Crystals of the enzyme were grown under strict exclusion of dioxygen by sitting-drop vapour diffusion using 2R,3R-butanediol as a cryoprotectant. N 2 OR crystallized in either space group P1 or P6 5 . Interestingly, the key determinant for the resulting space group was the crystallization temperature. Crystals belonging to space group P1 contained four 130 kDa dimers in the asymmetric unit, while crystals belonging to space group P6 5 contained a single dimer in the asymmetric unit. Diffraction data were collected to resolutions better than 2 Å

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

Science.gov (United States)

Plancarte, Agustin; Nava, Gabriela

2015-02-01

Thioredoxin glutathione reductases (TGRs) (EC 1.8.1.9) 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

Pyrrolidine dithiocarbamate inhibits superoxide anion-induced pain and inflammation in the paw skin and spinal cord by targeting NF-κB and oxidative stress.

Science.gov (United States)

Pinho-Ribeiro, Felipe A; Fattori, Victor; Zarpelon, Ana C; Borghi, Sergio M; Staurengo-Ferrari, Larissa; Carvalho, Thacyana T; Alves-Filho, Jose C; Cunha, Fernando Q; Cunha, Thiago M; Casagrande, Rubia; Verri, Waldiceu A

2016-06-01

We evaluated the effect of pyrrolidine dithiocarbamate (PDTC) in superoxide anion-induced inflammatory pain. Male Swiss mice were treated with PDTC and stimulated with an intraplantar or intraperitoneal injection of potassium superoxide, a superoxide anion donor. Subcutaneous PDTC treatment attenuated mechanical hyperalgesia, thermal hyperalgesia, paw oedema and leukocyte recruitment (neutrophils and macrophages). Intraplantar injection of superoxide anion activated NF-κB and increased cytokine production (IL-1β, TNF-α and IL-10) and oxidative stress (nitrite and lipid peroxidation levels) at the primary inflammatory foci and in the spinal cord (L4-L6). PDTC treatment inhibited superoxide anion-induced NF-κB activation, cytokine production and oxidative stress in the paw and spinal cord. Furthermore, intrathecal administration of PDTC successfully inhibited superoxide anion-induced mechanical hyperalgesia, thermal hyperalgesia and inflammatory response in peripheral foci (paw). These results suggest that peripheral stimulus with superoxide anion activates the local and spinal cord oxidative- and NF-κB-dependent inflammatory nociceptive mechanisms. PDTC targets these events, therefore, inhibiting superoxide anion-induced inflammatory pain in mice.

Role of Lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase

International Nuclear Information System (INIS)

Huang, Shaoming; Tan, Xuehai; Thompson, P.D.; Freisheim, J.H.; Appleman, J.R.; Blakley, R.L.; Sheridan, R.P.; Venkataraghavan, R.

1990-01-01

Lysine-54 of human dihydrofolate reductase (hDHFR) appears to be involved in the interaction with the 2'-phosphate of NADPH and is conserved as a basic residue in other species. Studies have suggested that in Lactobacillus casei dihydrofolate reductase Arg-43, the homologous residue at this position, plays an important role in the binding of NADPH and in the differentiation of K m values for NADPH and NADH. A Lys-54 to Gln-54 mutant (K54Q) of hDHFR has been constructed by oligodeoxynucleotide-directed mutagenesis in order to study the role of Lys-54 in differentiating K m and k cat values for NADPH and NADH as well as in other functions of hDHFR. The purpose of this paper is to delineate in quantitative terms the magnitude of the effect of the Lys-54 to Gln-54 replacement on the various kinetic parameters of hDHFR. Such quantitative effects cannot be predicted solely on the basis of X-ray structures. The ratio of K m (NADH)/K m (NADPH) decreases from 69 in the wild-type enzyme to 4.7 in the K54Q enzyme, suggesting that Lys-54, among other interactions between protein side-chain residues and the 2'-phosphate, makes a major contribution in terms of binding energy and differentiation of K m values for NADPH and NADH. Agents at concentrations that show activating effects on the wild-type enzyme such as potassium chloride and urea all inactivate the K54Q enzyme. There appear to be no gross conformational differences between wild-type and K54Q enzyme molecules as judged by competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase and from protease susceptibility studies on both wild-type and K54Q mutant enzymes. The pH-rate profiles using NADPH for K54Q and wild-type enzymes show divergences at certain pH values, suggesting the possibility of alteration(s) in the steps of the catalytic pathway for the K54Q enzyme

Real-time investigation of antibiotics-induced oxidative stress and superoxide release in bacteria using an electrochemical biosensor.

Science.gov (United States)

Liu, Xiaobo; Marrakchi, Mouna; Jahne, Michael; Rogers, Shane; Andreescu, Silvana

2016-02-01

The involvement of oxidative stress in the mechanism of antibiotics-meditated cell death is unclear and subject to debate. The kinetic profile and a quantitative relationship between the release of reactive oxygen species (ROS), bacteria and antibiotic type remain elusive. Here we report direct measurements and analytical quantification of the release of superoxide radicals (O2(·-)), a major contributor to ROS, in antibiotics-treated bacterial cultures using a cytochrome c electrochemical biosensor. The specificity of electrochemical measurements was established by the addition of superoxide dismutase (SOD) which decreased the O2(·-) signal. Measurements using a general ROS-specific fluorescence dye and colony forming units (CFU) assays were performed side-by-side to determine the total ROS and establish the relationship between ROS and the degree of lethality. Exposure of Escherichia coli and Listeria monocytogenes cultures to antibiotics increased the release of O2(·-) radicals in a dose-dependent manner, suggesting that the transmembrane generation of ROS may occur as part of the antibiotic action. The study provides a quantitative methodology and fundamental knowledge to further explore the role of oxidative stress in antibiotics-meditated bacterial death and to assess physiological changes associated with the complex metabolic events related to oxidative stress and bacterial resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase.

Science.gov (United States)

Huppke, Peter; Brendel, Cornelia; Korenke, Georg Christoph; Marquardt, Iris; Donsante, Anthony; Yi, Ling; Hicks, Julia D; Steinbach, Peter J; Wilson, Callum; Elpeleg, Orly; Møller, Lisbeth Birk; Christodoulou, John; Kaler, Stephen G; Gärtner, Jutta

2012-08-01

Copper (Cu) is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular Cu metabolism, an elaborate system of chaperones and transporters has evolved, although no human Cu chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a highly conserved arginine residue at position 163, with tryptophan in domain II of CCS, which interacts directly with superoxide dismutase 1 (SOD1). Biochemical analyses of the patient's fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result, this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal Cu homeostasis. CCS-Arg163Trp represents the primary example of a human mutation in a gene coding for a Cu chaperone. © 2012 Wiley Periodicals, Inc.

Profiling of antioxidant superoxide dismutase in saliva of oral submucous fibrosis patients to categorize its diagnosis in varying stages

International Nuclear Information System (INIS)

Sirohi, Y.; Shetty, D.C.; Urs, A.B.; Rai, H.C.

2011-01-01

Background: Oral submucous fibrosis is a pre malignant condition in Indian and South-East Asia. Role of oxidant-antioxidant in causation and progression of cancer and pre cancers is known. Reactive oxygen species are generated in the oral cavity during chewing areca nut, the major etiological agent in oral submucous fibrosis. Objectives: To see the alterations in the salivary superoxide dismutase levels in various clinical and histopathological grades of oral submucous fibrosis. Materials and Methods: Unstimulated saliva was collected from 25 oral submucous fibrosis patients and age and gender matched controls. The saliva was assessed for superoxide dismutase value by spectrophotometric method using assay kit (Bio Vision Catalog number K335-100). The oral submucous fibrosis cases were grouped into clinical stages and histopathological grades and superoxide dismutase values were compared in different clinical stages and histopathological grades. Results: The superoxide dismutase levels were reduced in oral submucous fibrosis as compared to controls. A steady decline in the levels was seen as the clinical stage and histopathological grade of oral submucous fibrosis advanced. Conclusions: Salivary superoxide dismutase levels can be alternatively used as a surrogate marker for the diagnosis of oral submucous fibrosis. Policy message: Oral physicians should advise the pan chewers to regularly check their salivary superoxide dismutase levels so as to ease the early diagnosis of oral submucous fibrosis. (author)

Role of aldose reductase C-106T polymorphism among diabetic Egyptian patients with different microvascular complications

Directory of Open Access Journals (Sweden)

Nermine Hossam Zakaria

2014-04-01

Full Text Available The aldose reductase pathway proves that elevated blood glucose promotes cellular dysfunction. The polyol pathway converts excess intracellular glucose into alcohols via activity of the aldose reductase. This enzyme catalyzes the conversion of glucose to sorbitol which triggers variety of intracellular changes in the tissues. Among diabetes, activity is drastically increased in association with three main consequences inside the cells. The aim of this study was to detect the association of the C-106 T polymorphism of the aldose reductase gene and its frequency among a sample of 150 Egyptian adults with type 2 diabetic patients having diabetic microvascular. The detection of the aldose reductase C-106 T polymorphism gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. The genotype distribution of the C-106 T polymorphism showed that CC genotype was statistically significantly higher among patients with retinopathy compared to nephropathy. Patients with nephropathy had significant association with the TT genotype when compared with diabetic retinopathy patients. Follow up study after the genotype detection among recently diagnosed diabetic patients in order to give a prophylactic aldose reductase inhibitors; studying the microvascular complications and its relation to the genotype polymorphisms. The study may include multiple gene polymorphisms to make the relation between the gene and the occurrence of these complications more evident.

Nitrate reductase and nitrous oxide production by Fusarium oxysporum 11dn1 under aerobic and anaerobic conditions.

Science.gov (United States)

Kurakov, A V; Nosikov, A N; Skrynnikova, E V; L'vov, N P

2000-08-01

The fungus Fusarium oxysporum 11dn1 was found to be able to grow and produce nitrous oxide on nitrate-containing medium in anaerobic conditions. The rate of nitrous oxide formation was three to six orders of magnitude lower than the rates of molecular nitrogen production by common denitrifying bacteria. Acetylene and ammonia did not affect the release of nitrous oxide release. It was shown that under anaerobic conditions fast increase of nitrate reductase activity occurred, caused by the synthesis of enzyme de novo and protein dephosphorylation. Reverse transfer of the mycelium to aerobic conditions led to a decline in nitrate reductase activity and stopped nitrous oxide production. The presence of two nitrate reductases was shown, which differed in molecular mass, location, temperature optima, and activity in nitrate- and ammonium-containing media. Two enzymes represent assimilatory and dissimilatory nitrate reductases, which are active in aerobic and anaerobic conditions, respectively.

Furin proteolytically processes the heparin-binding region of extracellular superoxide dismutase

DEFF Research Database (Denmark)

Bowler, Russell P; Nicks, Mike; Olsen, Dorte Aa

2002-01-01

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that attenuates brain and lung injury from oxidative stress. A polybasic region in the carboxyl terminus distinguishes EC-SOD from other superoxide dismutases and determines EC-SOD's tissue half-life and affinity for heparin....... There are two types of EC-SOD that differ based on the presence or absence of this heparin-binding region. It has recently been shown that proteolytic removal of the heparin-binding region is an intracellular event (Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo, J. D...... of intracellular proteases implicate furin as a processing protease. In vitro experiments using furin and purified EC-SOD suggest that furin proteolytically cleaves EC-SOD in the middle of the polybasic region and then requires an additional carboxypeptidase to remove the remaining lysines and arginines...

Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

International Nuclear Information System (INIS)

Muid, Khandaker Ashfaqul; Karakaya, Hüseyin Çaglar; Koc, Ahmet

2014-01-01

Highlights: • Aging process increases ROS accumulation. • Aging process increases DNA damage levels. • Absence of SOD activity does not cause DNA damage in young cells. • Absence of SOD activity accelerate aging and increase oxidative DNA damages during the aging process. - Abstract: Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1Δ and sod2Δ strains. However, ccs1Δ mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1Δ, sod2Δ, and ccs1Δ mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process

Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

Energy Technology Data Exchange (ETDEWEB)

Muid, Khandaker Ashfaqul; Karakaya, Hüseyin Çaglar; Koc, Ahmet, E-mail: ahmetkoc@iyte.edu.tr

2014-02-07

Highlights: • Aging process increases ROS accumulation. • Aging process increases DNA damage levels. • Absence of SOD activity does not cause DNA damage in young cells. • Absence of SOD activity accelerate aging and increase oxidative DNA damages during the aging process. - Abstract: Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1Δ and sod2Δ strains. However, ccs1Δ mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1Δ, sod2Δ, and ccs1Δ mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process.

Transcriptional modulation of genes encoding nitrate reductase in ...

African Journals Online (AJOL)

The free aluminum (Al) content in soil can reach levels that are toxic to plants, and this has frequently limited increased productivity of cultures. Four genes encoding nitrate reductase (NR) were identified, named ZmNR1–4. With the aim of evaluating NR activity and the transcriptional modulation of the ZmNR1, ZmNR2, ...

Functional and crystallographic characterization of Salmonella typhimurium Cu,Zn superoxide dismutase coded by the sodCI virulence gene

NARCIS (Netherlands)

Pesce, A; Battistoni, A; Stroppolo, ME; Polizio, F; Nardini, M; Kroll, JS; Langford, PR; O'Neill, P; Sette, M; Desideri, A; Bolognesi, M

2000-01-01

The functional and three-dimensional structural features of Cu,Zn superoxide dismutase coded by the Salmonella typhimurium sodCI gene, have been characterized. Measurements of the catalytic rate indicate that this enzyme is the most efficient superoxide dismutase analyzed so far, a feature that may

DNA damage induction of ribonucleotide reductase.

OpenAIRE

Elledge, S J; Davis, R W

1989-01-01

RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidas...

Cell death by SecTRAPs: thioredoxin reductase as a prooxidant killer of cells.

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Karin Anestål

Full Text Available BACKGROUND: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins can be formed from the selenoprotein thioredoxin reductase (TrxR by targeting of its selenocysteine (Sec residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function. PRINCIPAL FINDINGS: Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS and consequently antioxidants could protect against the cell killing by SecTRAPs. CONCLUSIONS: We conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity.

Differences in associations between markers of antioxidative defense and asthma are sex specific

DEFF Research Database (Denmark)

Malling, Tine Halsen; Sigsgaard, Torben; Andersen, Helle R

2010-01-01

on a screening questionnaire, random sampling, or both. Serum selenium concentrations and antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase [GPX], glutathione reductase [GR], and glucose-6-phosphate dehydrogenase [G6PD]) in erythrocytes were measured. Asthma was defined as either...

Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra

International Nuclear Information System (INIS)

Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2009-01-01

The NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra was expressed, purified, and crystallized and X-ray diffraction data of this crystal were collected to 2.2 Å resolution. (R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P4 1 2 1 2, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%

Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

DEFF Research Database (Denmark)

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

2010-01-01

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...

Crystal Structure of Cu/Zn Superoxide Dismutase from Taenia Solium Reveals Metal-mediated Self-assembly

Energy Technology Data Exchange (ETDEWEB)

A Hernandez-Santoyo; A Landa; E Gonzalez-Mondragon; M Pedraza-Escalona; R Parra-Unda; A Rodriguez-Romero

2011-12-31

Taenia solium is the cestode responsible for porcine and human cysticercosis. The ability of this parasite to establish itself in the host is related to its evasion of the immune response and its antioxidant defence system. The latter includes enzymes such as cytosolic Cu/Zn superoxide dismutase. In this article, we describe the crystal structure of a recombinant T. solium Cu/Zn superoxide dismutase, representing the first structure of a protein from this organism. This enzyme shows a different charge distribution at the entrance of the active channel when compared with human Cu/Zn superoxide dismutase, giving it interesting properties that may allow the design of specific inhibitors against this cestode. The overall topology is similar to other superoxide dismutase structures; however, there are several His and Glu residues on the surface of the protein that coordinate metal ions both intra- and intermolecularly. Interestingly, one of these ions, located on the {beta}2 strand, establishes a metal-mediated intermolecular {beta}-{beta} interaction, including a symmetry-related molecule. The factors responsible for the abnormal protein-protein interactions that lead to oligomerization are still unknown; however, high metal levels have been implicated in these phenomena, but exactly how they are involved remains unclear. The present results suggest that this structure could be useful as a model to explain an alternative mechanism of protein aggregation commonly observed in insoluble fibrillar deposits.

Relationship between nitrate reductase and nitrate uptake in phytoplankton in the Peru upwelling region

International Nuclear Information System (INIS)

Blasco, D.; MacIsaac, J.J.; Packard, T.T.; Dugdale, R.C.

1984-01-01

Nitrate reductase (NR) activity and 15 NO 3 - uptake in phytoplankton were compared under different environmental conditions on two cruises in the upwelling region off Peru. The NR activity and NO 3 - uptake rates responded differently to light and nutrients and the differences led to variations in the uptake: reductase ratio. Analysis of these variations suggests that the re-equilibration time of the two processes in response to environmental perturbation is an important source of variability. The nitrate uptake system responds faster than the nitrate reductase system. Considering these differences in response time the basic differences in the two processes, and the differences in their measurement, the authors conclude that the Nr activity measures the current nitrate-reducing potential, which reflects NO 3 - assimilation before the sampling time, while 15 NO 3 - uptake measures NO 3 - assimilation in the 6-h period following sampling

Superoxide radical (O2-) reactivity with respect to glutathione

International Nuclear Information System (INIS)

Sekaki, A.; Gardes-Albert, M.; Ferradini, C.

1984-01-01

Influence of superoxide radicals formed during gamma irradiation of glutathione in aerated aqueous solutions is examined. Solutions are buffered at pH7 and contain sodium formate for capture of H and OH radicals which are transformed in COO - radicals and then O 2 - radicals. G value of glutathione disparition vs glutathione concentration are given with and without enzyme or catalase. Reaction mechanism are interpreted [fr

Cloning and characterization of a nitrite reductase gene related to ...

African Journals Online (AJOL)

STORAGESEVER

2010-03-01

Mar 1, 2010 ... Alexander et al., 2005) and heme-type nitrite reductase gene (Smith and ... owing to a genotype-dependent response (Zhang et al.,. 1991; Sakhanokho et al., ..... Improvement of cell culture conditions for rice. Jpn. Agric. Res.

Effect of ammonium and nitrate on ferric chelate reductase and nitrate reductase in Vaccinium species.

Science.gov (United States)

Poonnachit, U; Darnell, R

2004-04-01

Most Vaccinium species have strict soil requirements for optimal growth, requiring low pH, high iron availability and nitrogen primarily in the ammonium form. These soils are limited and are often located near wetlands. Vaccinium arboreum is a wild species adapted to a wide range of soils, including high pH, low iron, and nitrate-containing soils. This broader soil adaptation in V. arboreum may be related to increased efficiency of iron or nitrate uptake compared with the cultivated Vaccinium species. Nitrate, ammonium and iron uptake, and nitrate reductase (NR) and ferric chelate reductase (FCR) activities were compared in two Vaccinium species grown hydroponically in either nitrate or ammonia, with or without iron. The species studied were the wild V. arboreum and the cultivated V. corymbosum interspecific hybrid, which exhibits the strict soil requirements of most Vaccinium species. Ammonium uptake was significantly greater than nitrate uptake in both species, while nitrate uptake was greater in the wild species, V. arboreum, compared with the cultivated species, V. corymbosum. The increased nitrate uptake in V. arboreum was correlated with increased root NR activity compared with V. corymbosum. The lower nitrate uptake in V. corymbosum was reflected in decreased plant dry weight in this species compared with V. arboreum. Root FCR activity increased significantly in V. corymbosum grown under iron-deficient conditions, compared with the same species grown under iron-sufficient conditions or with V. arboreum grown under either iron condition. V. arboreum appears to be more efficient in acquiring nitrate compared with V. corymbosum, possibly due to increased NR activity and this may partially explain the wider soil adaptation of V. arboreum.

Superoxide dismutase from Trichuris ovis--inhibition by benzimidazoles and pyrimidine derivatives.

Science.gov (United States)

Sanchez-Moreno, M; Garcia-Rejon, L; Salas, I; Osuna, A; Monteoliva, M

1992-01-01

Three superoxide dismutase isoenzymes of different cellular location were detected in an homogenate of Trichuris ovis. Each of these molecular forms was purified by differential centrifugation and precipitation with ammonium sulphate, followed by chromatography on DEAE-cellulose and Sephadex G-75 columns. The activity levels of the two molecular forms detected in the mitochondrial (one cyanide sensitive Cu-Zn-SOD and the other cyanide insensitive Mn-SOD) were higher than that of the superoxide dismutase detected in the cytoplasmic fraction (cyanide sensitive Cu-Zn-SOD). All molecular forms present evident differences to the SODs contained in the host liver. Molecular mass and some of the physical and chemical properties of the enzyme was determined for all three molecular forms. An inhibitory effect on the SOD of the parasite an the host was detected with a series of compounds, some of which markedly inhibited parasite enzyme but not host enzyme.

Superoxide dismutase levels and peak expiratory flow in asthmatic children

Directory of Open Access Journals (Sweden)

Arie Kurniasih

2016-11-01

Full Text Available Background Asthma is a chronic inflammatory process which involve variety of cells such as inflammatory mediators, reactive oxygen species (ROS, and cytokines. The inflammatory process would be exacerbated in the presence of oxidative stress. Superoxide dismutase (SOD is the first important enzyme to protect the respiratory tract against oxidative stress. The decreased of SOD has a correlation with increased of airway obstruction and bronchospasm. Objective To assess for a correlation between superoxide dismutase (SOD levels and peak expiratory flow, as well as to determine the impact of SOD levels for predicting asthma attacks. Methods We conducted a prospective cohort study at Dr. Sardjito Hospital, Yogyakarta, between February and April 2011 involving asthmatic children aged 5-18 years. Subjects’ serum SOD levels and peak expiratory flow were measured at the same time point. We then performed a prospective study following up on the same subjects to find out if they had a recurrent asthma attack within one month of the tests. We also reassessed their peak expiratory flow one month after blood specimens were obtained. Results Thirty-nine patients were enrolled in this study. There was no significant correlation between SOD level and peak expiratory flow [r=0.289; 95%CI -0.025 to 0.47; P=0.074]. However, older age was significantly associated with higher peak expiratory flow (=0.5; 95%CI 3.10 to 11.57; P=0.01. Lower levels of SOD increased the risk of asthma attacks in a month following the initial measurements (RR=5.5; 95%CI 1.6 to 18.9; P=0.009. Conclusion Superoxide dismutase (SOD level is not significantly associated with peak expiratory flow. However, we find a relationship between older age and higher peak expiratory flow and a relationship between lower SOD levels and risk of asthma attacks within one month following the tests.

The In Vitro Influence of a Genetic Superoxide-Hydrogen Peroxide Imbalance on Immunosenescence.

Science.gov (United States)

Barbisan, Fernanda; Azzolin, Verônica Farina; Ribeiro, Euler Esteves; Duarte, Marta Maria Medeiros Frescura; da Cruz, Ivana Beatrice Mânica

2017-08-01

As superoxide is a key molecule of inflammatory activation, superoxide-hydrogen peroxide (S-HP) imbalance genetically caused could alter immunosenescence patterns. To test this hypothesis, we collected and cultured peripheral blood mononuclear cells (PBMCs) carrier's different genotypes of a genetic polymorphism located in the superoxide dismutase manganese-dependent gene (Val16Ala-SOD2). We used an in vitro genetic model based on previous studies, which suggested an association between homozygous genotypes (AA and VV) and alterations in oxidative-inflammatory mediators. PBMCs collected from young healthy volunteers were cultured in the presence of phytohemagglutinin, as well as the following cell culture passages obtained from the 72-hour initial culture. Each follow passage started with the same cell concentration (1 × 10 5 cells). The general immunosenescence pattern was observed independent of SOD2 genotypes: cellular proliferation until the 15th passage, when cellular arrestment occurred in the G0/G1 phase. From the 10th passage, a higher proliferative state was observed, indicating inflammatory hyperactivation, with an increase in the levels of inflammatory cytokines (IL-1, IL-6, and TNFα), nitric oxide, superoxide, lipoperoxidation, protein carbonylation, reactive oxygen species, and DNA damage. The S-HP imbalance affected the intensity of some immunosenescence parameters. AA cells, which present basal high HP levels, were associated with higher DNA damage and lipoperoxidation levels, whereas VV, which present basal high S levels, was associated with higher proinflammatory cytokine levels. In summary, the results suggested that a basal S-HP imbalance could affect the intensity of some immunosenescence markers, and this influence could explain the potential association between an imbalance of genotypes (AA and VV) and the risk of developing some chronic diseases.

Effect of Food Sources of Natural Chemo preventive Agents on ...

African Journals Online (AJOL)

Objective: The work attempted to evaluate the potential of natural products containing cancer chemopreventive agents in increasing the level of some endogenous antioxidant enzymes such as Glutathione STransferase (GST), Glutathione reductase (GR), catalase, superoxide dismutase(SOD-1,2) in brain and kidney ...

The uptake and accumulation of phosphorous and nitrates and the activity of nitrate reductase in cucumber seedlings treated with PbCl2 or CdCl2

Directory of Open Access Journals (Sweden)

Marek Burzyński

2014-01-01

Full Text Available Treatment of 4-day-old cucumber (Cucumis sativus L. seedlings with PbCl2 or CdCl2 caused a significant increase in the accumulation of heavy metals by the plants, especially in the roots. The accumulated Pb initially enhanced the uptake of phosphorous after the plants had been transferred to a nutrient medium (6, 24 hrs, but after only 48 Ins the uptake had dropped to below control level. The plants treated with Cd exhibited a constant decreased phosphorous uptake level. The accumulated lead and cadmium also inhibited nitrate uptake and the activity of nitrate reductase. It is suggested that the reason for the decreased nitrate reductase activity lay rather in the lower nitrate uptake than in a direct effect of the heavy metals on the enzyme.

Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis

International Nuclear Information System (INIS)

Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting

2012-01-01

Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K 3 ) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ∼ 12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. -- Highlights: ► Menadione causes mitochondrial superoxide accumulation and injury. ► Menadione-induced cell death is caspase-independent, due to rapid depletion of ATP

Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis

Energy Technology Data Exchange (ETDEWEB)

Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting, E-mail: BTZhu@kumc.edu

2012-07-15

Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K{sub 3}) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ∼ 12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. -- Highlights: ► Menadione causes mitochondrial superoxide accumulation and injury. ► Menadione-induced cell death is caspase-independent, due to rapid depletion of

Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase

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Margit Winkler

2013-08-01

Full Text Available Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S-selectivity and together with a highly (R-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.

Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase.

Science.gov (United States)

Napora-Wijata, Kamila; Strohmeier, Gernot A; Sonavane, Manoj N; Avi, Manuela; Robins, Karen; Winkler, Margit

2013-08-12

Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.

The NADPH thioredoxin reductase C functions as an electron donor to 2-Cys peroxiredoxin in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

International Nuclear Information System (INIS)

Sueoka, Keigo; Yamazaki, Teruaki; Hiyama, Tetsuo; Nakamoto, Hitoshi

2009-01-01

An NADPH thioredoxin reductase C was co-purified with a 2-Cys peroxiredoxin by the combination of anion exchange chromatography and electroelution from gel slices after native PAGE from a thermophilic cyanobacterium Thermosynechococcus elongatus as an NAD(P)H oxidase complex induced by oxidative stress. The result provided a strong evidence that the NADPH thioredoxin reductase C interacts with the 2-Cys peroxiredoxin in vivo. An in vitro reconstitution assay with purified recombinant proteins revealed that both proteins were essential for an NADPH-dependent reduction of H 2 O 2 . These results suggest that the reductase transfers the reducing power from NADPH to the peroxiredoxin, which reduces peroxides in the cyanobacterium under oxidative stress. In contrast with other NADPH thioredoxin reductases, the NADPH thioredoxin reductase C contains a thioredoxin-like domain in addition to an NADPH thioredoxin reductase domain in the same polypeptide. Each domain contains a conserved CXYC motif. A point mutation at the CXYC motif in the NADPH thioredoxin reductase domain resulted in loss of the NADPH oxidation activity, while a mutation at the CXYC motif in the thioredoxin-like domain did not affect the electron transfer, indicating that this motif is not essential in the electron transport from NADPH to the 2-Cys peroxiredoxin.

Alpha 1-blockers vs 5 alpha-reductase inhibitors in benign prostatic hyperplasia. A comparative review

DEFF Research Database (Denmark)

Andersen, J T

1995-01-01

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...... and improvement in objective parameters for bladder outflow obstruction. Approximately 30 to 50% of patients will respond to treatment with 5 alpha-reductase inhibitors. The definitive role of pharmacological treatment in symptomatic BPH remains to be established, although it seems that patients unfit...

High glucose impairs superoxide production from isolated blood neutrophils

DEFF Research Database (Denmark)

Perner, A; Nielsen, S E; Rask-Madsen, J

2003-01-01

Superoxide (O(2)(-)), a key antimicrobial agent in phagocytes, is produced by the activity of NADPH oxidase. High glucose concentrations may, however, impair the production of O(2)(-) through inhibition of glucose-6-phosphate dehydrogenase (G6PD), which catalyzes the formation of NADPH. This study...... measured the acute effects of high glucose or the G6PD inhibitor dehydroepiandrosterone (DHEA) on the production of O(2)(-) from isolated human neutrophils....

Experimental study of antiradiation properties of recombinant superoxide dismutase

International Nuclear Information System (INIS)

Derimedvyid', L.V.; Simonova, L.Yi.; Gertman, V.Z.

2003-01-01

The study involved 250 mongrel white male mice weighing 18-22 g. It was shown that the superoxide dismutase had a marked radioprotective effect. The experiments on animals exposed to ionizing radiation at a absolute and mean lethal doses demonstrate considerable increase of survival rate, mean life span of the dead animals, shifts in the peaks of lethality to later terms, reduction in the percentage of animals with intestinal syndrome,

Reconstitution of FMN-free NADPH-cytochrome P-450 reductase with a phosphorothioate analog of FMN: 31P NMR studies of the reconstituted protein

International Nuclear Information System (INIS)

Krum, D.P.; Otvos, J.D.; Calhoun, J.P.; Miziorko, H.M.; Masters, B.S.S.

1987-01-01

A phosphorothioate analog of FMN (FMNS) has been synthesized and shown to be completely competent in reconstituting the FMN-free form of NADPH-cytochrome P-450 reductase as evidenced by flavin determinations and cytochrome c reductase activity assays. The FMNS-reconstituted FMN-free reductase gives rise to an air-stable semiquinone, and the fluorescence of FMNS is quenched upon addition of FMN-free reductase. 31 P NMR spectra of the FMN-free reductase reveal only two resonances (-7.3 and -11.3 ppm), which are attributable to FAD. This result confirms the assignments of Otvos et al, and demonstrates unequivocally that there are no phosphate residues other than those of FMN and FAD attached to the steapsin-solubilized reductase. The addition of FMN to the FMN-free reductase resulted in the appearance of one additional resonance at 3.9 ppm. Addition of FMNS to the FMN-free reductase caused no change, surprisingly, in the 31 P NMR spectrum until Mn(II) was added, after which a peak centered at ∼ 45 ppm was observed. This unexpected result may be explained if the T 1 for the phosphate of FMNS is significantly longer than that of FMN, and suggests that the sulfur atom of FMNS may perturb the interaction of the phosphate with its protein environment. These results demonstrate the utility of phosphorothioate analogs as mechanistic probes for proteins containing nucleotide cofactors

Directed Evolution of Carbonyl Reductase from Rhodosporidium toruloides and Its Application in Stereoselective Synthesis of tert-Butyl (3R,5S)-6-Chloro-3,5-dihydroxyhexanoate.

Science.gov (United States)

Liu, Zhi-Qiang; Wu, Lin; Zhang, Xiao-Jian; Xue, Ya-Ping; Zheng, Yu-Guo

2017-05-10

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key intermediate of atorvastatin and rosuvastatin synthesis. Carbonyl reductase RtSCR9 from Rhodosporidium toruloides exhibited excellent activity toward tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH). For the activity of RtSCR9 to be improved, random mutagenesis and site-saturation mutagenesis were performed. Three positive mutants were obtained (mut-Gln95Asp, mut-Ile144Lys, and mut-Phe156Gln). These mutants exhibited 1.94-, 3.03-, and 1.61-fold and 1.93-, 3.15-, and 1.97-fold improvement in the specific activity and k cat /K m , respectively. Asymmetric reduction of (S)-CHOH by mut-Ile144Lys coupled with glucose dehydrogenase was conducted. The yield and enantiomeric excess of (3R,5S)-CDHH reached 98 and 99%, respectively, after 8 h bioconversion in a single batch reaction with 1 M (S)-CHOH, and the space-time yield reached 542.83 mmol L -1 h -1 g -1 wet cell weight. This study presents a new carbonyl reductase for efficient synthesis of (3R,5S)-CDHH.

Cyclophosphamide as a potent inhibitor of tumor thioredoxin reductase in vivo

International Nuclear Information System (INIS)

Wang Xufang; Zhang Jinsong; Xu Tongwen

2007-01-01

Cyclophosphamide (CTX) is in the nitrogen mustard group of alkylating antineoplastic chemotherapeutic agents. It is one of the most frequently used antitumor agents for the treatment of a broad spectrum of human cancers. Thioredoxin reductase (TrxR) catalyze the NADPH-dependent reduction of thioredoxin and play an important role in multiple cellular events related to carcinogenesis including cell proliferation, apoptosis, and cell signaling. This enzyme represents a promising target for the development of cytostatic agents. The purpose of this study is to determine whether CTX could target TrxR in vivo. Lewis lung carcinoma and solid H22 hepatoma treated with 50-250 mg/kg CTX for 3 h lost TrxR activity in a dose-dependent fashion. Over 75% and 95% of TrxR activity was lost at the dose of 250 mg/kg. There was, however, a recovery of TrxR activity such that it attained normal levels by 120 h after a dose of 250 mg/kg. In addition, we found that CTX caused a preferential TrxR inhibition over other antioxidant enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase. We also used ascites H22 cells to investigate cancer cells response after TrxR was inhibited by CTX in vivo since CTX is needed to be activated by liver cytochrome P450 enzymes. The time course and dose-dependent changes of cellular TrxR activity were similar with those in tumor tissue. CTX caused a dose-dependent cellular proliferation inhibition which was positively correlated with TrxR inhibition at 3 h. Furthermore, when 3 h CTX-treated cells with various TrxR backgrounds, harvested from ascites-bearing mice, were implanted into mice, the proliferations of these cells were again proportionally dependent on TrxR activity. The TrxR inhibition could thereby be considered as a crucial mechanism contributing to anticancer effect seen upon clinical use of CTX

Evaluation of the conserve flavin reductase gene from three ...

African Journals Online (AJOL)

STORAGESEVER

2009-12-15

Dec 15, 2009 ... means of PCR technique. The nucleic acid sequences of the PCR primers were designed using conserved nucleic acid sequences of the flavin reductase enzyme from. Rhodococcus sp. strain IGTS8. The oligonucleotide primers were as follows: 5'-GAA TTC ATG TCT GAC. AAG CCG AAT GCC-3' (forward) ...

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

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Hahn-Hägerdal Bärbel

2008-10-01

Full Text Available Abstract Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells-1 h-1 compared with 0.01 g (g cells-1 h-1

Exogenous superoxide dismutase may lose its antidotal ability on rice leaves

Science.gov (United States)

Leaf diffusates of the resistant rice cultivars suppressed spore germination of blast fungus (Magnaporthe grisea). Bovine Cu-Zn superoxide dismutase (SOD) added to the diffusate abolished its toxicity. However, the enzyme added to the inoculum did not affect the toxicity of the diffusate. Even the s...

Radioprotective effects of bacterial superoxide dismutase on mice

International Nuclear Information System (INIS)

Hu Tianxi

1992-01-01

The radioprotective effects of bacterial superoxide dismutase (b-SOD) on the mice irradiated by 8 Gy γ-ray were investigated. The results showed that when b-SOD was injected before and after irradiation, the survival fraction of mice is increased 50% and 30% respectively. The former treatment could increase the DNA synthesis of the myeloid cells and spleen's lymphocytes, decrease the LPO of tissue homogenates and the hemolysis of erythrocytes significantly. The mechanism that b-SOD can drop the radiation injury of the mice was discussed

Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis☆

Science.gov (United States)

Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting

2013-01-01

Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K3) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ~12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. PMID:22575170

Superoxide dismutase from Trichuris ovis, inhibiton by benzimidazoles and pyrimidine derivatives

Directory of Open Access Journals (Sweden)

M. Sanchez-Moreno

1992-01-01

Full Text Available Three superoxide dismutase isoenzymes of different cellular location were detected in an homogenate of Thrichuris ovis. Each of these molecular forms was purified by differential centrifugation and precipitation with ammonium sulphate, followed by chromatography on DEAE-cellulose and Sephadex G-75 columns. The activity levels of the two molecular forms detected in the mitochondrial (one cyanide sensitive Cu-Zn-SOD and the other cyanide intensitive Mn-Sod were higher than that of the superoxide dismutase detected in the cytoplasmic fraction (cyanid sensitive Cu-Zn-SOD. All the mollecular forms present evident differences to the SODs contained in the host liver. Molecular mass and some of the physical and chemical aproperties of the enzyme was determined for all three molecular forms. An inhibitory effect on the SOD of the parasite an the host was detected with a series of compounds, some of wich markedly inhibited parasite ensyme but not host enzyme.

NMR evaluation of total statin content and HMG-CoA reductase inhibition in red yeast rice (Monascus spp. food supplements

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Lachenmeier Dirk W

2012-03-01

Full Text Available Abstract Background Red yeast rice (i.e., rice fermented with Monascus spp., as a food supplement, is claimed to be blood cholesterol-lowering. The red yeast rice constituent monacolin K, also known as lovastatin, is an inhibitor of the hydroxymethylglutaryl-CoA (HMG-CoA reductase. This article aims to develop a sensitive nuclear magnetic resonance (NMR method to determine the total statin content of red yeast rice products. Methods The total statin content was determined by a 400 MHz 1H NMR spectroscopic method, based on the integration of the multiplet at δ 5.37-5.32 ppm of a hydrogen at the hexahydronaphthalene moiety in comparison to an external calibration with lovastatin. The activity of HMG-CoA reductase was measured by a commercial spectrophotometric assay kit. Results The NMR detection limit for total statins was 6 mg/L (equivalent to 0.3 mg/capsule, if two capsules are dissolved in 50 mL ethanol. The relative standard deviations were consistently lower than 11%. The total statin concentrations of five red yeast rice supplements were between 1.5 and 25.2 mg per specified daily dose. A dose-dependent inhibition of the HMG-CoA reductase enzyme activity by the red yeast rice products was demonstrated. Conclusion A simple and direct NMR assay was developed to determine the total statin content in red yeast rice. The assay can be applied for the determination of statin content for the regulatory control of red yeast rice products.

Aortic superoxide production at the early hyperglycemic stage in a rat type 2 diabetes model and the effects of pravastatin.

Science.gov (United States)

Kikuchi, Chigusa; Kajikuri, Junko; Hori, Eisei; Nagami, Chie; Matsunaga, Tamihide; Kimura, Kazunori; Itoh, Takeo

2014-01-01

Endothelium-derived superoxide induces vascular dysfunctions. The aim of this study was to examine the activity of protein kinase C (PKC) isoforms and endothelial nitric oxide synthase (eNOS), which leads to vascular superoxide production in type 2 diabetes, in addition to the effects of pravastatin. We studied these mechanisms in Otsuka Long-Evans Tokushima Fatty (OLETF) rats (type 2 diabetes model) at the early hyperglycemic stage (vs. non-diabetic Long-Evans Tokushima Otsuka [LETO] rats). Superoxide production and catalase activity were measured in aortas, as were the protein expressions of PKCδ and phospho-Ser(1177) eNOS. Superoxide production was increased in OLETF rats, and this increase was inhibited by the selective conventional PKC (cPKC) inhibitor Gö6976 and by the non-selective cPKC and novel PKC inhibitor GF109203X. Phospho-Ser(1177) eNOS was significantly increased in OLETF rats, whereas the protein expressions of PKCδ and phosopho-Thr(505) PKCδ and catalase activity were all greatly reduced. Pravastatin administration to OLETF rats in vivo had normalizing effects on all of these variables. The increment in superoxide production seen in OLETF rats (but not the production in pravastatin-treated OLETF rats) was abolished by high concentration of N(ω)-nitro-L-arginine methyl ester (electron transport inhibitor of eNOS), by sepiapterin (precursor of tetrahydrobiopterin), and by LY294002 (phosphatidylinositol 3-kinase [PI3-kinase] inhibitor). In OLETF rats at the early hyperglycemic stage, aortic superoxide production is increased owing to activation of uncoupled eNOS through phosphorylation by PI3-kinase/Akt. This may be related to the observed reduction in PKCδ/catalase activities. Pravastatin inhibited endothelial superoxide production via normalization of PKCδ/catalase activities.

Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages.

Science.gov (United States)

Kim, Jun Sub; Kim, Jae Gyu; Jeon, Chan Young; Won, Ha Young; Moon, Mi Young; Seo, Ji Yeon; Kim, Jong Il; Kim, Jaebong; Lee, Jae Yong; Choi, Soo Young; Park, Jinseu; Yoon Park, Jung Han; Ha, Kwon Soo; Kim, Pyeung Hyeun; Park, Jae Bong

2005-12-31

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47(PHOX). Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47(PHOX) may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.

Structural insights into the neuroprotective-acting carbonyl reductase Sniffer of Drosophila melanogaster.

Science.gov (United States)

Sgraja, Tanja; Ulschmid, Julia; Becker, Katja; Schneuwly, Stephan; Klebe, Gerhard; Reuter, Klaus; Heine, Andreas

2004-10-01

In vivo studies with the fruit-fly Drosophila melanogaster have shown that the Sniffer protein prevents age-dependent and oxidative stress-induced neurodegenerative processes. Sniffer is a NADPH-dependent carbonyl reductase belonging to the enzyme family of short-chain dehydrogenases/reductases (SDRs). The crystal structure of the homodimeric Sniffer protein from Drosophila melanogaster in complex with NADP+ has been determined by multiple-wavelength anomalous dispersion and refined to a resolution of 1.75 A. The observed fold represents a typical dinucleotide-binding domain as detected for other SDRs. With respect to the cofactor-binding site and the region referred to as substrate-binding loop, the Sniffer protein shows a striking similarity to the porcine carbonyl reductase (PTCR). This loop, in both Sniffer and PTCR, is substantially shortened compared to other SDRs. In most enzymes of the SDR family this loop adopts a well-defined conformation only after substrate binding and remains disordered in the absence of any bound ligands or even if only the dinucleotide cofactor is bound. In the structure of the Sniffer protein, however, the conformation of this loop is well defined, although no substrate is present. Molecular modeling studies provide an idea of how binding of substrate molecules to Sniffer could possibly occur.

Formation and disappearance of superoxide radicals in aqueous solutions

International Nuclear Information System (INIS)

Allen, A.O.; Bielski, B.H.J.

1980-01-01

A literature review of superoxide radicals in aqueous solutions is presented covering the following: history; methods of formation of aqueous HO 2 /HO 2 - by radiolysis and photolysis, electrolysis, mixing nonaqueous solutions into water, chemical reactions, enzymatic generation of O 2 - , and photosensitization; and properties of HO 2 /O 2 - in aqueous solution, which cover spontaneous dismutation rates, pk and absorption spectra, catalyzed dismutation, thermodynamics and the so-called Haber-Weiss Reaction

Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.

Science.gov (United States)

Subburaj, Saminathan; Chung, Sung Jin; Lee, Choongil; Ryu, Seuk-Min; Kim, Duk Hyoung; Kim, Jin-Soo; Bae, Sangsu; Lee, Geung-Joo

2016-07-01

Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

Mitochondria-derived superoxide and voltage-gated sodium channels in baroreceptor neurons from chronic heart-failure rats.

Science.gov (United States)

Tu, Huiyin; Liu, Jinxu; Zhu, Zhen; Zhang, Libin; Pipinos, Iraklis I; Li, Yu-Long

2012-01-01

Our previous study has shown that chronic heart failure (CHF) reduces expression and activation of voltage-gated sodium (Na(v)) channels in baroreceptor neurons, which are involved in the blunted baroreceptor neuron excitability and contribute to the impairment of baroreflex in the CHF state. The present study examined the role of mitochondria-derived superoxide in the reduced Na(v) channel function in coronary artery ligation-induced CHF rats. CHF decreased the protein expression and activity of mitochondrial complex enzymes and manganese SOD (MnSOD) and elevated the mitochondria-derived superoxide level in the nodose neurons compared with those in sham nodose neurons. Adenoviral MnSOD (Ad.MnSOD) gene transfection (50 multiplicity of infection) into the nodose neurons normalized the MnSOD expression and reduced the elevation of mitochondrial superoxide in the nodose neurons from CHF rats. Ad.MnSOD also partially reversed the reduced protein expression and current density of the Na(v) channels and the suppressed cell excitability (the number of action potential and the current threshold for inducing action potential) in aortic baroreceptor neurons from CHF rats. Data from the present study indicate that mitochondrial dysfunction, including decreased protein expression and activity of mitochondrial complex enzymes and MnSOD and elevated mitochondria-derived superoxide, contributes to the reduced Na(v) channel activation and cell excitability in the aortic baroreceptor neurons in CHF rats.

Disinfection of titanium dioxide nanotubes using super-oxidized water decrease bacterial viability without disrupting osteoblast behavior

Energy Technology Data Exchange (ETDEWEB)

Beltrán-Partida, Ernesto [Department of Biomaterials, Dental Materials and Tissue Engineering, Faculty of Dentistry Mexicali, Autonomous University of Baja California, Av. Zotoluca and Chinampas St., 21040 Mexicali, Baja California (Mexico); Department of Corrosion and Materials, Engineering Institute, Autonomous University of Baja California, Blvd. Benito Juarez and Normal St., 21280 Mexicali, Baja California (Mexico); Valdez-Salas, Benjamín, E-mail: benval@uabc.edu.mx [Department of Corrosion and Materials, Engineering Institute, Autonomous University of Baja California, Blvd. Benito Juarez and Normal St., 21280 Mexicali, Baja California (Mexico); Escamilla, Alan; Curiel, Mario [Department of Corrosion and Materials, Engineering Institute, Autonomous University of Baja California, Blvd. Benito Juarez and Normal St., 21280 Mexicali, Baja California (Mexico); Valdez-Salas, Ernesto [Ixchel Medical Centre, Av. Bravo y Obregón, 21000 Mexicali, Baja California (Mexico); Nedev, Nicola [Department of Corrosion and Materials, Engineering Institute, Autonomous University of Baja California, Blvd. Benito Juarez and Normal St., 21280 Mexicali, Baja California (Mexico); Bastidas, Jose M. [National Centre for Metallurgical Research, CSIC, Av. Gregorio del Amo 8, 28040 Madrid (Spain)

2016-03-01

Amorphous titanium dioxide (TiO{sub 2}) nanotubes (NTs) on Ti6Al4V alloy were synthesized by anodization using a commercially available super-oxidized water (SOW). The NT surfaces were sterilized by ultraviolet (UV) irradiation and disinfected using SOW. The adhesion and cellular morphology of pig periosteal osteoblast (PPO) cells and the behavior of Staphylococcus aureus (S. aureus) cultured on the sterilized and disinfected surfaces were investigated. A non-anodized Ti6Al4V disc sterilized by UV irradiation (without SOW) was used as control. The results of this study reveal that the adhesion, morphology and filopodia development of PPO cells in NTs are dramatically improved, suggesting that SOW cleaning may not disrupt the benefits obtained by NTs. Significantly decreased bacterial viability in NTs after cleaning with SOW and comparing with non-cleaned NTs was seen. The results suggest that UV and SOW could be a recommendable method for implant sterilization and disinfection without altering osteoblast behavior while decreasing bacterial viability. - Highlights: • The effect of super-oxidized water cleaning was studied on Ti6Al4V nanotubes. • Super oxidized-water cleaning caused a decline in S. aureus viability. • Osteoblast behavior was not disrupted after super-oxidized water disinfection. • Super-oxidized water is suggested as a cleaning protocol for TiO{sub 2} nanotubes.

Disinfection of titanium dioxide nanotubes using super-oxidized water decrease bacterial viability without disrupting osteoblast behavior

International Nuclear Information System (INIS)

Beltrán-Partida, Ernesto; Valdez-Salas, Benjamín; Escamilla, Alan; Curiel, Mario; Valdez-Salas, Ernesto; Nedev, Nicola; Bastidas, Jose M.

2016-01-01

Amorphous titanium dioxide (TiO_2) nanotubes (NTs) on Ti6Al4V alloy were synthesized by anodization using a commercially available super-oxidized water (SOW). The NT surfaces were sterilized by ultraviolet (UV) irradiation and disinfected using SOW. The adhesion and cellular morphology of pig periosteal osteoblast (PPO) cells and the behavior of Staphylococcus aureus (S. aureus) cultured on the sterilized and disinfected surfaces were investigated. A non-anodized Ti6Al4V disc sterilized by UV irradiation (without SOW) was used as control. The results of this study reveal that the adhesion, morphology and filopodia development of PPO cells in NTs are dramatically improved, suggesting that SOW cleaning may not disrupt the benefits obtained by NTs. Significantly decreased bacterial viability in NTs after cleaning with SOW and comparing with non-cleaned NTs was seen. The results suggest that UV and SOW could be a recommendable method for implant sterilization and disinfection without altering osteoblast behavior while decreasing bacterial viability. - Highlights: • The effect of super-oxidized water cleaning was studied on Ti6Al4V nanotubes. • Super oxidized-water cleaning caused a decline in S. aureus viability. • Osteoblast behavior was not disrupted after super-oxidized water disinfection. • Super-oxidized water is suggested as a cleaning protocol for TiO_2 nanotubes.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol biosynthesis by oxylanosterols

Energy Technology Data Exchange (ETDEWEB)

Panini, S.R.; Sexton, R.C.; Gupta, A.K.; Parish, E.J.; Chitrakorn, S.; Rudney, H.

1986-11-01

Treatment of rat intestinal epithelial cell cultures with the oxidosqualene cyclase inhibitor, 3 beta-(2-(diethylamino)-ethoxy)androst-5-en-17-one (U18666A), resulted in an accumulation of squalene 2,3:22,23-dioxide (SDO). When U18666A was withdrawn and the cells were treated with the sterol 14 alpha-demethylase inhibitor, ketoconazole, SDO was metabolized to a product identified as 24(S),25-epoxylanosterol. To test the biological effects and cellular metabolism of this compound, we prepared 24(RS),25-epoxylanosterol by chemical synthesis. The epimeric mixture of 24,25-epoxylanosterols could be resolved by high performance liquid chromatography on a wide-pore, non-endcapped, reverse phase column. Both epimers were effective suppressors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity of IEC-6 cells. The suppressive action of the natural epimer, 24(S),25-epoxylanosterol, but not that of 24(R),25-epoxylanosterol could be completely prevented by ketoconazole. IEC-6 cells could efficiently metabolize biosynthetic 24(S),25-epoxy(/sup 3/H)anosterol mainly to the known reductase-suppressor 24(S),25-epoxycholesterol. This metabolism was substantially reduced by ketoconazole. These data support the conclusion that 24(S),25-epoxylanosterol per se is not a suppressor of HMG-CoA reductase activity but is a precursor to a regulatory oxysterol(s). It has recently been reported that 25-hydroxycholesterol can occur naturally in cultured cells in amounts sufficient to effect regulation of HMG-CoA reductase. In order to investigate the biological effects of possible precursors of 25-hydroxycholesterol, we chemically synthesized 25-hydroxylanosterol and 25-hydroxylanostene-3-one. Both oxylanosterol derivatives suppressed cellular sterol synthesis at the level of HMG-CoA reductase. (Abstract Truncated)

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol biosynthesis by oxylanosterols

International Nuclear Information System (INIS)

Panini, S.R.; Sexton, R.C.; Gupta, A.K.; Parish, E.J.; Chitrakorn, S.; Rudney, H.

1986-01-01

Treatment of rat intestinal epithelial cell cultures with the oxidosqualene cyclase inhibitor, 3 beta-[2-(diethylamino)-ethoxy]androst-5-en-17-one (U18666A), resulted in an accumulation of squalene 2,3:22,23-dioxide (SDO). When U18666A was withdrawn and the cells were treated with the sterol 14 alpha-demethylase inhibitor, ketoconazole, SDO was metabolized to a product identified as 24(S),25-epoxylanosterol. To test the biological effects and cellular metabolism of this compound, we prepared 24(RS),25-epoxylanosterol by chemical synthesis. The epimeric mixture of 24,25-epoxylanosterols could be resolved by high performance liquid chromatography on a wide-pore, non-endcapped, reverse phase column. Both epimers were effective suppressors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity of IEC-6 cells. The suppressive action of the natural epimer, 24(S),25-epoxylanosterol, but not that of 24(R),25-epoxylanosterol could be completely prevented by ketoconazole. IEC-6 cells could efficiently metabolize biosynthetic 24(S),25-epoxy[ 3 H]anosterol mainly to the known reductase-suppressor 24(S),25-epoxycholesterol. This metabolism was substantially reduced by ketoconazole. These data support the conclusion that 24(S),25-epoxylanosterol per se is not a suppressor of HMG-CoA reductase activity but is a precursor to a regulatory oxysterol(s). It has recently been reported that 25-hydroxycholesterol can occur naturally in cultured cells in amounts sufficient to effect regulation of HMG-CoA reductase. In order to investigate the biological effects of possible precursors of 25-hydroxycholesterol, we chemically synthesized 25-hydroxylanosterol and 25-hydroxylanostene-3-one. Both oxylanosterol derivatives suppressed cellular sterol synthesis at the level of HMG-CoA reductase. (Abstract Truncated)

Conjugates of Superoxide Dismutase 1 with Amphiphilic Poly(2-oxazoline) Block Copolymers for Enhanced Brain Delivery: Synthesis, Characterization and Evaluation in Vitro and in Vivo

KAUST Repository

Tong, Jing; Yi, Xiang; Luxenhofer, Robert; Banks, William A.; Jordan, Rainer; Zimmerman, Matthew C.; Kabanov, Alexander V.

2013-01-01

Superoxide dismutase 1 (SOD1) efficiently catalyzes dismutation of superoxide, but its poor delivery to the target sites in the body, such as brain, hinders its use as a therapeutic agent for superoxide-associated disorders. Here to enhance

Ubiquinol-cytochrome c reductase (Complex III) electrochemistry at multi-walled carbon nanotubes/Nafion modified glassy carbon electrodes

International Nuclear Information System (INIS)

Pelster, Lindsey N.; Minteer, Shelley D.

2012-01-01

Highlights: ► The electron transport chain is important to the understanding of metabolism in the living cell. ► Ubiquinol-cytochrome c reductase is a membrane bound complex of the electron transport chain (Complex III). ► The paper details the first bioelectrochemical characterization of ubiquinol-cytochrome c reductase at an electrode. - Abstract: Electron transport chain complexes are critical to metabolism in living cells. Ubiquinol-cytochrome c reductase (Complex III) is responsible for carrying electrons from ubiquinol to cytochrome c, but the complex has not been evaluated electrochemically. This work details the bioelectrochemistry of ubiquinol-cytochrome c reductase of the electron transport chain of tuber mitochondria. The characterization of the electrochemistry of this enzyme is investigated in carboxylated multi-walled carbon nanotube/tetrabutyl ammonium bromide-modified Nafion ® modified glassy carbon electrodes by cyclic voltammetry. Increasing concentrations of cytochrome c result in a catalytic response from the active enzyme in the nanotube sandwich. The experiments show that the enzyme followed Michaelis–Menten kinetics with a K m for the immobilized enzyme of 2.97 (±0.11) × 10 −6 M and a V max of 6.31 (±0.82) × 10 −3 μmol min −1 at the electrode, but the K m and V max values decreased compared to the free enzyme in solution, which is expected for immobilized redox proteins. This is the first evidence of ubiquinol-cytochrome c reductase bioelectrocatalysis.

Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene

DEFF Research Database (Denmark)

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

1989-01-01

Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) 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...

The 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase gene from ...

African Journals Online (AJOL)

The 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase gene from Taxus media: Cloning, characterization and functional identification. Y Sun, M Chen, J Tang, W Liu, C Yang, Y Yang, X Lan, M Hsieh, Z Liao ...

To Analyze the Amelioration of Phenobarbital Induced Oxidative Stress by Erucin, as Indicated by Biochemical and Histological Alterations.

Science.gov (United States)

Arora, Rohit; Bhushan, Sakshi; Kumar, Rakesh; Mannan, Rahul; Kaur, Pardeep; Singh, Bikram; Sharma, Ritika; Vig, Adarsh Pal; Singh, Balbir; Singh, Amrit Pal; Arora, Saroj

2016-01-01

Phenobarbital is a commonly employed antidepressant and anti-epileptic drug. The cancer promoting activity of this genotoxic xenobiotic is often ignored. It is responsible for oxidative stress leading to modulation in xenobiotic and antioxidative enzymes. Glucosinolates and more specifically their hydrolytic products are known for their antioxidative and anticancer activities. The present study involves the analysis of hepatoprotective effect of erucin (isolated from Eruca sativa (Mill.) Thell.) against phenobarbital mediated hepatic damage in male wistar rats. The liver homogenate was analyzed for oxidative stress (superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione reductase and lactate dehydrogenase), other oxidative parameters (thiobarbituric acid reactive species, conjugated dienes and lipid hydroperoxide), phase I enzymes (NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase, cytochrome P420, cytochrome P450 and cytochrome b5), phase II enzymes (γ-glutamyl transpeptidase, DT-diaphorase and glutathione-S-transferase), serum parameters (alkaline phosphatase, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, direct bilirubin and total bilirubin) and certain histological parameters. Erucin accorded protection from phenobarbital induced hepatic damage by normalizing antioxidative enzymes, other oxidative parameters, phase I, II, and serum parameters. Erucin, an analogue of sulforaphane has the potential to act as an anticancer agent by regulating various biochemical parameters.

Nitroxyl Modified Tobacco Mosaic Virus as a Metal-Free High-Relaxivity MRI and EPR Active Superoxide Sensor.

Science.gov (United States)

Dharmarwardana, Madushani; Martins, André F; Chen, Zhuo; Palacios, Philip M; Nowak, Chance M; Welch, Raymond P; Li, Shaobo; Luzuriaga, Michael A; Bleris, Leonidas; Pierce, Brad S; Sherry, A Dean; Gassensmith, Jeremiah J

2018-05-29

Superoxide overproduction is known to occur in multiple disease states requiring critical care; yet, noninvasive detection of superoxide in deep tissue remains a challenge. Herein, we report a metal-free magnetic resonance imaging (MRI) and electron paramagnetic resonance (EPR) active contrast agent prepared by "click conjugating" paramagnetic organic radical contrast agents (ORCAs) to the surface of tobacco mosaic virus (TMV). While ORCAs are known to be reduced in vivo to an MRI/EPR silent state, their oxidation is facilitated specifically by reactive oxygen species-in particular, superoxide-and are largely unaffected by peroxides and molecular oxygen. Unfortunately, single molecule ORCAs typically offer weak MRI contrast. In contrast, our data confirm that the macromolecular ORCA-TMV conjugates show marked enhancement for T 1 contrast at low field (<3.0 T) and T 2 contrast at high field (9.4 T). Additionally, we demonstrated that the unique topology of TMV allows for a "quenchless fluorescent" bimodal probe for concurrent fluorescence and MRI/EPR imaging, which was made possible by exploiting the unique inner and outer surface of the TMV nanoparticle. Finally, we show TMV-ORCAs do not respond to normal cellular respiration, minimizing the likelihood for background, yet still respond to enzymatically produced superoxide in complicated biological fluids like serum.

Extracellular superoxide dismutase is necessary to maintain renal blood flow during sepsis development.

Science.gov (United States)

Constantino, Larissa; Galant, Letícia Selinger; Vuolo, Francieli; Guarido, Karla Lorena; Kist, Luiza Wilges; de Oliveira, Giovanna Medeiros Tavares; Pasquali, Matheus Augusto de Bittencourt; de Souza, Cláudio Teodoro; da Silva-Santos, José Eduardo; Bogo, Maurício Reis; Moreira, José Cláudio Fonseca; Ritter, Cristiane; Dal-Pizzol, Felipe

2017-12-01

Extracellular superoxide dismutase (ECSOD) protects nitric oxide (NO) bioavailability by decreasing superoxide levels and preventing peroxynitrite generation, which is important in maintaining renal blood flow and in preventing acute kidney injury. However, the profile of ECSOD expression after sepsis is not fully understood. Therefore, we intended to evaluate the content and gene expression of superoxide dismutase (SOD) isoforms in the renal artery and their relation to renal blood flow. Sepsis was induced in Wistar rats by caecal ligation and perforation. Several times after sepsis induction, renal blood flow (12, 24 and 48 h); the renal arterial content of SOD isoforms, nitrotyrosine, endothelial and inducible nitric oxide synthase (e-NOS and i-NOS), and phosphorylated vasodilator-stimulated phosphoprotein (pVASP); and SOD activity (3, 6 and 12 h) were measured. The influence of a SOD inhibitor was also evaluated. An increase in ECSOD content was associated with decreased 3-nitrotyrosine levels. These events were associated with an increase in pVASP content and maintenance of renal blood flow. Moreover, previous treatment with a SOD inhibitor increased nitrotyrosine content and reduced renal blood flow. ECSOD appears to have a major role in decreasing peroxynitrite formation in the renal artery during the early stages of sepsis development, and its application can be important in renal blood flow control and maintenance during septic insult.

Relationship between nitrate reductase and nitrate uptake in phytoplankton in the Peru upwelling region

International Nuclear Information System (INIS)

Blasco, D.; MacIsaac, J.J.; Packard, T.T.; Dugdale, R.C.

1984-01-01

Nitrate reductase (NR) activity and 15 NO 3 - uptake in phytoplankton were compared under different environmental conditions on two cruises in the upwelling region off Peru. The NR activity and NO 3 - uptake rates responded differently to light and nutrients and the differences led to variations in the uptake:reductase ratio. Analysis of these variations suggests that the re-equilibration time of the two processes in response to environmental perturbation is an important source of variability. The nitrate uptake system responds faster than the nitrate reductase system. Considering these differences in response time, the basic differences in the two processes, and the differences in their measurement, the authors conclude that the NR activity measures the current nitrate-reducing potential, which relfects NO 3 - assimilation before the sampling time, while 15 NO 3 - uptake measures NO 3 - assimilation in the 6-h period following sampling. Thus, considering the sampling time as a point of reference, the former is a measure of the past and the latter is a measure of the future

Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation

DEFF Research Database (Denmark)

Björnberg, Olof; Viennet, Thibault; Skjoldager, Nicklas

2015-01-01

Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two...... enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s-1) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli Trx......-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass...

Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates

DEFF Research Database (Denmark)

Chen, Z-W; Liu, Y-Y; Wu, J-F

2007-01-01

The microbial community and sulfur oxygenase reductases of metagenomic DNA from bioreactors treating gold-bearing concentrates were studied by 16S rRNA library, real-time polymerase chain reaction (RT-PCR), conventional cultivation, and molecular cloning. Results indicated that major bacterial......) of bacteria and archaea were 4.59 x 10(9) and 6.68 x 10(5), respectively. Bacterial strains representing Acidithiobacillus, Leptospirillum, and Sulfobacillus were isolated from the bioreactors. To study sulfur oxidation in the reactors, pairs of new PCR primers were designed for the detection of sulfur...... oxygenase reductase (SOR) genes. Three sor-like genes, namely, sor (Fx), sor (SA), and sor (SB) were identified from metagenomic DNAs of the bioreactors. The sor (Fx) is an inactivated SOR gene and is identical to the pseudo-SOR gene of Ferroplasma acidarmanus. The sor (SA) and sor (SB) showed...

Influence of rete testis fluid deprivation on the kinetic parameters of goat epididymal 5 alpha-reductase.

Science.gov (United States)

Kelce, W R; Lubis, A M; Braun, W F; Youngquist, R S; Ganjam, V K

1990-01-01

A surgical technique to cannulate the rete testis of the goat was utilized to examine the effects of rete testis fluid (RTF) deprivation on the enzymatic activity of epididymal 5 alpha-reductase. Kinetic techniques were used to determine whether the regional enzymatic effect of RTF deprivation is to decrease the apparent number of 5 alpha-reductase active sites or the catalytic activity of each active site within the epididymal epithelium. Paired comparisons of (Vmax)app and (Km)app values between control and RTF-deprived epididymides indicated that RTF deprivation affected the value of (Vmax)app with no apparent change in the values of (Km)app in caput, corpus, and cauda epididymal regions. We conclude that RTF deprivation in the goat epididymis for 7 days results in a decreased number of apparent 5 alpha-reductase active sites within the epididymal epithelium.

Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra

Science.gov (United States)

Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2009-01-01

(R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-­quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P41212, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%. PMID:19478454

Ferric reductase genes involved in high-affinity iron uptake are differentially regulated in yeast and hyphae of Candida albicans.

Science.gov (United States)

Jeeves, Rose E; Mason, Robert P; Woodacre, Alexandra; Cashmore, Annette M

2011-09-01

The pathogenic yeast Candida albicans possesses a reductive iron uptake system which is active in iron-restricted conditions. The sequestration of iron by this mechanism initially requires the reduction of free iron to the soluble ferrous form, which is catalysed by ferric reductase proteins. Reduced iron is then taken up into the cell by a complex of a multicopper oxidase protein and an iron transport protein. Multicopper oxidase proteins require copper to function and so reductive iron and copper uptake are inextricably linked. It has previously been established that Fre10 is the major cell surface ferric reductase in C. albicans and that transcription of FRE10 is regulated in response to iron levels. We demonstrate here that Fre10 is also a cupric reductase and that Fre7 also makes a significant contribution to cell surface ferric and cupric reductase activity. It is also shown, for the first time, that transcription of FRE10 and FRE7 is lower in hyphae compared to yeast and that this leads to a corresponding decrease in cell surface ferric, but not cupric, reductase activity. This demonstrates that the regulation of two virulence determinants, the reductive iron uptake system and the morphological form of C. albicans, are linked. Copyright © 2011 John Wiley & Sons, Ltd.

L-Arginine Attenuates Diabetic Nephropathy In Streptozotocin ...

African Journals Online (AJOL)

Egyptian Journal of Biochemistry and Molecular Biology ... On the other hand, no significant change was detected in body weight, kidney weight and relative kidney weight, levels of plasma fructosamine, MDA and NOx, renal activities of glutathione peroxidase(GPx),superoxide dismutase(SOD), LDH, aldose reductase(AR) ...

Intramolecular electron transfer in Pseudomonas aeruginosa cd(1) nitrite reductase

DEFF Research Database (Denmark)

Farver, Ole; Brunori, Maurizio; Cutruzzolà, Francesca

2009-01-01

) as the level of reduction increased in both the WT and the His mutant. Equilibrium standard enthalpy and entropy changes and activation parameters of this ET process were determined. We concluded that negative cooperativity is a common feature among the cd(1) nitrite reductases, and we discuss this control...

The potential of the superoxide dismutase inhibitor, diethyldithiocarbamate as an adjuvant to radiotherapy

International Nuclear Information System (INIS)

Kent, C.

1990-10-01

Oxygen has the potential to be toxic to biologic systems. This toxicity is not due to oxygen itself, but due to the production of oxygen radicals. One of these potentially toxic radicals, superoxide, can be generated as a result of ionizing radiation, and if not adequately removed can proceed to cause cell damage. Superoxide dismutase (SOD) is one of the key enzymes involved in the defence against oxygen toxicity. SOD activity can be inhibited by diethyldithiocarbamate (DDC), a powerful copper chelator. If inhibition of SOD by DDC increases the lifetime and effectiveness of radiation induced superoxide, it follows that the potential exists for DDC to enhance the effect of radiation. DDC is however also a thiol compound, and thus may act as a radioprotector by modifying tissue oxygenation status or by free radical scavenging. The inhibition of superoxide dismutase by diethyldithiocarbamate in order to sensitize tumours to ionizing radiation was studied. The use of DDC as an inhibitor of SOD has however meant that any sensitization resulting from SOD inhibition could be masked by a radioprotective effect by DDC. The inhibition of SOD by DDC was confirmed in a murine rhabdomyosarcoma, and this inhibition can be maintained for up to twenty-four hours after DDC administration. It was shown that DDC could act as both a radiosensitizer and as a radioprotector in the same experiment. The dominant action of DDC was found to be dependent on the time allowed between DDC administration and irradiation. The time modulation effect of DDC was shown in larger tumours, rather than smaller tumours, which could indicate that tumour oxygenation is an important criterion in determining the response to radiation of DDC treated cells. Some caution should be exercised when DDC is put forward as either a radiosensitizer or a radioprotector in the clinic, but DDC may have potential as a thermosensitizer. 37 figs., 23 tabs., 208 refs

Identification of a Novel Epoxyqueuosine Reductase Family by Comparative Genomics.

Science.gov (United States)

Zallot, Rémi; Ross, Robert; Chen, Wei-Hung; Bruner, Steven D; Limbach, Patrick A; de Crécy-Lagard, Valérie

2017-03-17

The reduction of epoxyqueuosine (oQ) is the last step in the synthesis of the tRNA modification queuosine (Q). While the epoxyqueuosine reductase (EC 1.17.99.6) enzymatic activity was first described 30 years ago, the encoding gene queG was only identified in Escherichia coli in 2011. Interestingly, queG is absent from a large number of sequenced genomes that harbor Q synthesis or salvage genes, suggesting the existence of an alternative epoxyqueuosine reductase in these organisms. By analyzing phylogenetic distributions, physical gene clustering, and fusions, members of the Domain of Unknown Function 208 (DUF208) family were predicted to encode for an alternative epoxyqueuosine reductase. This prediction was validated with genetic methods. The Q modification is present in Lactobacillus salivarius, an organism missing queG but harboring the duf208 gene. Acinetobacter baylyi ADP1 is one of the few organisms that harbor both QueG and DUF208, and deletion of both corresponding genes was required to observe the absence of Q and the accumulation of oQ in tRNA. Finally, the conversion oQ to Q was restored in an E. coli queG mutant by complementation with plasmids harboring duf208 genes from different bacteria. Members of the DUF208 family are not homologous to QueG enzymes, and thus, duf208 is a non-orthologous replacement of queG. We propose to name DUF208 encoding genes as queH. While QueH contains conserved cysteines that could be involved in the coordination of a Fe/S center in a similar fashion to what has been identified in QueG, no cobalamin was identified associated with recombinant QueH protein.

Nitrate reductase activity of Staphylococcus carnosus affecting the color formation in cured raw ham.

Science.gov (United States)

Bosse Née Danz, Ramona; Gibis, Monika; Schmidt, Herbert; Weiss, Jochen

2016-07-01

The influence of the nitrate reductase activity of two Staphylococcus carnosus strains used as starter cultures on the formation of nitrate, nitrite and color pigments in cured raw ham was investigated. In this context, microbiological, chemical and multivariate image analyses were carried out on cured raw hams, which were injected with different brines containing either nitrite or nitrate, with or without the S. carnosus starter cultures. During processing and storage, the viable counts of staphylococci remained constant at 6.5logcfu/g in the hams inoculated with starter cultures, while the background microbiota of the hams processed without the starter cultures developed after 14days. Those cured hams inoculated with S. carnosus LTH 7036 (high nitrate reductase activity) showed the highest decrease in nitrate and high nitrite concentrations in the end product, but were still in the range of the legal European level. The hams cured with nitrate and without starter culture or with the other strain, S. carnosus LTH 3838 (low nitrate reductase activity) showed higher residual nitrate levels and a lower nitrite content in the end product. The multivariate image analysis identified spatial and temporal differences in the meat pigment profiles of the differently cured hams. The cured hams inoculated with S. carnosus LTH 3838 showed an uncured core due to a delay in pigment formation. Therefore, the selection of starter cultures based on their nitrate reductase activity is a key point in the formation of curing compounds and color pigments in cured raw ham manufacture. Copyright © 2016 Elsevier Ltd. All rights reserved.

EFFECT OF POLYPHENOLIC COMPLEX FROM WINE ON RATS ANTIOXIDANT ENZYMES ACTIVITY AT X-RAY IRRADIATION LOW DOSES

Directory of Open Access Journals (Sweden)

U. V. Datsyuk

2014-04-01

Full Text Available It is shown that the consumption of natural polyphenolic complex from grape wine in drinking water in the daily dose 2.5 ± 1.1 mg polyphenols/kg body mass of rats during the 10 day before exposure to radiation leads to increased of superoxide dismutase and gluthathione reductase activities in peripheral blood on 24 and 48 hours after full body X-ray irradiation (30 cGy. The of catalase, gluthathione peroxidase activities and the of the reactive thiobarbituric acid substances content in total lysates of peripheral blood within 72 hours after exposure are comparable to those in control rats. Marked decreased of catalase and superoxide dismutase activities at 24, 48 and 24 hours, respectively, was observed after exposure to ionizing radiation and increased content of lipid peroxidation products in all above mentioned time points. The decreased of superoxide dismutase and gluthathione peroxidase activities in lysates of rats aorta at 48 hour and increased content of the reactive thiobarbituric acid substances during 72 hours after radiation exposure were observed. The consumption of polyphenolic complex from wine did not change the superoxide dismutase and catalase activities in lysates of aorta rats treated with ionizing radiation, whereas gluthathione reductase and gluthathione peroxidase activities was increased during 72 hours after radiation influence. The content of TBA reactive substances was significantly decreased in lysates of aorta rats that were exposed to radiation and polyphenols of grape wine, compared with those of animals that were exposed to radiation alone.

Biochemical analysis of reactive oxygen species production and antioxidative responses in unripe avocado (Persea americana Mill var Hass) fruits in response to wounding.

Science.gov (United States)

Castro-Mercado, E; Martinez-Diaz, Y; Roman-Tehandon, N; Garcia-Pineda, E

2009-03-01

We analyzed the production of reactive oxygen species (ROS) and of detoxifying enzymes and enzymes of the ascorbate (ASC) acid cycle in avocado fruit (Pesea Americana Mill cv Hass) in response to wounding. The levels of superoxide anion (O(2-), hydroxyl radicals (OH.) and hydrogen peroxide (H(2)O(2)) increased at 15 min and 2 and 15 h post-wounding. Peroxidase (POD) activity had increased to high levels 24 h after wounding; in contrast, catalase and superoxide dismutase (SOD) levels hat decreased significantly at 24 h post-treatment. Basic POD was the major POD form induced, and the levels of at least three apoplastic POD isozymes -increased following wounding. Using specific inhibitors, we characterized one MnSOD and two CuZnSOD isozymes. CuZnSOD activities decreased notably 12 h after treatment. The activities of dehydroascorbate reductase and glutathione reductase increased dramatically following the wounding treatment, possibly as a means to compensate for the redox changes due to ROS production.

Reaction of hypotaurine or taurine with superoxide produces the organic peroxysulfonic acid peroxytaurine.

Science.gov (United States)

Grove, Roxanna Q; Karpowicz, Steven J

2017-07-01

Hypotaurine and taurine are amino acid derivatives and abundant molecules in many eukaryotes. The biological reaction in which hypotaurine is converted to taurine remains poorly understood. Here, hypotaurine and taurine were observed to react with superoxide anion in vitro to form the novel molecule peroxytaurine. In contrast, hypotaurine reacts with hydrogen peroxide to form taurine, but taurine does not react with hydrogen peroxide in vitro. Mass and NMR spectrometry as well as FTIR and Raman spectroscopy support the molecular characterization of peroxytaurine. Gravitometric and spectroscopy experiments suggest a stoichiometry of two superoxide anions reacting with one hypotaurine or two taurines. The newly identified molecule is a semi-stable, organic peroxysulfonic acid that may be an intermediate metabolite in taurine synthesis. Copyright © 2017 Elsevier Inc. All rights reserved.

NADPH-Thioredoxin Reductase C Mediates the Response to Oxidative Stress and Thermotolerance in the Cyanobacterium Anabaena sp PCC7120

NARCIS (Netherlands)

Sanchez-Riego, Ana M.; Mata-Cabana, Alejandro; Galmozzi, CarlaV.; Florencio, Francisco J.

2016-01-01

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

Sterol-induced Dislocation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase from Endoplasmic Reticulum Membranes into the Cytosol through a Subcellular Compartment Resembling Lipid Droplets*

Science.gov (United States)

Hartman, Isamu Z.; Liu, Pingsheng; Zehmer, John K.; Luby-Phelps, Katherine; Jo, Youngah; Anderson, Richard G. W.; DeBose-Boyd, Russell A.

2010-01-01

Sterol-induced binding to Insigs in the endoplasmic reticulum (ER) allows for ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. This ubiquitination marks reductase for recognition by the ATPase VCP/p97, which mediates extraction and delivery of reductase from ER membranes to cytosolic 26 S proteasomes for degradation. Here, we report that reductase becomes dislocated from ER membranes into the cytosol of sterol-treated cells. This dislocation exhibits an absolute requirement for the actions of Insigs and VCP/p97. Reductase also appears in a buoyant fraction of sterol-treated cells that co-purifies with lipid droplets, cytosolic organelles traditionally regarded as storage depots for neutral lipids such as triglycerides and cholesteryl esters. Genetic, biochemical, and localization studies suggest a model in which reductase is dislodged into the cytosol from an ER subdomain closely associated with lipid droplets. PMID:20406816

Ghrelin-related peptides do not modulate vasodilator nitric oxide production or superoxide levels in mouse systemic arteries.

Science.gov (United States)

Ku, Jacqueline M; Sleeman, Mark W; Sobey, Christopher G; Andrews, Zane B; Miller, Alyson A

2016-04-01

The ghrelin gene is expressed in the stomach where it ultimately encodes up to three peptides, namely, acylated ghrelin, des-acylated ghrelin and obestatin, which all have neuroendocrine roles. Recently, the authors' reported that these peptides have important physiological roles in positively regulating vasodilator nitric oxide (NO) production in the cerebral circulation, and may normally suppress superoxide production by the pro-oxidant enzyme, Nox2-NADPH oxidase. To date, the majority of studies using exogenous peptides infer that they may have similar roles in the systemic circulation. Therefore, this study examined whether exogenous and endogenous ghrelin-related peptides modulate NO production and superoxide levels in mouse mesenteric arteries and/or thoracic aorta. Using wire myography, it was found that application of exogenous acylated ghrelin, des-acylated ghrelin or obestatin to mouse thoracic aorta or mesenteric arteries failed to elicit a vasorelaxation response, whereas all three peptides elicited vasorelaxation responses of rat thoracic aorta. Also, none of the peptides modulated mouse aortic superoxide levels as measured by L-012-enhanced chemiluminescence. Next, it was found that NO bioactivity and superoxide levels were unaffected in the thoracic aorta from ghrelin-deficient mice when compared with wild-type mice. Lastly, using novel GHSR-eGFP reporter mice in combination with double-labelled immunofluorescence, no evidence was found for the growth hormone secretagogue receptor (GHSR1a) in the throracic aorta, which is the only functional ghrelin receptor identified to date. Collectively these findings demonstrate that, in contrast to systemic vessels of other species (e.g. rat and human) and mouse cerebral vessels, ghrelin-related peptides do not modulate vasodilator NO production or superoxide levels in mouse systemic arteries. © 2016 John Wiley & Sons Australia, Ltd.

Biodegradable polycaprolactone (PCL) nanosphere encapsulating superoxide dismutase and catalase enzymes.

Science.gov (United States)

Singh, Sushant; Singh, Abhay Narayan; Verma, Anil; Dubey, Vikash Kumar

2013-12-01

Biodegradable polycaprolactone (PCL) nanosphere encapsulating superoxide dismutase (SOD) and catalase (CAT) were successfully synthesized using double emulsion (w/o/w) solvent evaporation technique. Characterization of the nanosphere using dynamic light scattering, field emission scanning electron microscope, and Fourier transform infrared spectroscopy revealed a spherical-shaped nanosphere in a size range of 812 ± 64 nm with moderate protein encapsulation efficiency of 55.42 ± 3.7 % and high in vitro protein release. Human skin HaCat cells were used for analyzing antioxidative properties of SOD- and CAT-encapsulated PCL nanospheres. Oxidative stress condition in HaCat cells was optimized with exposure to hydrogen peroxide (H2O2; 1 mM) as external stress factor and verified through reactive oxygen species (ROS) analysis using H2DCFDA dye. PCL nanosphere encapsulating SOD and CAT together indicated better antioxidative defense against H2O2-induced oxidative stress in human skin HaCat cells in comparison to PCL encapsulating either SOD or CAT alone as well as against direct supplement of SOD and CAT protein solution. Increase in HaCat cells SOD and CAT activities after treatment hints toward uptake of PCL nanosphere into the human skin HaCat cells. The result signifies the role of PCL-encapsulating SOD and CAT nanosphere in alleviating oxidative stress.

Prevention of hemodynamic and vascular albumin filtration changes in diabetic rats by aldose reductase inhibitors

International Nuclear Information System (INIS)

Tilton, R.G.; Chang, K.; Pugliese, G.; Eades, D.M.; Province, M.A.; Sherman, W.R.; Kilo, C.; Williamson, J.R.

1989-01-01

This study investigated hemodynamic changes in diabetic rats and their relationship to changes in vascular albumin permeation and increased metabolism of glucose to sorbitol. The effects of 6 wk of streptozocin-induced diabetes and three structurally different inhibitors of aldose reductase were examined on (1) regional blood flow (assessed with 15-microns 85Sr-labeled microspheres) and vascular permeation by 125I-labeled bovine serum albumin (BSA) and (2) glomerular filtration rate (assessed by plasma clearance of 57Co-labeled EDTA) and urinary albumin excretion (determined by radial immunodiffusion assay). In diabetic rats, blood flow was significantly increased in ocular tissues (anterior uvea, posterior uvea, retina, and optic nerve), sciatic nerve, kidney, new granulation tissue, cecum, and brain. 125I-BSA permeation was increased in all of these tissues except brain. Glomerular filtration rate and 24-h urinary albumin excretion were increased 2- and 29-fold, respectively, in diabetic rats. All three aldose reductase inhibitors completely prevented or markedly reduced these hemodynamic and vascular filtration changes and increases in tissue sorbitol levels in the anterior uvea, posterior uvea, retina, sciatic nerve, and granulation tissue. These observations indicate that early diabetes-induced hemodynamic changes and increased vascular albumin permeation and urinary albumin excretion are aldose reductase-linked phenomena. Discordant effects of aldose reductase inhibitors on blood flow and vascular albumin permeation in some tissues suggest that increased vascular albumin permeation is not entirely attributable to hemodynamic change

Cloning, expression and antigenicity of the L. donovani reductase

DEFF Research Database (Denmark)

Jensen, A T; Kemp, K; Theander, T G

2001-01-01

(K). Only 2 of 22 plasma samples from patients with visceral leishmaniasis were found to have detectable anti-reductase antibodies and peripheral blood mononuclear cells (PBMC) from one of three individuals previously infected with visceral leishmaniasis proliferated in the presence of recombinant...

The superoxide scavenger TEMPOL induces urokinase receptor (uPAR expression in human prostate cancer cells

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Francis Joseph

2006-06-01

Full Text Available Abstract There is little understanding of the effect that reactive oxygen metabolites have on cellular behavior during the processes of invasion and metastasis. These oxygen metabolites could interact with a number of targets modulating their function such as enzymes involved in basement membrane dissolution, adhesion molecules involved in motility or receptors involved in proliferation. We investigated the effect of increased scavenging of superoxide anions on the expression of the urokinase receptor (uPAR in PC-3M human prostate cancer cells. Urokinase receptor is a GPI-linked cell surface molecule which mediates multiple functions including adhesion, proliferation and pericellular proteolysis. Addition of the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL to PC-3M cultures stimulated expression of uPAR protein peaking between 48 and 72 hours. Cell surface expression of the uPAR was also increased. Surprisingly, uPAR transcript levels increased only slightly and this mild increase did not coincide with the striking degree of protein increase. This disparity indicates that the TEMPOL effect on uPAR occurs through a post-transcriptional mechanism. TEMPOL presence in PC-3M cultures reduced intracellular superoxide-type species by 75% as assayed by NBT dye conversion; however this reduction significantly diminished within hours following TEMPOL removal. The time gap between TEMPOL treatment and peak uPAR protein expression suggests that reduction of reactive oxygen metabolites in prostate cancer cells initiates a multistep pathway which requires several hours to culminate in uPAR induction. These findings reveal a novel pathway for uPAR regulation involving reactive oxygens such as superoxide anion.

Impact of the application of humic acid and sodium nitroprusside on ...

African Journals Online (AJOL)

Nickel (Ni) is an essential micronutrient for plants but in high concentrations may turn toxic. This paper discusses the potential role of humic acid (HA) and sodium nitroprusside in modulating or preventing oxidative stress in rice plants. Three genes [superoxide dismutase (SOD) glutathione reductase (GR) and ascorbate ...

A New Type of YumC-Like Ferredoxin (Flavodoxin) Reductase Is Involved in Ribonucleotide Reduction

DEFF Research Database (Denmark)

Chen, Jun; Shen, Jing; Solem, Christian

2015-01-01

. subtilis but that the addition of deoxynucleosides cannot compensate for the lethal phenotype displayed by the B. subtilis yumC knockout mutant. Ferredoxin (flavodoxin) reductase (FdR) is involved in many important reactions in both eukaryotes and prokaryotes, such as photosynthesis, nitrate reduction, etc. The recently...... ribonucleotide reductase, which represents the workhorse for the bioconversion of nucleotides to deoxynucleotides in many prokaryotes and eukaryotic pathogens under aerobic conditions. As the partner of the flavodoxin (NrdI), the key FdR is missing in the current model describing the class Ib system...

Improvement of operational stability of Ogataea minuta carbonyl reductase for chiral alcohol production.

Science.gov (United States)

Honda, Kohsuke; Inoue, Mizuha; Ono, Tomohiro; Okano, Kenji; Dekishima, Yasumasa; Kawabata, Hiroshi

2017-06-01

Directed evolution of enantio-selective carbonyl reductase from Ogataea minuta was conducted to improve the operational stability of the enzyme. A mutant library was constructed by an error-prone PCR and screened using a newly developed colorimetric assay. The stability of a mutant with two amino acid substitutions was significantly higher than that of the wild type at 50°C in the presence of dimethyl sulfoxide. Site-directed mutagenesis analysis showed that the improved stability of the enzyme can be attributed to the amino acid substitution of V166A. The half-lives of the V166A mutant were 11- and 6.1-times longer than those of the wild type at 50°C in the presence and absence, respectively, of 20% (v/v) dimethyl sulfoxide. No significant differences in the substrate specificity and enantio-selectivity of the enzyme were observed. The mutant enzyme converted 60 mM 2,2,2-trifluoroacetophenone to (R)-(-)-α-(trifluoromethyl)benzyl alcohol in a molar yield of 71% whereas the conversion yield with an equivalent concentration of the wild-type enzyme was 27%. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization.

Science.gov (United States)

Fu, Yuan; Lin, Hongyu; Wisitpitthaya, Somsinee; Blessing, William A; Aye, Yimon

2014-11-24

Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger α-dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We therefore developed a fluorimetric assay that reliably reports on oligomeric state changes of α with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-α, covalently labeled with two fluorophores, allows for direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of α self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional characterization of Dihydroflavonol-4-reductase in anthocyanin biosynthesis of purple sweet potato underlies the direct evidence of anthocyanins function against abiotic stresses.

Science.gov (United States)

Wang, Hongxia; Fan, Weijuan; Li, Hong; Yang, Jun; Huang, Jirong; Zhang, Peng

2013-01-01

Dihydroflavonol-4-reductase (DFR) is a key enzyme in the catalysis of the stereospecific reduction of dihydroflavonols to leucoanthocyanidins in anthocyanin biosynthesis. In the purple sweet potato (Ipomoea batatas Lam.) cv. Ayamurasaki, expression of the IbDFR gene was strongly associated with anthocyanin accumulation in leaves, stems and roots. Overexpression of the IbDFR in Arabidopsis tt3 mutants fully complemented the pigmentation phenotype of the seed coat, cotyledon and hypocotyl. Downregulation of IbDFR expression in transgenic sweet potato (DFRi) using an RNAi approach dramatically reduced anthocyanin accumulation in young leaves, stems and storage roots. In contrast, the increase of flavonols quercetin-3-O-hexose-hexoside and quercetin-3-O-glucoside in the leaves and roots of DFRi plants is significant. Therefore, the metabolic pathway channeled greater flavonol influx in the DFRi plants when their anthocyanin and proanthocyanidin accumulation were decreased. These plants also displayed reduced antioxidant capacity compared to the wild type. After 24 h of cold treatment and 2 h recovery, the wild-type plants were almost fully restored to the initial phenotype compared to the slower recovery of DFRi plants, in which the levels of electrolyte leakage and hydrogen peroxide accumulation were dramatically increased. These results provide direct evidence of anthocyanins function in the protection against oxidative stress in the sweet potato. The molecular characterization of the IbDFR gene in the sweet potato not only confirms its important roles in flavonoid metabolism but also supports the protective function of anthocyanins of enhanced scavenging of reactive oxygen radicals in plants under stressful conditions.

Functional characterization of Dihydroflavonol-4-reductase in anthocyanin biosynthesis of purple sweet potato underlies the direct evidence of anthocyanins function against abiotic stresses.

Directory of Open Access Journals (Sweden)

Hongxia Wang

Full Text Available Dihydroflavonol-4-reductase (DFR is a key enzyme in the catalysis of the stereospecific reduction of dihydroflavonols to leucoanthocyanidins in anthocyanin biosynthesis. In the purple sweet potato (Ipomoea batatas Lam. cv. Ayamurasaki, expression of the IbDFR gene was strongly associated with anthocyanin accumulation in leaves, stems and roots. Overexpression of the IbDFR in Arabidopsis tt3 mutants fully complemented the pigmentation phenotype of the seed coat, cotyledon and hypocotyl. Downregulation of IbDFR expression in transgenic sweet potato (DFRi using an RNAi approach dramatically reduced anthocyanin accumulation in young leaves, stems and storage roots. In contrast, the increase of flavonols quercetin-3-O-hexose-hexoside and quercetin-3-O-glucoside in the leaves and roots of DFRi plants is significant. Therefore, the metabolic pathway channeled greater flavonol influx in the DFRi plants when their anthocyanin and proanthocyanidin accumulation were decreased. These plants also displayed reduced antioxidant capacity compared to the wild type. After 24 h of cold treatment and 2 h recovery, the wild-type plants were almost fully restored to the initial phenotype compared to the slower recovery of DFRi plants, in which the levels of electrolyte leakage and hydrogen peroxide accumulation were dramatically increased. These results provide direct evidence of anthocyanins function in the protection against oxidative stress in the sweet potato. The molecular characterization of the IbDFR gene in the sweet potato not only confirms its important roles in flavonoid metabolism but also supports the protective function of anthocyanins of enhanced scavenging of reactive oxygen radicals in plants under stressful conditions.

Microbial production of branched-chain dicarboxylate 2-methylsuccinic acid via enoate reductase-mediated bioreduction.

Science.gov (United States)

Wang, Jian; Yang, Yaping; Zhang, Ruihua; Shen, Xiaolin; Chen, Zhenya; Wang, Jia; Yuan, Qipeng; Yan, Yajun

2018-01-01

2-Methylsuccinic acid (2-MSA) is a C5 branched-chain dicarboxylate that serves as an attractive synthon for the synthesis of polymers with extensive applications in coatings, cosmetic solvents and bioplastics. However, the lack of natural pathways for 2-MSA biosynthesis has limited its application as a promising bio-replacement. Herein, we conceived a non-natural three-step biosynthetic route for 2-MSA, via employing the citramalate pathway in combination with enoate reductase-mediated bioreduction of the pathway intermediate citraconate. First, over-expression of codon-optimized citramalate synthase variant CimA* from Methanococcus jannaschii, endogenous isopropylmalate isomerase EcLeuCD and enoate reductase YqjM from Bacillus subtilis allowed the production of 2-MSA in Escherichia coli for the first time, with a titer of 0.35g/L in shake flask experiments. Subsequent screening of YqjM-like enoate reductases of different bacterial origins enabled identification and characterization of a new NAD(P)H-dependent enoate reductase KpnER from Klebsiella pneumoniae, which exhibited higher activity towards citraconate than YqjM. Incorporation of KpnER into the 2-MSA biosynthetic pathway led to 2-MSA production improvement to a titer of 0.96g/L in aerobic condition. Subsequent optimizations including cofactor regeneration, microaerobic cultivation and host strain engineering, boosted 2-MSA titer to 3.61g/L with a molar yield of 0.36 in shake flask experiments. This work established a promising platform for 2-MSA bioproduction, which enabled the highest titer of 2-MSA production in microbial hosts so far. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

Energy Technology Data Exchange (ETDEWEB)

Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas; Joachimiak, Andrzej

2017-08-02

In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of δ1-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

Energy Technology Data Exchange (ETDEWEB)

Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas; Joachimiak, Andrzej

2017-08-02

In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of delta(1)-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

Directory of Open Access Journals (Sweden)

Giuseppe Forlani

2017-08-01

Full Text Available In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of δ1-pyrroline-5-carboxylate (P5C catalyzed by P5C reductase (EC 1.5.1.2. In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

Overexpression of Soybean Isoflavone Reductase (GmIFR) Enhances Resistance to Phytophthora sojae in Soybean.

Science.gov (United States)

Cheng, Qun; Li, Ninghui; Dong, Lidong; Zhang, Dayong; Fan, Sujie; Jiang, Liangyu; Wang, Xin; Xu, Pengfei; Zhang, Shuzhen

2015-01-01

Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET), abscisic acid (placeCityABA), salicylic acid (SA). It is located in the cytoplasm when transiently expressed in soybean protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while the relative content of glyceollins in transgenic plants was significantly higher than that of non-transgenic plants. Furthermore, we found that the relative expression levels of reactive oxygen species (ROS) of transgenic soybean plants were significantly lower than those of non-transgenic plants after incubation with P. sojae, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean. The enzyme activity assay suggested that GmIFR has isoflavone reductase activity.

Association study of methylenetetrahydrofolate reductase C677T mutation with cerebral venous thrombosis in an Iranian population.

Science.gov (United States)

Ghaznavi, Habib; Soheili, Zahra; Samiei, Shahram; Soltanpour, Mohammad S

2015-12-01

There are limited data on the role of methylenetetrahydrofolate reductase C677T polymorphism and hyperhomocysteinemia as risk factors for cerebral venous thrombosis in Iranian population. We examined a possible association between fasting plasma homocysteine levels, methylenetetrahydrofolate reductase C677T polymorphism, and cerebral venous thrombosis in 50 patients with a diagnosis of cerebral venous thrombosis (20-63 years old) and 75 healthy controls (18-65 years old). Genotyping of the methylenetetrahydrofolate reductase C677T gene polymorphism was performed by PCR-restriction fragment length polymorphism analysis, and homocysteine levels were measured by enzyme immunoassay. Fasting plasma homocysteine levels were significantly higher in cerebral venous thrombosis patients than in controls (P = 0.015). Moreover, plasma homocysteine levels were significantly higher in methylenetetrahydrofolate reductase 677TT genotype compared to 677CT and 677CC genotypes in both cerebral venous thrombosis patients (P = 0.01) and controls (P = 0.03). Neither 677CT heterozygote genotype [odds ratio (OR) 1.35, 95% confidence interval (CI) 0.64-2.84, P = 0.556] nor 677TT homozygote genotype (OR 1.73, 95% CI 0.32-9.21, P = 0.833) was significantly associated with cerebral venous thrombosis. Additionally, no significant differences in the frequency of 677T allele between cerebral venous thrombosis patients and controls were identified (OR 1.31, 95% CI 0.69-2.50, P = 0.512). In conclusion, our study demonstrated that elevated plasma homocysteine levels are significant risk factors for cerebral venous thrombosis. Also, methylenetetrahydrofolate reductase 677TT genotype is not linked with cerebral venous thrombosis, but is a determinant of elevated plasma homocysteine levels.

Physical and chemical stability of different formulations with superoxide dismutase.

Science.gov (United States)

Di Mambro, V M; Campos, P M B G Maia; Fonseca, M J V

2004-10-01

Topical formulations with superoxide dismutase (SOD), a scavenger of superoxide radicals, have proved to be effective against some skin diseases. Nevertheless, formulations with proteins are susceptible to both chemical and physical instability. Three different formulations (anionic and non-ionic gel and emulsion) were developed and supplemented with SOD in order to determine the most stable formulation that would maintain SOD activity. Physical stability was evaluated by assessing the rheological behavior of the formulations stored at room temperature, 37 and 45 degrees C. Chemical stability was evaluated by the measurement of enzymatic activity in the formulations stored at room temperature and at 45 degrees C. Formulations showed a flow index less than one, characterizing pseudoplastic behavior. There was no significant difference in initial values of flow index, tixotropy or minimum apparent viscosity. Neither gel showed significant changes in minimum apparent viscosity concerning storage time or temperature, as well, SOD presence and its activity. The emulsion showed decreased viscosity by the 28th day, but no significant changes concerning storage temperature or SOD presence, although it showed a decreased activity. The addition of SOD to the formulations studied did not affect their physical stability but gel formulations seem to be better bases for enzyme addition.

Neuronal uptake and intracellular superoxide scavenging of a fullerene (C60)-poly(2-oxazoline)s nanoformulation

KAUST Repository

Tong, Jing

2011-05-01

Fullerene, the third allotrope of carbon, has been referred to as a "radical sponge" because of its powerful radical scavenging activities. However, the hydrophobicity and toxicity associated with fullerene limits its application as a therapeutic antioxidant. In the present study, we sought to overcome these limitations by generating water-soluble nanoformulations of fullerene (C(60)). Fullerene (C(60)) was formulated with poly(N-vinyl pyrrolidine) (PVP) or poly(2-alkyl-2-oxazoline)s (POx) homopolymer and random copolymer to form nano-complexes. These C(60)-polymer complexes were characterized by UV-vis spectroscopy, infrared spectroscopy (IR), dynamic light scattering (DLS), atomic force microscopy (AFM) and transmission electron microscopy (TEM). Cellular uptake and intracellular distribution of the selected formulations in catecholaminergic (CATH.a) neurons were examined by UV-vis spectroscopy, immunofluorescence and immunogold labeling. Electron paramagnetic resonance (EPR) spectroscopy was used to determine the ability of these C(60)-polymer complexes to scavenge superoxide. Their cytotoxicity was evaluated in three different cell lines. C(60)-POx and C(60)-PVP complexes exhibited similar physicochemical properties and antioxidant activities. C(60)-poly(2-ethyl-2-oxazoline) (PEtOx) complex, but not C(60)-PVP complex, were efficiently taken up by CATH.a neurons and attenuated the increase in intra-neuronal superoxide induced by angiotensin II (Ang II) stimulation. These results show that C(60)-POx complexes are non-toxic, neuronal cell permeable, superoxide scavenging antioxidants that might be promising candidates for the treatment of brain-related diseases associated with increased levels of superoxide.

The structure of Lactococcus lactis thioredoxin reductase reveals molecular features of photo-oxidative damage

DEFF Research Database (Denmark)

Skjoldager, Nicklas; Bang, Maria Blanner; Rykær, Martin

2017-01-01

The NADPH-dependent homodimeric flavoenzyme thioredoxin reductase (TrxR) provides reducing equivalents to thioredoxin, a key regulator of various cellular redox processes. Crystal structures of photo-inactivated thioredoxin reductase (TrxR) from the Gram-positive bacterium Lactococcus lactis have...... been determined. These structures reveal novel molecular features that provide further insight into the mechanisms behind the sensitivity of this enzyme toward visible light. We propose that a pocket on the si-face of the isoalloxazine ring accommodates oxygen that reacts with photo-excited FAD...... thus be a widespread feature among bacterial TrxR with the described characteristics, which affords applications in clinical photo-therapy of drug-resistant bacteria....

Hydroxyurea-resistant vaccinia virus: overproduction of ribonucleotide reductase

International Nuclear Information System (INIS)

Slabaugh, M.B.; Mathews, C.K.

1986-01-01

Repeated passage of vaccinia virus in increasing concentrations of hydroxyurea followed by plaque purification resulted in the isolation of variants capable of growth in 5 mM hydroxyurea, a drug concentration which inhibited the reproduction of wild-type vaccinia virus 1000-fold. Analyses of viral protein synthesis by using [ 35 S]methionine pulse-labeling at intervals throughout the infection cycle revealed that all isolates overproduced a 34,000-molecular-weight (MW) early polypeptide. Measurement of ribonucleoside-diphosphate reductase activity after infection indicated that 4- to 10-fold more activity was induced by hydroxyurea-resistant viruses than by the wild-type virus. A two-step partial purification resulted in a substantial enrichment for the 34,000-MW protein from extracts of wild-type and hydroxyurea-resistant-virus-infected, but not mock-infected, cells. In the presence of the drug, the isolates incorporated [ 3 H]thymidine into DNA earlier and a rate substantially greater than that of the wild type, although the onset of DNA synthesis was delayed in both cases. The drug resistance trait was markedly unstable in all isolates. In the absence of selective pressure, plaque-purified isolated readily segregated progeny that displayed a wide range of resistance phenotypes. The results of this study indicate that vaccinia virus encodes a subunit of ribonucleotide reductase which is 34,000-MW early protein whose overproduction confers hydroxyurea resistance on reproducing viruses

Boletus edulis Nitrite Reductase Reduces Nitrite Content of Pickles and Mitigates Intoxication in Nitrite-intoxicated Mice.

Science.gov (United States)

Zhang, Weiwei; Tian, Guoting; Feng, Shanshan; Wong, Jack Ho; Zhao, Yongchang; Chen, Xiao; Wang, Hexiang; Ng, Tzi Bun

2015-10-08

Pickles are popular in China and exhibits health-promoting effects. However, nitrite produced during fermentation adversely affects health due to formation of methemoglobin and conversion to carcinogenic nitrosamine. Fruiting bodies of the mushroom Boletus edulis were capable of inhibiting nitrite production during pickle fermentation. A 90-kDa nitrite reductase (NiR), demonstrating peptide sequence homology to fungal nitrite reductase, was isolated from B. edulis fruiting bodies. The optimum temperature and pH of the enzyme was 45 °C and 6.8, respectively. B. edulis NiR was capable of prolonging the lifespan of nitrite-intoxicated mice, indicating that it had the action of an antidote. The enzyme could also eliminate nitrite from blood after intragastric administration of sodium nitrite, and after packaging into capsule, this nitrite-eliminating activity could persist for at least 120 minutes thus avoiding immediate gastric degradation. B. edulis NiR represents the first nitrite reductase purified from mushrooms and may facilitate subsequent applications.

Streptococcus sanguinis Class Ib Ribonucleotide Reductase

Science.gov (United States)

Makhlynets, Olga; Boal, Amie K.; Rhodes, DeLacy V.; Kitten, Todd; Rosenzweig, Amy C.; Stubbe, JoAnne

2014-01-01

Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (MnIII2-Y•) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and β subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with FeII and O2 can self-assemble a diferric-tyrosyl radical (FeIII2-Y•) cofactor (1.2 Y•/β2) and with the help of NrdI can assemble a MnIII2-Y• cofactor (0.9 Y•/β2). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdIox and MnII2-NrdF are reported and provide a possible rationale for the weak affinity (2.9 μm) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR. PMID:24381172

Structural studies of cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase, key enzymes of monolignol biosynthesis.

Science.gov (United States)

Pan, Haiyun; Zhou, Rui; Louie, Gordon V; Mühlemann, Joëlle K; Bomati, Erin K; Bowman, Marianne E; Dudareva, Natalia; Dixon, Richard A; Noel, Joseph P; Wang, Xiaoqiang

2014-09-01

The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. © 2014 American Society of Plant Biologists. All rights reserved.

Cancer cell death induced by phosphine gold(I) compounds targeting thioredoxin reductase.

Science.gov (United States)

Gandin, Valentina; Fernandes, Aristi Potamitou; Rigobello, Maria Pia; Dani, Barbara; Sorrentino, Francesca; Tisato, Francesco; Björnstedt, Mikael; Bindoli, Alberto; Sturaro, Alberto; Rella, Rocco; Marzano, Cristina

2010-01-15

The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH (nicotinamide adenine dinucleotide phosphate), plays a central role in regulating cellular redox homeostasis and signaling pathways. TrxR, overexpressed in many tumor cells and contributing to drug resistance, has emerged as a new target for anticancer drugs. Gold complexes have been validated as potent TrxR inhibitors in vitro in the nanomolar range. In order to obtain potent and selective TrxR inhibitors, we have synthesized a series of linear, 'auranofin-like' gold(I) complexes all containing the [Au(PEt(3))](+) synthon and the ligands: Cl(-), Br(-), cyanate, thiocyanate, ethylxanthate, diethyldithiocarbamate and thiourea. Phosphine gold(I) complexes efficiently inhibited cytosolic and mitochondrial TrxR at concentrations that did not affect the two related oxidoreductases glutathione reductase (GR) and glutathione peroxidase (GPx). The inhibitory effect of the redox proteins was also observed intracellularly in cancer cells pretreated with gold(I) complexes. Gold(I) compounds were found to induce antiproliferative effects towards several human cancer cells some of which endowed with cisplatin or multidrug resistance. In addition, they were able to activate caspase-3 and induce apoptosis observed as nucleosome formation and sub-G1 cell accumulation. The complexes with thiocyanate and xanthate ligands were particularly effective in inhibiting thioredoxin reductase and inducing apoptosis. Pharmacodynamic studies in human ovarian cancer cells allowed for the correlation of intracellular drug accumulation with TrxR inhibition that leads to the induction of apoptosis via the mitochondrial pathway.

Characterisation of PduS, the pdu metabolosome corrin reductase, and evidence of substructural organisation within the bacterial microcompartment.

Directory of Open Access Journals (Sweden)

Joshua B Parsons

2010-11-01

Full Text Available PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(IIIalamin to cob(IIalamin and cob(IIalamin to cob(Ialamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment.

X-ray crystal structure of GarR-tartronate semialdehyde reductase from Salmonella typhimurium.

Science.gov (United States)

Osipiuk, J; Zhou, M; Moy, S; Collart, F; Joachimiak, A

2009-09-01

Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related beta-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 A resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme.

Aerobic Swim Training Restores Aortic Endothelial Function by Decreasing Superoxide Levels in Spontaneously Hypertensive Rats

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Camila P. Jordão

Full Text Available OBJECTIVE: We aimed to determine whether aerobic training decreases superoxide levels, increases nitric oxide levels, and improves endothelium-dependent vasodilation in the aortas of spontaneously hypertensive rats. METHODS: Spontaneously hypertensive rats (SHR and Wistar Kyoto rats (WKY were distributed into 2 groups: sedentary (SHRsd and WKYsd, n=10 each and swimming-trained (SHRtr, n=10 and WKYtr, n=10, respectively. The trained group participated in training sessions 5 days/week for 1 h/day with an additional work load of 4% of the animal’s body weight. After a 10-week sedentary or aerobic training period, the rats were euthanized. The thoracic aortas were removed to evaluate the vasodilator response to acetylcholine (10-10 to 10-4 M with or without preincubation with L-NG-nitro-L-arginine methyl ester hydrochloride (L-NAME; 10-4 M in vitro. The aortic tissue was also used to assess the levels of the endothelial nitric oxide synthase and nicotinamide adenine dinucleotide oxidase subunit isoforms 1 and 4 proteins, as well as the superoxide and nitrite contents. Blood pressure was measured using a computerized tail-cuff system. RESULTS: Aerobic training significantly increased the acetylcholine-induced maximum vasodilation observed in the SHRtr group compared with the SHRsd group (85.9±4.3 vs. 71.6±5.2%. Additionally, in the SHRtr group, superoxide levels were significantly decreased, nitric oxide bioavailability was improved, and the levels of the nicotinamide adenine dinucleotide oxidase subunit isoform 4 protein were decreased compared to the SHRsd group. Moreover, after training, the blood pressure of the SHRtr group decreased compared to the SHRsd group. Exercise training had no effect on the blood pressure of the WKYtr group. CONCLUSIONS: In SHR, aerobic swim training decreased vascular superoxide generation by nicotinamide adenine dinucleotide oxidase subunit isoform 4 and increased nitric oxide bioavailability, thereby improving

[Aldose reductase gene polymorphism and rate of appearance of retinopathy in non insulin dependent diabetics].

Science.gov (United States)

Olmos, P; Acosta, A M; Schiaffino, R; Díaz, R; Alvarado, D; O'Brien, A; Muñoz, X; Arriagada, P; Claro, J C; Vega, R; Vollrath, V; Velasco, S; Emmerich, M; Maiz, A

1999-04-01

Recent studies suggest that polymorphisms associated to the aldose reductase gene could be related to early retinopathy in noninsulin dependent diabetics (NIDDM). There is also new interest on the genetic modulation of coagulation factors in relation to this complication. To look for a possible relationship between the rate of appearance of retinopathy and the genotype of (AC)n polymorphic marker associated to aldose reductase gene. A random sample of 27 NIDDM, aged 68.1 +/- 10.6 years, with a mean diabetes duration of 20.7 +/- 4.8 years and a mean glycosilated hemoglobin of 10.6 +/- 1.6%, was studied. The genotype of the (AC)n, polymorphic marker associated to the 5' end of the aldose reductase (ALR2) gene was determined by 32P-PCR plus sequenciation. Mutations of the factor XIII-A gene were studied by single stranded conformational polymorphism, sequenciation and restriction fragment length polymorphism. Four patients lacked the (AC)24 and had a higher rate of appearance of retinopathy than patients with the (AC)24 allele (0.0167 and 0.0907 score points per year respectively, p = 0.047). Both groups had similar glycosilated hemoglobin (11.7 +/- 0.2 and 10.5 +/- 1.6% respectively). Factor XIII gene mutations were not related to the rate of appearance of retinopathy. Our data suggest that the absence of the (AC)24 allele of the (AC)n polymorphic marker associated to the 5' end of the aldose reductase gene, is associated to a five fold reduction of retinopathy appearance rate.

A novel amperometric biosensor for superoxide anion based on superoxide dismutase immobilized on gold nanoparticle-chitosan-ionic liquid biocomposite film

International Nuclear Information System (INIS)

Wang Lu; Wen Wei; Xiong Huayu; Zhang Xiuhua; Gu Haoshuang; Wang Shengfu

2013-01-01

Graphical abstract: Schematic representation of the assembly process of SOD/GNPs-CS-IL/GCE. Highlights: ► SOD was immobilized in gold nanoparticles-chitosan-ionic liquid (GNPs-CS-IL) film. ► The biosensor was constructed by one-step ultrasonic electrodeposition of GNPs-CS-IL onto GCE. ► The biosensor showed excellent analytical performance for O 2 · − real-time analysis. - Abstract: A novel superoxide anion (O 2 · − ) biosensor is proposed based on the immobilization of copper-zinc superoxide dismutase (SOD) in a gold nanoparticle-chitosan-ionic liquid (GNPs-CS-IL) biocomposite film. The SOD-based biosensor was constructed by one-step ultrasonic electrodeposition of GNP-CS-IL composite onto glassy carbon electrode (GCE), followed by immobilization of SOD on the modified electrode. Surface morphologies of a set of representative films were characterized by scanning electron microscopy. The electrochemical performance of the biosensor was evaluated by cyclic voltammetry and chronoamperometry. A pair of quasi-reversible redox peaks of SOD with a formal potential of 0.257 V was observed at SOD/GNPs-CS-IL/GCE in phosphate buffer solution (PBS, 0.1 M, pH 7.0). The effects of varying test conditions on the electrochemical behavior of the biosensor were investigated. Furthermore, several electrochemical parameters were calculated in detail. Based on the biomolecule recognition of the specific reactivity of SOD toward O 2 · − , the developed biosensor exhibited a fast amperometric response ( 3 nM), low detection limit (1.7 nM), and excellent selectivity for the real-time measurement of O 2 · − . The proposed method is promising for estimating quantitatively the dynamic changes of O 2 · − in biological systems.

Superoxide produced in the matrix of mitochondria enhances methylmercury toxicity in human neuroblastoma cells

Energy Technology Data Exchange (ETDEWEB)

Mailloux, Ryan J.; Yumvihoze, Emmanuel; Chan, Hing Man, E-mail: laurie.chan@uottawa.ca

2015-12-15

The mechanism of intracellular metabolism of methylmercury (MeHg) is not fully known. It has been shown that superoxide (O{sub 2}·{sup −}), the proximal reactive oxygen species (ROS) generated by mitochondria, is responsible for MeHg demethylation. Here, we investigated the impact of different mitochondrial respiratory inhibitors, namely rotenone and antimycin A, on the O{sub 2}·{sup −} mediated degradation of MeHg in human neuroblastoma cells SH-K-SN. We also utilized paraquat (PQ) which generates O{sub 2}·{sup −} in the mitochondrial matrix. We found that the cleavage of the carbon-metal bond in MeHg was highly dependent on the topology of O{sub 2}·{sup −} production by mitochondria. Both rotenone and PQ, which increase O{sub 2}·{sup −} in the mitochondrial matrix at a dose-dependent manner, enhanced the conversion of MeHg to inorganic mercury (iHg). Surprisingly, antimycin A, which prompts emission of O{sub 2}·{sup −} into the intermembrane space, did not have the same effect even though antimycin A induced a dose dependent increase in O{sub 2}·{sup −} emission. Rotenone and PQ also enhanced the toxicity of sub-toxic doses (0.1 μM) MeHg which correlated with the accumulation of iHg in mitochondria and depletion of mitochondrial protein thiols. Taken together, our results demonstrate that MeHg degradation is mediated by mitochondrial O{sub 2}·{sup −}, specifically within the matrix of mitochondria when O{sub 2}·{sup −} is in adequate supply. Our results also show that O{sub 2}·{sup −} amplifies MeHg toxicity specifically through its conversion to iHg and subsequent interaction with protein cysteine thiols (R-SH). The implications of our findings in mercury neurotoxicity are discussed herein. - Highlights: • Superoxide produced in the matrix of mitochondria degrades MeHg. • Superoxide produced in intermembrane space does not degrade MeHg. • Matrix-generated superoxide enhances Hg toxicity by converting MeHg to iHg.

HMG-CoA reductase inhibitory activity and phytocomponent investigation of Basella alba leaf extract as a treatment for hypercholesterolemia

Directory of Open Access Journals (Sweden)

Baskaran G

2015-01-01

Full Text Available Gunasekaran Baskaran,1 Shamala Salvamani,1 Siti Aqlima Ahmad,1 Noor Azmi Shaharuddin,1 Parveen Devi Pattiram,2 Mohd Yunus Shukor1 1Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, 2Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, Selangor, Malaysia Abstract: The enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA reductase is the key enzyme of the mevalonate pathway that produces cholesterol. Inhibition of HMG-CoA reductase reduces cholesterol biosynthesis in the liver. Synthetic drugs, statins, are commonly used for the treatment of hypercholesterolemia. Due to the side effects of statins, natural HMG-CoA reductase inhibitors of plant origin are needed. In this study, 25 medicinal plant methanol extracts were screened for anti-HMG-CoA reductase activity. Basella alba leaf extract showed the highest inhibitory effect at about 74%. Thus, B. alba was examined in order to investigate its phytochemical components. Gas chromatography with tandem mass spectrometry and reversed phase high-performance liquid chromatography analysis revealed the presence of phenol 2,6-bis(1,1-dimethylethyl, 1-heptatriacotanol, oleic acid, eicosyl ester, naringin, apigenin, luteolin, ascorbic acid, and a-tocopherol, which have been reported to possess antihypercholesterolemic effects. Further investigation of in vivo models should be performed in order to confirm its potential as an alternative treatment for hypercholesterolemia and related cardiovascular diseases. Keywords: HMG-CoA reductase, Basella alba, phytochemical, GC-MS/MS, RP-HPLC, hypercholesterolemia

Manganese superoxide dismutase (MnSOD catalyzes NO-dependent tyrosine residue nitration

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SRDJAN STOJANOVIC

2005-04-01

Full Text Available The peroxynitrite-induced nitration of manganese superoxide dismutase (MnSOD tyrosine residue, which causes enzyme inactivation, is well established. This led to suggestions that MnSOD nitration and inactivation in vivo, detected in various diseases associated with oxidative stress and overproduction of nitric monoxide (NO, conditions which favor peroxynitrite formation, is also caused by peroxynitrite. However, our previous in vitro study demonstrated that exposure of MnSOD to NO led to NO conversion into nitrosonium (NO+ and nitroxyl (NO– species, which caused enzyme modifications and inactivation. Here it is reported that MnSOD is tyrosine nitrated upon exposure to NO, as well as that MnSOD nitration contributes to inactivation of the enzyme. Collectively, these observations provide a compelling argument supporting the generation of nitrating species in MnSOD exposed to NO and shed a new light on MnSOD tyrosine nitration and inactivation in vivo. This may represent a novel mechanism by which MnSOD protects cell from deleterious effects associated with overproduction of NO. However, extensive MnSOD modification and inactivation associated with prolonged exposure to NO will amplify the toxic effects caused by increased cell superoxide and NO levels.

Kinetics of mercury reduction by Serratia marcescens mercuric reductase expressed by pseudomonas putida strains

Energy Technology Data Exchange (ETDEWEB)

Schneider, M.; Deckwer, W.D. [GBF-Gesellschaft fuer Biotechnologische Forschung mbH, Abteilung TU-BCE, Mascheroder Weg 1, D-38124 Braunschweig (Germany)

2005-10-01

Mercury (Hg) resistance is widespread among microorganisms and is based on the intracellular transformation of Hg(II) to less toxic elemental Hg(0). The use of microbial consortia to demercurize polluted wastewater streams and environments has been demonstrated. To develop efficient and versatile microbial cleanup strategies requires detailed knowledge of transport and reaction rates. This study focuses on the kinetics of the key enzyme of the microbial transformation, e.g., the mercuric reductase (MerA) under conditions closely resembling the cell interior. To this end, previously constructed and characterized Pseudomonas putida strains expressing MerA from Serratia marcescens were applied. Of the P. putida strains considered in this study P. putida KT2442::mer73 constitutively expressing broad spectrum mercury resistance (merTPAB) yielded the highest mercuric reductase (MerA) activity directly after cell disruption. MerA in the raw extract was further purified (about 100 fold). Reduction rates were measured for various substrates (HgCl{sub 2}, Hg{sub 2}SO{sub 4}, Hg(NO{sub 3}){sub 2} and phenyl mercury acetate) up to high concentrations dependent on the purification grade. In all cases, a pronounced substrate inhibition was found. The kinetic constants determined for the cell raw extract are in agreement with those measured for intact cells. However, the rate data exhibit reduced affinity and inhibition with rising purification grade (specific activity). Therefore, the findings seemingly point to reactions preceding the catalytic reduction. Based on simplified assumptions, a kinetic model is suggested which reasonably describes the experimental findings and can advantageously be applied to the bioreactor design. (Abstract Copyright [2005], Wiley Periodicals, Inc.)

Two-stage gene regulation of the superoxide stress response soxRS system in Escherichia coli.

Science.gov (United States)

Nunoshiba, T

1996-01-01

All organisms have adapted to environmental changes by acquiring various functions controlled by gene regulation. In bacteria, a number of specific responses have been found to confer cell survival in various nutrient-limited conditions, and under physiological stresses such as high or low temperature, extreme pH, radiation, and oxidation (for review, see Neidhardt et al., 1987). In this article, I introduce an Escherichia coli (E. coli) global response induced by superoxide stress, the soxRS regulon. The functions controlled by this system consist of a wide variety of enzymes such as manganese-containing SOD (Mn-SOD); glucose 6-phosphate dehydrogenase (G6PD), the DNA repair enzyme endonuclease IV, fumarase C, NADPH:ferredoxin oxidoreductase, and aconitase. This response is positively regulated by a two-stage control system in which SoxR iron-sulfur protein senses exposure to superoxide and nitric oxide, and then activates transcription of the soxS gene, whose product stimulates the expression of the regulon genes. Our recent finding indicates that soxS transcription is initiated in a manner dependent on the rpoS gene encoding RNA polymerase sigma factor, theta s, in response to entering the stationary phase of growth. With this information, mechanisms for prokaryotic coordinating gene expression in response to superoxide stress and in stationary phase are discussed.

Genetic and Biochemical Analysis of Intragenic Complementation Events among Nitrate Reductase Apoenzyme-Deficient Mutants of Nicotiana Plumbaginifolia

OpenAIRE

Pelsy, F.; Gonneau, M.

1991-01-01

Intragenic complementation has been observed between apoenzyme nitrate reductase-deficient mutants (nia) of Nicotiana plumbaginifolia. In vivo as in vitro, the NADH-nitrate reductase (NR) activity in plants heterozygous for two different nia alleles was lower than in the wild type plant, but the plants were able to grow on nitrate as a sole nitrogen source. NR activity, absent in extracts of homozygous nia mutants was restored by mixing extracts from two complementing nia mutants. These obser...

Expression, purification, crystallization and preliminary X-ray analysis of maleylacetate reductase from Burkholderia sp. strain SJ98

International Nuclear Information System (INIS)

Chauhan, Archana; Islam, Zeyaul; Jain, Rakesh Kumar; Karthikeyan, Subramanian

2009-01-01

Purification and preliminary X-ray crystallographic analysis of maleylacetate reductase encoded by the pnpD gene is reported. Maleylacetate reductase (EC 1.3.1.32) is an important enzyme that is involved in the degradation pathway of aromatic compounds and catalyzes the reduction of maleylacetate to 3-oxoadipate. The gene pnpD encoding maleylacetate reductase in Burkholderia sp. strain SJ98 was cloned, expressed in Escherichia coli and purified by affinity chromatography. The enzyme was crystallized in both native and SeMet-derivative forms by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant at 293 K. The crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 72.91, b = 85.94, c = 53.07 Å. X-ray diffraction data for the native and SeMet-derivative crystal were collected to 2.7 and 2.9 Å resolution, respectively

Intracellular implantation of enzymes in hollow silica nanospheres for protein therapy: cascade system of superoxide dismutase and catalase.

Science.gov (United States)

Chang, Feng-Peng; Chen, Yi-Ping; Mou, Chung-Yuan

2014-11-01

An approach for enzyme therapeutics is elaborated with cell-implanted nanoreactors that are based on multiple enzymes encapsulated in hollow silica nanospheres (HSNs). The synthesis of HSNs is carried out by silica sol-gel templating of water-in-oil microemulsions so that polyethyleneimine (PEI) modified enzymes in aqueous phase are encapsulated inside the HSNs. PEI-grafted superoxide dismutase (PEI-SOD) and catalase (PEI-CAT) encapsulated in HSNs are prepared with quantitative control of the enzyme loadings. Excellent activities of superoxide dismutation by PEI-SOD@HSN are found and transformation of H2 O2 to water by PEI-CAT@HSN. When PEI-SOD and PEI-CAT are co-encapsulated, cascade transformation of superoxide through hydrogen peroxide to water was facile. Substantial fractions of HSNs exhibit endosome escape to cytosol after their delivery to cells. The production of downstream reactive oxygen species (ROS) and COX-2/p-p38 expression show that co-encapsulated SOD/CAT inside the HSNs renders the highest cell protection against the toxicant N,N'-dimethyl-4,4'-bipyridinium dichloride (paraquat). The rapid cell uptake and strong detoxification effect on superoxide radicals by the SOD/CAT-encapsulated hollow mesoporous silica nanoparticles demonstrate the general concept of implanting catalytic nanoreactors in biological cells with designed functions. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel twist on molecular interactions between thioredoxin and nicotinamide adenine dinucleotide phosphate-dependent thioredoxin reductase

DEFF Research Database (Denmark)

Kirkensgaard, Kristine Groth; Hägglund, Per; Shahpiri, Azar

2013-01-01

The ubiquitous disulfide reductase thioredoxin (Trx) regulates several important biological processes such as seed germination in plants. Oxidized cytosolic Trx is regenerated by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (NTR) in a multistep transfer...... dinucleotide (FAD)-binding domain of HvNTR2 to strongly affect the interaction with Trx. In particular, Trp42 and Met43 play key roles for recognition of the endogenous HvTrxh2. Trx from Arabidopsis thaliana is also efficiently recycled by HvNTR2 but turnover in this case appears to be less dependent...

Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis[C][W

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Pan, Haiyun; Zhou, Rui; Louie, Gordon V.; Mühlemann, Joëlle K.; Bomati, Erin K.; Bowman, Marianne E.; Dudareva, Natalia; Dixon, Richard A.; Noel, Joseph P.; Wang, Xiaoqiang

2014-01-01

The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. PMID:25217505

Understanding Free Radicals: Isolating Active Thylakoid Membranes and Purifying the Cytochrome b6f Complex for Superoxide Generation Studies

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Jason Stofleth

2012-01-01

Full Text Available All life persists in an environment that is rich in molecular oxygen. The production of oxygen free radicals, or superoxide, is a necessary consequence of the biogenesis of energy in cells. Both mitochondrial and photosynthetic electron transport chains have been found to produce superoxide associated with cell differentiation, proliferation, and cell death, thereby contributing to the effects of aging. Aerobic respiration in mitochondria consumes oxygen, whereas photosynthesis in chloroplasts or cyanobacteria produces oxygen. The increased concentration of molecular oxygen may serve to allow greater availability for the production of superoxide by cytochrome bc complexes in photosynthetic membranes compared to those of mitochondrial membranes. The isolation of well-coupled chloroplasts, containing the cytochrome b6f complex of oxygenic photosynthesis, is a vital initial step in the process of comparing the rate of production of superoxide to those of the homologous cytochrome bc1 complex of aerobic respiration. It is necessary to determine if the isolated chloroplasts have retained their oxygengenerating capability after isolation by an oxygen evolution assay with a Clark-type electrode. A necessary second step, which is the isolation of cytochrome b6f from spinach, has yet to be successfully performed. Oxygen measurements taken from chloroplasts in the presence of the uncoupler, NH4Cl, exhibited a rate of oxygen evolution over three times greater at 344 +/- 18 μmol O2/mg Chlorophyll a/hr than the rate of oxygen evolution without uncoupler at 109 +/- 29 μmol O2/mg Chlorophyll a/hr. These data demonstrate that the technique used to isolate spinach chloroplasts preserves their light-driven electron-transport activity, making them reliable for future superoxide assays.

Co-Immobilization of Superoxide Dismutase with Catalase on Soft Microparticles Formed by Self-Assembly of Amphiphilic Poly(Aspartic Acid

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Siyu Mao

2017-07-01

Full Text Available Through genetic engineering technology, catalase (CAT and superoxide dismutase (SOD have been separately fused to an elastin-like polypeptide (ELP. Thus, the enzymes can be purified through phase transition. Hexadecylamine-modified poly(aspartic acid (HPASP is able to self-assemble, forming soft microparticles. The HPASP microparticles were used to co-immobilize SOD-ELP and CAT-ELP through amidation reaction. Circular dichroism (CD confirmed that the secondary structures of the co-immobilized enzymes have been preserved. Fluorescence spectra showed that the co-immobilized enzymes exhibited a higher stability than the free enzymes. Dismutation of superoxide by superoxide dismutase (SOD generates hydrogen peroxide. By using the co-immobilized enzymes (SOD-ELP/CAT-ELP@HPASP, the generated hydrogen peroxide of SOD-ELP can be decomposed in situ by CAT-ELP. Activity assay results demonstrated that the superoxide anion (•O2− scavenging ability is 63.15 ± 0.75% for SOD-ELP/CAT-ELP@HPASP. The advantages of the approach of enzyme co-immobilization include the fact that the soft support HPASP itself is a polypeptide in nature, the stability of immobilized enzymes is improved, and a high activity has been achieved. Potentially SOD-ELP/CAT-ELP@HPASP can be applied in the cosmetic industry.

Pichia stipitis xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF

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Röder Anja

2008-06-01

Full Text Available Abstract Background Pichia stipitis xylose reductase (Ps-XR has been used to design Saccharomyces cerevisiae strains that are able to ferment xylose. One example is the industrial S. cerevisiae xylose-consuming strain TMB3400, which was constructed by expression of P. stipitis xylose reductase and xylitol dehydrogenase and overexpression of endogenous xylulose kinase in the industrial S. cerevisiae strain USM21. Results In this study, we demonstrate that strain TMB3400 not only converts xylose, but also displays higher tolerance to lignocellulosic hydrolysate during anaerobic batch fermentation as well as 3 times higher in vitro HMF and furfural reduction activity than the control strain USM21. Using laboratory strains producing various levels of Ps-XR, we confirm that Ps-XR is able to reduce HMF both in vitro and in vivo. Ps-XR overexpression increases the in vivo HMF conversion rate by approximately 20%, thereby improving yeast tolerance towards HMF. Further purification of Ps-XR shows that HMF is a substrate inhibitor of the enzyme. Conclusion We demonstrate for the first time that xylose reductase is also able to reduce the furaldehyde compounds that are present in undetoxified lignocellulosic hydrolysates. Possible implications of this newly characterized activity of Ps-XR on lignocellulosic hydrolysate fermentation are discussed.

Transgenic mice with increased Cu/Zn-superoxide dismutase activity: animal model of dosage effects in Down syndrome

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Epstein, C.J.; Avraham, K.B.; Lovett, M.; Smith, S.; Elroy-Stein, O.; Rotman, G.; Bry, C.; Groner, Y.

1987-01-01

Down syndrome, the phenotypic expression of human trisomy 21, is presumed to result from a 1.5-fold increase in the expression of the genes on human chromosome 21. As an approach to the development of an animal model for Down syndrome, several strains of transgenic mice that carry the human Cu/Zn-superoxide dismutase gene have been prepared. The animals express the transgene in a manner similar to that of humans, with 0.9- and 0.7-kilobase transcripts in a 1:4 ratio, and synthesize the human enzyme in an active form capable of forming human-mouse enzyme heterodimers. Cu/Zn-superoxide dismutase activity is increased from 1.6- to 6.0-fold in the brains of four transgenic strains and to an equal or lesser extent in several other tissues. These animals provide a unique system for studying the consequences of increased dosage of the Cu/Zn-superoxide dismutase gene in Down syndrome and the role of this enzyme in a variety of other pathological processes

X-Ray crystal structure of GarR—tartronate semialdehyde reductase from Salmonella typhimurium

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Osipiuk, J.; Zhou, M.; Moy, S.; Collart, F.

2009-01-01

Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 Å resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme. PMID:19184529

Normal bone density in male pseudohermaphroditism due to 5a- reductase 2 deficiency

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Costa Elaine Maria Frade

2001-01-01

Full Text Available Bone is an androgen-dependent tissue, but it is not clear whether the androgen action in bone depends on testosterone or on dihydrotestosterone. Patients with 5alpha-reductase 2 deficiency present normal levels of testosterone and low levels of dihydrotestosterone, providing an in vivo human model for the analysis of the effect of testosterone on bone. OBJECTIVE: To analyze bone mineral density in 4 adult patients with male pseudohermaphroditism due to 5alpha-reductase 2 deficiency. RESULTS: Three patients presented normal bone mineral density of the lumbar column (L1-L4 and femur neck, and the other patient presented a slight osteopenia in the lumbar column. CONCLUSION: Patients with dihydrotestosterone deficiency present normal bone mineral density, suggesting that dihydrotestosterone is not the main androgen acting in bone.

The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism.

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Stenmark, Pål; Moche, Martin; Gurmu, Daniel; Nordlund, Pär

2007-10-12

We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.

Activation of CuZn superoxide dismutases from Caenorhabditis elegans does not require the copper chaperone CCS.

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Jensen, Laran T; Culotta, Valeria Cizewski

2005-12-16

Reactive oxygen species are produced as the direct result of aerobic metabolism and can cause damage to DNA, proteins, and lipids. A principal defense against reactive oxygen species involves the superoxide dismutases (SOD) that act to detoxify superoxide anions. Activation of CuZn-SODs in eukaryotic cells occurs post-translationally and is generally dependent on the copper chaperone for SOD1 (CCS), which inserts the catalytic copper cofactor and catalyzes the oxidation of a conserved disulfide bond that is essential for activity. In contrast to other eukaryotes, the nematode Caenorhabditis elegans does not contain an obvious CCS homologue, and we have found that the C. elegans intracellular CuZn-SODs (wSOD-1 and wSOD-5) are not dependent on CCS for activation when expressed in Saccharomyces cerevisiae. CCS-independent activation of CuZn-SODs is not unique to C. elegans; however, this is the first organism identified that appears to exclusively use this alternative pathway. As was found for mammalian SOD1, wSOD-1 exhibits a requirement for reduced glutathione in CCS-independent activation. Unexpectedly, wSOD-1 was inactive even in the presence of CCS when glutathione was depleted. Our investigation of the cysteine residues that form the disulfide bond in wSOD-1 suggests that the ability of wSODs to readily form this disulfide bond may be the key to obtaining high levels of activation through the CCS-independent pathway. Overall, these studies demonstrate that the CuZn-SODs of C. elegans have uniquely evolved to acquire copper without the copper chaperone and this may reflect the lifestyle of this organism.

Determination of superoxide dismutase mimetic activity in common culinary herbs.

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Chohan, Magali; Naughton, Declan P; Opara, Elizabeth I

2014-01-01

Under conditions of oxidative stress, the removal of superoxide, a free radical associated with chronic inflammation, is catalysed by superoxide dismutase (SOD). Thus in addition to acting as an antioxidant, SOD may also be utilized as an anti-inflammatory agent. Some plant derived foods have been shown to have SOD mimetic (SODm) activity however it is not known if this activity is possessed by culinary herbs which have previously been shown to possess both antioxidant and anti-inflammatory properties. The aim of the study was to ascertain if the culinary herbs rosemary, sage and thyme possess SODm activity, and to investigate the influence of cooking and digestion on this activity. Transition metal ion content was also determined to establish if it could likely contribute to any SODm activity detected. All extracts of uncooked (U), cooked (C) and cooked and digested (C&D) herbs were shown to possess SODm activity, which was significantly correlated with previously determined antioxidant and anti-inflammatory activities of these herbs. SODm activity was significantly increased following (C) and (C&D) for rosemary and sage only. The impact of (C) and (C&D) on the SODm for thyme may have been influenced by its transition metal ion content. SODm activity may contribute to the herbs' antioxidant and anti-inflammatory activities however the source and significance of this activity need to be established.

Oxygen-dependent radiosensitivity of Escherichia coli and mitigation in lethality by superoxide dismutase

International Nuclear Information System (INIS)

Niwa, Taeko; Yamaguchi, Hikoyuki; Yano, Keiji

1978-01-01

Oxygen-dependent radiosensitivity of Escherichia coli W3623 his - was confirmed. Regarding cellular superoxide dismutase (SOD), cells grown oxically gained higher activity than those anoxically, however, the reinforced enzyme level could not compensate the oxygen effect, i.e., the enhanced lethal effect of oxic γ-irradiation. Rather, the enhancement of oxygen effect was found in cells grown oxically compared with those anoxically. Oxygen enhanced lethality was mitigated to the extent by the amount of added SOD into the cell suspension to be irradiated. The results supported a proposal that superoxide anion, O 2 - , is involved in the oxygen effect, with the most likely site of the damage in the outer structure of cell but not in the cell matrix. Reverse oxygen effect could be found with lambda phage DNA in transfecting ability. Added SOD protected phage DNA somewhat in oxic irradiation. While considerable protections were found in anoxic one with the added SOD even autoclaved but their function was still unknown. (auth.)

Superoxide dismutase: an industrial perspective.

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Bafana, Amit; Dutt, Som; Kumar, Sanjay; Ahuja, Paramvir S

2011-03-01

The application of enzyme technologies to industrial research, development, and manufacturing has become a very important field. Since the production of crude rennet in 1874, several enzymes have been commercialized, and used for therapeutic, supplementary, and other applications. Recent advancements in biotechnology now allow companies to produce safer and less expensive enzymes with enhanced potency and specificity. Antioxidant enzymes are emerging as a new addition to the pool of industrial enzymes and are surpassing all other enzymes in terms of the volume of research and production. In the 1990s, an antioxidant enzyme--superoxide dismutase (SOD)--was introduced into the market. Although the enzyme initially showed great promise in therapeutic applications, it did not perform up to expectations. Consequently, its use was limited to non-drug applications in humans and drug applications in animals. This review summarizes the rise and fall of SOD at the industrial level, the reasons for this, and potential future thrust areas that need to be addressed. The review also focuses on other industrially relevant aspects of SOD such as industrial importance, enzyme engineering, production processes, and process optimization and scale-up.

ADP-ribosylation of dinitrogenase reductase in Rhodobacter capsulatus

International Nuclear Information System (INIS)

Jouanneau, Y.; Roby, C.; Meyer, C.M.; Vignais, P.M.

1989-01-01

In the photosynthetic bacterium Rhodobacter capsulatus, nitrogenase is regulated by a reversible covalent modification of Fe protein or dinitrogenase reductase (Rc2). The linkage of the modifying group to inactive Rc2 was found to be sensitive to alkali and to neutral hydroxylamine. Complete release of the modifying group was achieved by incubation of inactive Rc2 in 0.4 or 1 M hydroxylamine. After hydroxylamine treatment of the Rc2 preparation, the modifying group could be isolated and purified by affinity chromatography and ion-exchange HPLC. The modifying group comigrated with ADP-ribose on both ion-exchange HPLC and thin-layer chromatography. Analyses by 31 P NMR spectroscopy and mass spectrometry provided further evidence that the modifying group was ADP-ribose. The NMR spectrum of inactive Rc2 exhibited signals characteristic of ADP-ribose; integration of these signals allowed calculation of a molar ration ADP-ribose/Rc2 of 0.63. A hexapeptide carrying the ADP-ribose moiety was purified from a subtilisin digest of inactive Rc2. The structure of this peptide, determined by amino acid analysis and sequencing, is Gly-Arg(ADP-ribose)-Gly-Val-Ile-Thr. This structure allows identification of the binding site for ADP-ribose as Arg 101 of the polypeptide chain of Rc2. It is concluded that nitrogenase activity in R. capsulatus is regulated by reversible ADP-ribosylation of a specific arginyl residue of dinitrogenase reductase

Inhibition of 5α-Reductase, IL-6 Secretion, and Oxidation Process of Equisetum debile Roxb. ex Vaucher Extract as Functional Food and Nutraceuticals Ingredients

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Wantida Chaiyana

2017-10-01

Full Text Available This study aims to investigate the biological activities related to hair loss of Equisetum debile extracts, including 5α-reductase inhibition, interleukin-6 (IL-6 secretion reduction, and anti-oxidation. E. debile extracts were obtained by maceration in various solvents. Crude extract (CE was obtained by maceration in 95% ethanol. Chlorophyll-free extract (CF was the CE which of the chlorophyll has been removed by electrocoagulation. Hexane extract (HE, ethyl acetate extract (EA, and ethanolic extract (ET were fraction extracts obtained from maceration in hexane, ethyl acetate, and 95% ethanol, respectively. The extracts were investigated for inhibitory activity against 5α-reductase and IL-6 secretion. Total phenolic contents (TPC were investigated and antioxidant activities were determined by means of 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, 2,2′-diphenyl-1-picrylhydrazyl (DPPH, and ferric reducing antioxidant power (FRAP assays. The inhibition of lipid peroxidation was determined by the ferric thiocyanate method. The cytotoxicity of the extracts on dermal papilla cells and irritation test by hen's egg test chorioallantoic membrane assay were also investigated. All extracts could inhibit 5α-reductase and decrease IL-6 secretion in lipopolysaccharide-stimulated macrophage. The antioxidant activity of E. debile extracts was directly related to their TPC. ET which contained the highest TPC (68.8 ± 6.7 mg GA/g showed the highest equivalent concentration (EC1 of 289.1 ± 26.4 mM FeSO4/g, TEAC of 156.6 ± 34.6 mM Trolox/g, and 20.0 ± 6.0% DPPH inhibition. However, EA exhibited the highest inhibition against lipid peroxidation (57.2 ± 0.4%. In addition, EA showed no cytotoxicity on dermal papilla cell line and no irritation on chorioallantoic membrane of hen’s eggs. In conclusion, EA was suggested as the most attractive ingredients for functional food and nutraceuticals because of the high inhibitory activity against 5α-reductase

Superoxide Anions and NO in the Paraventricular Nucleus Modulate the Cardiac Sympathetic Afferent Reflex in Obese Rats

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Qing-Bo Lu

2017-12-01

Full Text Available This study was conducted to explore the hypothesis that the endogenous superoxide anions (O2− and nitric oxide (NO system of the paraventricular nucleus (PVN regulates the cardiac sympathetic afferent reflex (CSAR contributing to sympathoexcitation in obese rats induced by a high-fat diet (42% kcal as fat for 12 weeks. CSAR was evaluated by monitoring the changes of renal sympathetic nerve activity (RSNA and the mean arterial pressure (MAP responses to the epicardial application of capsaicin (CAP in anaesthetized rats. In obese rats with hypertension (OH group or without hypertension (OB group, the levels of PVN O2−, angiotensinII (Ang II, Ang II type 1 receptor (AT1R, and nicotinamide adenine dinucleotide phosphate (NADPH oxidase were elevated, whereas neural NO synthase (nNOS and NO were significantly reduced. Moreover, CSAR was markedly enhanced, which promoted the elevation of plasma norepinephrine levels. The enhanced CSAR was attenuated by PVN application of the superoxide scavenger polyethylene glycol-superoxide dismutase (PEG-SOD and the NO donor sodium nitroprusside (SNP, and was strengthened by the superoxide dismutase inhibitor diethyldithiocarbamic acid (DETC and the nNOS inhibitor N(ω-propyl-l-arginine hydrochloride (PLA; conversely, there was a smaller CSAR response to PLA or SNP in rats that received a low-fat (12% kcal diet. Furthermore, PVN pretreatment with the AT1R antagonist losartan or with PEG-SOD, but not SNP, abolished Ang II-induced CSAR enhancement. These findings suggest that obesity alters the PVN O2− and NO system that modulates CSAR and promotes sympathoexcitation.

A genetic screen reveals a periplasmic copper chaperone required for nitrite reductase activity in pathogenic Neisseria.

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Jen, Freda E-C; Djoko, Karrera Y; Bent, Stephen J; Day, Christopher J; McEwan, Alastair G; Jennings, Michael P

2015-09-01

Under conditions of low oxygen availability, Neisseria meningitidis and Neisseria gonorrhoeae are able to respire via a partial denitrification pathway in which nitrite is converted to nitrous oxide. In this process, nitrite reductase (AniA), a copper (Cu)-containing protein converts nitrite to NO, and this product is converted to nitrous oxide by nitric oxide reductase (NorB). NorB also confers protection against toxic NO, and so we devised a conditional lethal screen, using a norB mutant, to identify mutants that were resistant to nitrite-dependent killing. After random-deletion mutagenesis of N. meningitidis, this genetic screen identified a gene encoding a Cu chaperone that is essential for AniA function, AccA. Purified AccA binds one Cu (I) ion and also possesses a second binding site for Cu (II). This novel periplasmic Cu chaperone (AccA) appears to be essential for provision of Cu ions to AniA of pathogenic Neisseria to generate an active nitrite reductase. Apart from the Neisseria genus, AccA is distributed across a wide range of environmental Proteobacteria species. © FASEB.

Increased superoxide accumulation in pyruvate dehydrogenase complex deficient fibroblasts.

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Glushakova, Lyudmyla G; Judge, Sharon; Cruz, Alex; Pourang, Deena; Mathews, Clayton E; Stacpoole, Peter W

2011-11-01

The pyruvate dehydrogenase complex (PDC) oxidizes pyruvate to acetyl CoA and is critically important in maintaining normal cellular energy homeostasis. Loss-of-function mutations in PDC give rise to congenital lactic acidosis and to progressive cellular energy failure. However, the subsequent biochemical consequences of PDC deficiency that may contribute to the clinical manifestations of the disorder are poorly understood. We postulated that altered flux through PDC would disrupt mitochondrial electron transport, resulting in oxidative stress. Compared to cells from 4 healthy subjects, primary cultures of skin fibroblasts from 9 patients with variable mutations in the gene encoding the alpha subunit (E1α) of pyruvate dehydrogenase (PDA1) demonstrated reduced growth and viability. Superoxide (O(2)(.-)) from the Qo site of complex III of the electron transport chain accumulated in these cells and was associated with decreased activity of manganese superoxide dismutase. The expression of uncoupling protein 2 was also decreased in patient cells, but there were no significant changes in the expression of cellular markers of protein or DNA oxidative damage. The expression of hypoxia transcription factor 1 alpha (HIF1α) also increased in PDC deficient fibroblasts. We conclude that PDC deficiency is associated with an increase in O(2)(.-) accumulation coupled to a decrease in mechanisms responsible for its removal. Increased HIF1α expression may contribute to the increase in glycolytic flux and lactate production in PDC deficiency and, by trans-activating pyruvate dehydrogenase kinase, may further suppress residual PDC activity through phosphorylation of the E1α subunit. Copyright © 2011 Elsevier Inc. All rights reserved.

Aldose Reductase-Deficient Mice Develop Nephrogenic Diabetes Insipidus

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Ho, Horace T. B.; Chung, Sookja K.; Law, Janice W. S.; Ko, Ben C. B.; Tam, Sidney C. F.; Brooks, Heddwen L.; Knepper, Mark A.; Chung, Stephen S. M.

2000-01-01

Aldose reductase (ALR2) is thought to be involved in the pathogenesis of various diseases associated with diabetes mellitus, such as cataract, retinopathy, neuropathy, and nephropathy. However, its physiological functions are not well understood. We developed mice deficient in this enzyme and found that they had no apparent developmental or reproductive abnormality except that they drank and urinated significantly more than their wild-type littermates. These ALR2-deficient mice exhibited a partially defective urine-concentrating ability, having a phenotype resembling that of nephrogenic diabetes insipidus. PMID:10913167

Parasitization by Scleroderma guani influences expression of superoxide dismutase genes in Tenebrio molitor

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Superoxide dismutase (SOD) is an antioxidant enzyme involved in detoxifying reactive oxygen species. In this study, we identified genes encoding the extracellular and intracellular copper-zinc SODs (ecCuZnSOD and icCuZnSOD) and a manganese SOD (MnSOD) in the yellow mealworm beetle, Tenebrio molitor....

Low-temperature phase transformation in rubidium and cesium superoxides

International Nuclear Information System (INIS)

Alikhanov, R.A.; Toshich, B.S.; Smirnov, L.S.

1980-01-01

Crystal structures of rubidium and cesium superoxides which are two interpenetrating lattices of metal ions and oxygen molecule ions reveal a number of phase transformations with temperature decrease. Crystal-phase transformations in CsO 2 are 1-2, 2-3 and low temperature one 3-4 at 378, 190 and 10 K. Low temperature transition is considered as the instability of lattice quadrupoles of oxygen molecule ions to phase transformation of the order-disorder type. Calculated temperatures of low temperature phase transformations in PbO 2 and CsO 2 agree with experimental calculations satisfactory [ru

X-ray structural studies of quinone reductase 2 nanomolar range inhibitors

Energy Technology Data Exchange (ETDEWEB)

Pegan, Scott D.; Sturdy, Megan; Ferry, Gilles; Delagrange, Philippe; Boutin, Jean A.; Mesecar, Andrew D. (IdRS); (Purdue); (Colorado); (UIC)

2011-09-06

Quinone reductase 2 (QR2) is one of two members comprising the mammalian quinone reductase family of enzymes responsible for performing FAD mediated reductions of quinone substrates. In contrast to quinone reductase 1 (QR1) which uses NAD(P)H as its co-substrate, QR2 utilizes a rare group of hydride donors, N-methyl or N-ribosyl nicotinamide. Several studies have linked QR2 to the generation of quinone free radicals, several neuronal degenerative diseases, and cancer. QR2 has been also identified as the third melatonin receptor (MT3) through in cellulo and in vitro inhibition of QR2 by traditional MT3 ligands, and through recent X-ray structures of human QR2 (hQR2) in complex with melatonin and 2-iodomelatonin. Several MT3 specific ligands have been developed that exhibit both potent in cellulo inhibition of hQR2 nanomolar, affinity for MT3. The potency of these ligands suggest their use as molecular probes for hQR2. However, no definitive correlation between traditionally obtained MT3 ligand affinity and hQR2 inhibition exists limiting our understanding of how these ligands are accommodated in the hQR2 active site. To obtain a clearer relationship between the structures of developed MT3 ligands and their inhibitory properties, in cellulo and in vitro IC{sub 50} values were determined for a representative set of MT3 ligands (MCA-NAT, 2-I-MCANAT, prazosin, S26695, S32797, and S29434). Furthermore, X-ray structures for each of these ligands in complex with hQR2 were determined allowing for a structural evaluation of the binding modes of these ligands in relation to the potency of MT3 ligands.

Ebselen: A thioredoxin reductase-dependent catalyst for α-tocopherol quinone reduction

International Nuclear Information System (INIS)

Fang Jianguo; Zhong Liangwei; Zhao Rong; Holmgren, Arne

2005-01-01

The thioredoxin system, composed of thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH, is a powerful protein disulfide reductase system with a broad substrate specificity. Recently the selenazol drug ebselen was shown to be a substrate for both mammalian TrxR and Trx. We examined if α-tocopherol quinone (TQ), a product of α-tocopherol oxidation, is reduced by ebselen in the presence of TrxR, since TQ was not a substrate for the enzyme itself. Ebselen reduction of TQ in the presence of TrxR was caused by ebselen selenol, generated from fast reduction of ebselen by the enzyme. TQ has no intrinsic antioxidant activity, while the product of reduction of TQ, α-tocopherolhydroquinone (TQH 2 ), is a potent antioxidant. The thioredoxin system dependence of ebselen to catalyze reduction of other oxidized species, such as hydrogen peroxide, dehydroascorbate, and peroxynitrite, is discussed. The ability of ebselen to reduce TQ via the thioredoxin system is a novel mechanism to explain the effects of the drug as an antioxidant in vivo

Protective effect of Pterocarpus marsupium bark extracts against cataract through the inhibition of aldose reductase activity in streptozotocin-induced diabetic male albino rats.

Science.gov (United States)

Xu, YanLi; Zhao, Yongxia; Sui, YaNan; Lei, XiaoJun

2018-04-01

The present study was aimed to investigate the protective effect of Pterocarpus marsupium bark extracts against cataract in streptozotocin-induced diabetic male albino rats. Aldose reductase is a key enzyme in the intracellular polyol pathway, which plays a major role in the development of diabetic cataract. Rats were divided into five groups as normal control, diabetic control, and diabetic control treated with different concentrations of Pterocarpus marsupium bark extracts. Presence of major constituents in Pterocarpus marsupium bark extract was performed by qualitative analysis. Body weight changes, blood glucose, blood insulin, and reduced glutathione (GSH) and aldose reductase mRNA and protein expression were determined. Rat body weight gain was noted following treatment with bark extracts. The blood glucose was reduced up to 36% following treatment with bark extracts. The blood insulin and tissue GSH contents were substantially increased more than 100% in diabetic rats following treatment with extracts. Aldose reductase activity was reduced up to 79.3% in diabetic rats following treatment with extracts. V max , K m , and K i of aldose reductase were reduced in the lens tissue homogenate compared to the diabetic control. Aldose reductase mRNA and protein expression were reduced more than 50% following treatment with extracts. Treatment with Pterocarpus marsupium bark was able to normalize these levels. Taking all these data together, it is concluded that the use of Pterocarpus marsupium bark extracts could be the potential therapeutic approach for the reduction of aldose reductase against diabetic cataract.

Metal bacteriochlorins which act as dual singlet oxygen and superoxide generators.

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Fukuzumi, Shunichi; Ohkubo, Kei; Zheng, Xiang; Chen, Yihui; Pandey, Ravindra K; Zhan, Riqiang; Kadish, Karl M

2008-03-06

A series of stable free-base, Zn(II) and Pd(II) bacteriochlorins containing a fused six- or five-member diketo- or imide ring have been synthesized as good candidates for photodynamic therapy sensitizers, and their electrochemical, photophysical, and photochemical properties were examined. Photoexcitation of the palladium bacteriochlorin affords the triplet excited state without fluorescence emission, resulting in formation of singlet oxygen with a high quantum yield due to the heavy atom effect of palladium. Electrochemical studies revealed that the zinc bacteriochlorin has the smallest HOMO-LUMO gap of the investigated compounds, and this value is significantly lower than the triplet excited-state energy of the compound in benzonitrile. Such a small HOMO-LUMO gap of the zinc bacteriochlorin enables intermolecular photoinduced electron transfer from the triplet excited state to the ground state to produce both the radical cation and the radical anion. The radical anion thus produced can transfer an electron to molecular oxygen to produce superoxide anion which was detected by electron spin resonance. The same photosensitizer can also act as an efficient singlet oxygen generator. Thus, the same zinc bacteriochlorin can function as a sensitizer with a dual role in that it produces both singlet oxygen and superoxide anion in an aprotic solvent (benzonitrile).

Soybean and tempeh total isoflvones improved antioxidant activities in normal and scopolamine-induced rat brain

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Aliya Ahmad

2015-11-01

Full Text Available Objective: To highlight the comparative studies between total isoflavone extracts from soybean and tempeh on the neuronal oxidative stress and antioxidant activities. Methods: The total isoflavones were administered orally for 15 days with 3 selected doses (10, 20 and 40 mg/kg. Piracetam (400 mg/kg, p.o. was used as a standard drug while scopolamine (1 mg/kg, i.p. was used as a drug that promoted amnesia in selected groups. The oxidative markers (thiobarbituric acid reactive substances and nitric oxide were measured in brain homogenate. The antioxidant activities evaluated were catalase, superoxide dismutase, glutathione reductase and glutathione. Results: Our results showed that soybean and tempeh isoflavones significantly improved the levels of catalase, superoxide dismutase, glutathione reductase and glutathione while decreased levels of thiobarbituric acid reactive substances and nitric oxide in both the brain of normal as well as scopolamine-induced animals. Conclusions: Our findings suggested that soybean and tempeh isoflavones could be useful in the management and prevention of age-related neurodegenerative changes including Alzheimer’s disease through its antioxidant activities.

Manganese superoxide dismutase and breast cancer recurrence

DEFF Research Database (Denmark)

Cronin-Fenton, Deirdre P; Christensen, Mariann; Lash, Timothy L

2014-01-01

BACKGROUND: Manganese superoxide dismutase (MnSOD) inhibits oxidative damage and cancer therapy effectiveness. A polymorphism in its encoding gene (SOD2: Val16Ala rs4880) may confer poorer breast cancer survival, but data are inconsistent. We examined the association of SOD2 genotype and breast......-metastatic breast cancer from 1990-2001, received adjuvant Cyclo, and were registered in the Danish Breast Cancer Cooperative Group. We identified 118 patients with BCR and 213 matched breast cancer controls. We genotyped SOD2 and used conditional logistic regression to compute the odds ratio (OR) and associated 95...... cancer recurrence (BCR) among patients treated with cyclophosphamide-based chemotherapy (Cyclo). We compared our findings with published studies using meta-analyses. METHODS: We conducted a population-based case-control study of BCR among women in Jutland, Denmark. Subjects were diagnosed with non...

NADPH-dependent D-aldose reductases and xylose fermentation in Fusarium oxysporum

DEFF Research Database (Denmark)

Panagiotou, Gianni; Christakopoulos, P.

2004-01-01

Two aldose (xylose) reductases (ARI and ARII) from Fusarium oxysporum were purified and characterized. The native ARI was a monomer with M-r 41000, pI 5.2 and showed a 52-fold preference for NADPH over NADH, while ARII was homodimeric with a subunit of M-r 37000, pI 3.6 and a 60-fold preference...

Piper betle shows antioxidant activities, inhibits MCF-7 cell proliferation and increases activities of catalase and superoxide dismutase

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Abrahim Noor

2012-11-01

Full Text Available Abstract Background Breast cancer is the most common form of cancer and the focus on finding chemotherapeutic agents have recently shifted to natural products. Piper betle is a medicinal plant with various biological activities. However, not much data is available on the anti-cancer effects of P. betle on breast cancer. Due to the current interest in the potential effects of antioxidants from natural products in breast cancer treatment, we investigated the antioxidant activities of the leaves of P. betle and its inhibitory effect on the proliferation of the breast cancer cell line, MCF-7. Methods The leaves of P. betle were extracted with solvents of varying polarities (water, methanol, ethyl acetate and hexane and their phenolic and flavonoid content were determined using colorimetric assays. Phenolic composition was characterized using HPLC. Antioxidant activities were measured using FRAP, DPPH, superoxide anion, nitric oxide and hyroxyl radical scavenging assays. Biological activities of the extracts were analysed using MTT assay and antioxidant enzyme (catalase, superoxide dismutase, glutathione peroxidase assays in MCF-7 cells. Results Overall, the ethyl acetate extract showed the highest ferric reducing activity and radical scavenging activities against DPPH, superoxide anion and nitric oxide radicals. This extract also contained the highest phenolic content implying the potential contribution of phenolics towards the antioxidant activities. HPLC analyses revealed the presence of catechin, morin and quercetin in the leaves. The ethyl acetate extract also showed the highest inhibitory effect against the proliferation of MCF-7 cells (IC50=65 μg/ml. Treatment of MCF-7 cells with the plant extract increased activities of catalase and superoxide dismutase. Conclusions Ethyl acetate is the optimal solvent for the extraction of compounds with antioxidant and anti-proliferative activities. The increased activities of catalase and superoxide

Piper betle shows antioxidant activities, inhibits MCF-7 cell proliferation and increases activities of catalase and superoxide dismutase.

Science.gov (United States)

Abrahim, Noor Nazirahanie; Kanthimathi, M S; Abdul-Aziz, Azlina

2012-11-15

Breast cancer is the most common form of cancer and the focus on finding chemotherapeutic agents have recently shifted to natural products. Piper betle is a medicinal plant with various biological activities. However, not much data is available on the anti-cancer effects of P. betle on breast cancer. Due to the current interest in the potential effects of antioxidants from natural products in breast cancer treatment, we investigated the antioxidant activities of the leaves of P. betle and its inhibitory effect on the proliferation of the breast cancer cell line, MCF-7. The leaves of P. betle were extracted with solvents of varying polarities (water, methanol, ethyl acetate and hexane) and their phenolic and flavonoid content were determined using colorimetric assays. Phenolic composition was characterized using HPLC. Antioxidant activities were measured using FRAP, DPPH, superoxide anion, nitric oxide and hyroxyl radical scavenging assays. Biological activities of the extracts were analysed using MTT assay and antioxidant enzyme (catalase, superoxide dismutase, glutathione peroxidase) assays in MCF-7 cells. Overall, the ethyl acetate extract showed the highest ferric reducing activity and radical scavenging activities against DPPH, superoxide anion and nitric oxide radicals. This extract also contained the highest phenolic content implying the potential contribution of phenolics towards the antioxidant activities. HPLC analyses revealed the presence of catechin, morin and quercetin in the leaves. The ethyl acetate extract also showed the highest inhibitory effect against the proliferation of MCF-7 cells (IC50=65 μg/ml). Treatment of MCF-7 cells with the plant extract increased activities of catalase and superoxide dismutase. Ethyl acetate is the optimal solvent for the extraction of compounds with antioxidant and anti-proliferative activities. The increased activities of catalase and superoxide dismutase in the treated cells could alter the antioxidant defense

Influence of acute and chronic administration of methadone hydrochloride on NADPH-cytochrome c reductase and cytochrome P-450 of mouse liver microsomes.

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Datta, R K; Johnson, E A; Bhattacharjee, G; Stenger, R J

1976-03-01

Administration of a single acute dose (20 mg/kg body weight) of methadone hydrochloride to both male and female mice increased the specific activity of NADPH-cytochrome c reductase and did not change much the content of cytochrome P-450 of their liver microsomes. Administration of multiple acute doses of methadone in male mice increased the specific activity of cytochrome c reductase and the content of cytochrome P-450 of their liver microsomes. Chronic administration of progressively increasing doses of methadone (up to 40 mg/kg body weight) to male mice increased the specific activity of c reductase. Similar chronic administration of methadone up to 28 mg/kg body weight also increased the microsomal content of P-450, but with higher doses of methadone, the content of P-450 declined and finally dropped slightly below control levels. The levels of c reductase activity and P-450 content returned to normal about two weeks after discontinuation of methadone administration.

Unexpected ethical dilemmas in sex assignment in 46,XY DSD due to 5-alpha reductase type 2 deficiency.

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Byers, Heather M; Mohnach, Lauren H; Fechner, Patricia Y; Chen, Ming; Thomas, Inas H; Ramsdell, Linda A; Shnorhavorian, Margarett; McCauley, Elizabeth A; Amies Oelschlager, Anne-Marie E; Park, John M; Sandberg, David E; Adam, Margaret P; Keegan, Catherine E

2017-06-01

Sex assignment at birth remains one of the most clinically challenging and controversial topics in 46,XY disorders of sexual development (DSD). This is particularly challenging in deficiency of 5-alpha reductase type 2 given that external genitalia are typically undervirilized at birth but typically virilize at puberty to a variable degree. Historically, most individuals with 5-alpha reductase deficiency were raised females. However, reports that over half of patients who underwent a virilizing puberty adopted an adult male gender identity have challenged this practice. Consensus guidelines on assignment of sex of rearing at birth are equivocal or favor male assignment in the most virilized cases. While a male sex of rearing assignment may avoid lifelong hormonal therapy and/or allow the potential for fertility, female sex assignment may be more consistent with external anatomy in the most severely undervirilized cases. Herein, we describe five patients with 46,XY DSD due 5-alpha-reductase type 2 deficiency, all with a severe phenotype. An inter-disciplinary DSD medical team at one of two academic centers evaluated each patient. This case series illustrates the complicated decision-making process of assignment of sex of rearing at birth in 5-alpha reductase type 2 deficiency and the challenges that arise when the interests of the child, parental wishes, recommendations of the medical team, and state law collide. © 2017 Wiley Periodicals, Inc.

Acute Superoxide Radical Scavenging Reduces Blood Pressure but Does Not Influence Kidney Function in Hypertensive Rats with Postischemic Kidney Injury

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Zoran Miloradović

2014-01-01

Full Text Available Acute kidney injury (AKI is associated with significant morbidity and mortality in hypertensive surroundings. We investigated superoxide radical molecules influence on systemic haemodynamic and kidney function in spontaneously hypertensive rats (SHR with induced postischemic AKI. Experiment was performed in anesthetized adult male SHR. The right kidney was removed, and left renal artery was subjected to ischemia by clamping for 40 minutes. The treated group received synthetic superoxide dismutase mimetic TEMPOL in the femoral vein 5 minutes before, during, and 175 minutes after the period of reperfusion, while the control AKI group received the vehicle via the same route. All parameters were measured 24 h after renal reperfusion. TEMPOL treatment significantly decreased mean arterial pressure and total peripheral resistance P<0.05 compared to AKI control. It also increased cardiac output and catalase activity P<0.05. Lipid peroxidation and renal vascular resistance were decreased in TEMPOL P<0.05. Plasma creatinine and kidney morphological parameters were unchanged among TEMPOL treated and control groups. Our study shows that superoxide radicals participate in haemodynamic control, but acute superoxide scavenging is ineffective in glomerular and tubular improvement, probably due to hypertension-induced strong endothelial dysfunction which neutralizes beneficial effects of O2− scavenging.

Martian Superoxide and Peroxide O2 Release (OR) Assay: A New Technology for Terrestrial and Planetary Applications

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Georgiou, Christos D.; Zisimopoulos, Dimitrios; Panagiotidis, Konstantinos; Grintzalis, Kontantinos; Papapostolou, Ioannis; Quinn, Richard C.; McKay, Christopher P.; Sun, Henry J.

2015-01-01

This study presents an assay for the detection and quantification of soil metal superoxides and peroxides in regolith and soil. The O2 release (OR) assay is based on the enzymatic conversion of the hydrolysis products of metal oxides to O2, and their quantification by an O2 electrode based on the stoichiometry of the involved reactions: The intermediate product O2 from the hydrolysis of metal superoxides is converted by cytochrome c to O2, and also by superoxide dismutase (SOD) to 1/2 mol O2 and 1/2 mol H2O2, which is then converted by catalase (CAT) to 1/2 mol O2. The product H2O2 from the hydrolysis of metal peroxides and hydroperoxides is converted to 1/2 mol O2 by CAT. The assay-method was validated in a sealed sample chamber using a liquid-phase Clark-type O2 electrode with known concentrations of O2 and H2O2, and with commercial metal superoxide and peroxide mixed with Mars analogue Mojave and Atacama Desert soils. Carbonates and perchlorates, both present on Mars, do not interfere with the assay. The assay lower limit of detection, using luminescence quenching/optical sensing O2-electrodes, is 1 nmol O2 cm(exp. -3) or better. The activity of the assay enzymes SOD and cytochrome c was unaffected up to 6 Gy exposure by gamma-radiation, while CAT retained 100% and 40% of its activity at 3 and 6 Gy, respectively, demonstrating the suitability of these enzymes for planetary missions, e.g., in Mars or Europa.

The Cu,Zn Superoxide Dismutase: not only a dismutase enzyme

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Paolo Mondola

2016-11-01

Full Text Available The Cu,Zn superoxide dismutase (SOD1 is an ubiquitary cytosolic dimeric carbohydrate free molecule, belonging to a family of isoenzymes involved in the scavenger of superoxide anions. This effect certainly represents the main and well known function ascribed to this enzyme. Here we highlight new aspects of SOD1 physiology that point out some inedited effects of this enzyme in addition to the canonic role of oxygen radical enzymatic dismutation. In the last two decades our research group produced many data obtained in in vitro studies performed in many cellular lines, mainly neuroblastoma SK-N-BE cells, indicating that this enzyme is secreted either constitutively or after depolarization induced by high extracellular K+ concentration. In addition, we gave many experimental evidences showing that SOD1 is able to stimulate, through muscarinic M1 receptor, pathways involving ERK1/2 and AKT activation. These effects are accompanied with an intracellular calcium increase. In the last part of this review we describe researches that link deficient extracellular secretion of mutant SOD1G93A to its intracellular accumulation and toxicity in NSC-34 cells. Alternatively, SOD1G93A toxicity has been attributed to a decrease of Km for H2O2 with consequent OH. radical formation. Interestingly, this last inedited effect of SOD1G93A could represent a gain of function that could be involved in the pathogenesis of familial Amyotrophic Lateral Sclerosis (fALS.

Variation and inheritance of iron reductase activity in the roots of common bean (Phaseolus vulgaris L.) and association with seed iron accumulation QTL.

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Blair, Matthew W; Knewtson, Sharon Jb; Astudillo, Carolina; Li, Chee-Ming; Fernandez, Andrea C; Grusak, Michael A

2010-10-05

Iron deficiency anemia is a global problem which often affects women and children of developing countries. Strategy I plants, such as common bean (Phaseolus vulgaris L.) take up iron through a process that involves an iron reduction mechanism in their roots; this reduction is required to convert ferric iron to ferrous iron. Root absorbed iron is critical for the iron nutrition of the plant, and for the delivery of iron to the shoot and ultimately the seeds. The objectives of this study were to determine the variability and inheritance for iron reductase activity in a range of genotypes and in a low × high seed iron cross (DOR364 x G19833), to identify quantitative trait loci (QTL) for this trait, and to assess possible associations with seed iron levels. The experiments were carried out with hydroponically grown plants provided different amounts of iron varying between 0 and 20 μM Fe(III)-EDDHA. The parents, DOR364 and G19833, plus 13 other cultivated or wild beans, were found to differ in iron reductase activity. Based on these initial experiments, two growth conditions (iron limited and iron sufficient) were selected as treatments for evaluating the DOR364 × G19833 recombinant inbred lines. A single major QTL was found for iron reductase activity under iron-limited conditions (1 μM Fe) on linkage group b02 and another major QTL was found under iron sufficient conditions (15 μM Fe) on linkage group b11. Associations between the b11 QTL were found with several QTL for seed iron. Genes conditioning iron reductase activity in iron sufficient bean plants appear to be associated with genes contributing to seed iron accumulation. Markers for bean iron reductase (FRO) homologues were found with in silico mapping based on common bean synteny with soybean and Medicago truncatula on b06 and b07; however, neither locus aligned with the QTL for iron reductase activity. In summary, the QTL for iron reductase activity under iron limited conditions may be useful in

Variation and inheritance of iron reductase activity in the roots of common bean (Phaseolus vulgaris L. and association with seed iron accumulation QTL

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Fernandez Andrea C

2010-10-01

Full Text Available Abstract Background Iron deficiency anemia is a global problem which often affects women and children of developing countries. Strategy I plants, such as common bean (Phaseolus vulgaris L. take up iron through a process that involves an iron reduction mechanism in their roots; this reduction is required to convert ferric iron to ferrous iron. Root absorbed iron is critical for the iron nutrition of the plant, and for the delivery of iron to the shoot and ultimately the seeds. The objectives of this study were to determine the variability and inheritance for iron reductase activity in a range of genotypes and in a low × high seed iron cross (DOR364 × G19833, to identify quantitative trait loci (QTL for this trait, and to assess possible associations with seed iron levels. Results The experiments were carried out with hydroponically grown plants provided different amounts of iron varying between 0 and 20 μM Fe(III-EDDHA. The parents, DOR364 and G19833, plus 13 other cultivated or wild beans, were found to differ in iron reductase activity. Based on these initial experiments, two growth conditions (iron limited and iron sufficient were selected as treatments for evaluating the DOR364 × G19833 recombinant inbred lines. A single major QTL was found for iron reductase activity under iron-limited conditions (1 μM Fe on linkage group b02 and another major QTL was found under iron sufficient conditions (15 μM Fe on linkage group b11. Associations between the b11 QTL were found with several QTL for seed iron. Conclusions Genes conditioning iron reductase activity in iron sufficient bean plants appear to be associated with genes contributing to seed iron accumulation. Markers for bean iron reductase (FRO homologues were found with in silico mapping based on common bean synteny with soybean and Medicago truncatula on b06 and b07; however, neither locus aligned with the QTL for iron reductase activity. In summary, the QTL for iron reductase activity

Alteration in expression of defence genes in Pisum sativum after exposure to supplementary ultraviolet-B radiation

International Nuclear Information System (INIS)

Strid, A.

1993-01-01

Alterations in the amounts of mRNA for different types of defence genes after exposure of peas to supplementary ultraviolet-B radiation are demonstrated. The expression of the genes which encode the chalcone synthase of the flavonoid biosynthetic pathway and glutathione reductase was induced, while a decrease was found for the chloroplastic radical-scavenging enzyme, superoxide dismutase. (author)

Reduced superoxide dismutase activity in xeroderma pigmentosum fibroblasts

International Nuclear Information System (INIS)

Nishigori, C.; Miyachi, Y.; Imamura, S.; Takebe, H.

1989-01-01

This study was performed in order to assess the possible protective effect of superoxide dismutase (SOD) on ultraviolet (UV) damage in xeroderma pigmentosum (XP) fibroblasts. SOD activity in fibroblasts originating from seven xeroderma pigmentosum (XP) patients was significantly lower than that in normal cells (p less than 0.005). Average SOD activity in XP cells belonging to complementation group A was 3.68 +/- 0.54 (n = 7) and that in normal human cells was 5.79 +/- 1.59 (n = 6). Addition of SOD before and during UV irradiation (UVB and UVC) to the cells caused no change in the amount of unscheduled DNA synthesis and UV survival. A possible involvement of reduced SOD in XP and a possible protective effect by SOD on UV damage is discussed

Fatty acyl-CoA reductases of birds

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Hellenbrand Janine

2011-12-01

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.

Fatty acyl-CoA reductases of birds

Science.gov (United States)

2011-01-01

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. PMID:22151413

HV1 acts as a sodium sensor and promotes superoxide production in medullary thick ascending limb of Dahl salt-sensitive rats.

Science.gov (United States)

Jin, Chunhua; Sun, Jingping; Stilphen, Carly A; Smith, Susan M E; Ocasio, Hiram; Bermingham, Brent; Darji, Sandip; Guha, Avirup; Patel, Roshan; Geurts, Aron M; Jacob, Howard J; Lambert, Nevin A; O'Connor, Paul M

2014-09-01

We previously characterized a H(+) transport pathway in medullary thick ascending limb nephron segments that when activated stimulated the production of superoxide by nicotinamide adenine dinucleotide phosphate oxidase. Importantly, the activity of this pathway was greater in Dahl salt-sensitive rats than salt-resistant (SS.13(BN)) rats, and superoxide production was enhanced in low Na(+) media. The goal of this study was to determine the molecular identity of this pathway and its relationship to Na(+). We hypothesized that the voltage-gated proton channel, HV1, was the source of superoxide-stimulating H(+) currents. To test this hypothesis, we developed HV1(-/-) null mutant rats on the Dahl salt-sensitive rat genetic background using zinc-finger nuclease gene targeting. HV1 could be detected in medullary thick limb from wild-type rats. Intracellular acidification using an NH4Cl prepulse in 0 sodium/BaCl2 containing media resulted in superoxide production in thick limb from wild-type but not HV1(-/-) rats (Pthick limb and peritoneal macrophages only when HV1 was present. When fed a high-salt diet, blood pressure, outer medullary renal injury (tubular casts), and oxidative stress (4-hydroxynonenal staining) were significantly reduced in HV1(-/-) rats compared with wild-type Dahl salt-sensitive rats. We conclude that HV1 is expressed in medullary thick ascending limb and promotes superoxide production in this segment when intracellular Na(+) is low. HV1 contributes to the development of hypertension and renal disease in Dahl salt-sensitive rats. © 2014 American Heart Association, Inc.

Use of 5-alpha-reductase inhibitors did not increase the risk of cardiovascular diseases in patients with benign prostate hyperplasia: a five-year follow-up study.

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Teng-Fu Hsieh

Full Text Available This nationwide population-based study investigated the risk of cardiovascular diseases after 5-alpha-reductase inhibitor therapy for benign prostate hyperplasia (BPH using the National Health Insurance Research Database (NHIRD in Taiwan.In total, 1,486 adult patients newly diagnosed with BPH and who used 5-alpha-reductase inhibitors were recruited as the study cohort, along with 9,995 subjects who did not use 5-alpha-reductase inhibitors as a comparison cohort from 2003 to 2008. Each patient was monitored for 5 years, and those who subsequently had cardiovascular diseases were identified. A Cox proportional hazards model was used to compare the risk of cardiovascular diseases between the study and comparison cohorts after adjusting for possible confounding risk factors.The patients who received 5-alpha-reductase inhibitor therapy had a lower cumulative rate of cardiovascular diseases than those who did not receive 5-alpha-reductase inhibitor therapy during the 5-year follow-up period (8.4% vs. 11.2%, P=0.003. In subgroup analysis, the 5-year cardiovascular event hazard ratio (HR was lower among the patients older than 65 years with 91 to 365 cumulative defined daily dose (cDDD 5-alpha-reductase inhibitor use (HR=0.63, 95% confidence interval (CI 0.42 to 0.92; P=0.018, however there was no difference among the patients with 28 to 90 and more than 365 cDDD 5-alpha-reductase inhibitor use (HR=1.14, 95% CI 0.77 to 1.68; P=0.518 and HR=0.83, 95% CI 0.57 to 1.20; P=0.310, respectively.5-alpha-reductase inhibitor therapy did not increase the risk of cardiovascular events in the BPH patients in 5 years of follow-up. Further mechanistic research is needed.

Crystallization and preliminary crystallographic analysis of selenomethionine-labelled progesterone 5β-reductase from Digitalis lanata Ehrh

Energy Technology Data Exchange (ETDEWEB)

Egerer-Sieber, Claudia [Lehrstuhl für Biotechnik, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Henkestrasse 91, D-91052 Erlangen (Germany); Herl, Vanessa; Müller-Uri, Frieder; Kreis, Wolfgang [Lehrstuhl für Pharmazeutische Biologie, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen (Germany); Muller, Yves A., E-mail: ymuller@biologie.uni-erlangen.de [Lehrstuhl für Biotechnik, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Henkestrasse 91, D-91052 Erlangen (Germany)

2006-03-01

Progesterone 5β-reductase is the first stereospecific enzyme in the pathway for the synthesis of cardenolides. To elucidate the structural mechanism of this reaction, we crystallized the selenomethionine-labelled enzyme from D. lanata and report the preliminary analysis of a MAD data set collected from these crystals. Progesterone 5β-reductase (5β-POR) catalyzes the reduction of progesterone to 5β-pregnane-3,20-dione and is the first stereospecific enzyme in the putative biosynthetic pathway of Digitalis cardenolides. Selenomethionine-derivatized 5β-POR from D. lanata was successfully overproduced and crystallized. The crystals belong to space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = 71.73, c = 186.64 Å. A MAD data set collected at 2.7 Å resolution allowed the identification of six out of eight possible Se-atom positions. A first inspection of the MAD-phased electron-density map shows that 5β-POR is a Rossmann-type reductase and the quality of the map is such that it is anticipated that a complete atomic model of 5β-POR will readily be built.

Superoxide dismutases in chronic gastritis.

Science.gov (United States)

Švagelj, Dražen; Terzić, Velimir; Dovhanj, Jasna; Švagelj, Marija; Cvrković, Mirta; Švagelj, Ivan

2016-04-01

Human gastric diseases have shown significant changes in the activity and expression of superoxide dismutase (SOD) isoforms. The aim of this study was to detect Mn-SOD activity and expression in the tissue of gastric mucosa, primarily in chronic gastritis (immunohistochemical Helicobacter pylori-negative gastritis, without other pathohistological changes) and to evaluate their possible connection with pathohistological diagnosis. We examined 51 consecutive outpatients undergoing endoscopy for upper gastrointestinal symptoms. Patients were classified based on their histopathological examinations and divided into three groups: 51 patients (archive samples between 2004-2009) with chronic immunohistochemical Helicobacter pylori-negative gastritis (mononuclear cells infiltration were graded as absent, moderate, severe) divided into three groups. Severity of gastritis was graded according to the updated Sydney system. Gastric tissue samples were used to determine the expression of Mn-SOD with anti-Mn-SOD Ab immunohistochemically. The Mn-SOD expression was more frequently present in specimens with severe and moderate inflammation of gastric mucosa than in those with normal mucosa. In patients with normal histological finding, positive immunoreactivity of Mn-SOD was not found. Our results determine the changes in Mn-SOD expression occurring in the normal gastric mucosa that had undergone changes in the intensity of chronic inflammatory infiltrates in the lamina propria. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Brevetoxin-2, is a unique inhibitor of the C-terminal redox center of mammalian thioredoxin reductase-1.

Science.gov (United States)

Chen, Wei; Tuladhar, Anupama; Rolle, Shantelle; Lai, Yanhao; Rodriguez Del Rey, Freddy; Zavala, Cristian E; Liu, Yuan; Rein, Kathleen S

2017-08-15

Karenia brevis, the Florida red tide dinoflagellate produces a suite of neurotoxins known as the brevetoxins. The most abundant of the brevetoxins PbTx-2, was found to inhibit the thioredoxin-thioredoxin reductase system, whereas the PbTx-3 has no effect on this system. On the other hand, PbTx-2 activates the reduction of small disulfides such as 5,5'-dithio-bis-(2-nitrobenzoic acid) by thioredoxin reductase. PbTx-2 has an α, β-unsaturated aldehyde moiety which functions as an efficient electrophile and selenocysteine conjugates are readily formed. PbTx-2 blocks the inhibition of TrxR by the inhibitor curcumin, whereas curcumin blocks PbTx-2 activation of TrxR. It is proposed that the mechanism of inhibition of thioredoxin reduction is via the formation of a Michael adduct between selenocysteine and the α, β-unsaturated aldehyde moiety of PbTx-2. PbTx-2 had no effect on the rates of reactions catalyzed by related enzymes such as glutathione reductase, glutathione peroxidase or glutaredoxin. Copyright © 2017 Elsevier Inc. All rights reserved.

The 5 Alpha-Reductase Isozyme Family: A Review of Basic Biology and Their Role in Human Diseases

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Faris Azzouni

2012-01-01

Full Text Available Despite the discovery of 5 alpha-reduction as an enzymatic step in steroid metabolism in 1951, and the discovery that dihydrotestosterone is more potent than testosterone in 1968, the significance of 5 alpha-reduced steroids in human diseases was not appreciated until the discovery of 5 alpha-reductase type 2 deficiency in 1974. Affected males are born with ambiguous external genitalia, despite normal internal genitalia. The prostate is hypoplastic, nonpalpable on rectal examination and approximately 1/10th the size of age-matched normal glands. Benign prostate hyperplasia or prostate cancer does not develop in these patients. At puberty, the external genitalia virilize partially, however, secondary sexual hair remains sparse and male pattern baldness and acne develop rarely. Several compounds have been developed to inhibit the 5 alpha-reductase isozymes and they play an important role in the prevention and treatment of many common diseases. This review describes the basic biochemical properties, functions, tissue distribution, chromosomal location, and clinical significance of the 5 alpha-reductase isozyme family.

Cloning, Expression, Characterization, and Computational Approach for Cross-Reactivity Prediction of Manganese Superoxide Dismutase Allergen from Pistachio Nut

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Reihaneh Noorbakhsh

Full Text Available ABSTRACT: Background: Tree nut allergy is one of the common potentially life-threatening food allergies in children and adults. Recombinant food allergens offer new perspectives to solve problems of clinical and molecular allergology in diagnosis, research, and therapy of food allergies. So far, superoxide dismutase (s has been identified as a panallergen and studied in different allergenic sources. Manganese Superoxide Dismutase (MnSOD has also been reported in pistachio that may cause allergic reactions in atopic subjects. The aim of this study was to describe the cloning, expression, and purification of MnSOD from pistachio nut. Methods: The pistachio MnSOD was cloned and expressed in E. coli BL21 (DE3 using a vector pET-32b (+. A recombinant protein was purified by metal precipitation. The protein immunoreactivity was evaluated using patients' IgE binding by means of ELISA and immunoblotting assays. Results: The MnSOD gene from pistachio was successfully cloned and expressed in E. coli. The purified pistachio MnSOD was recognized by IgE in 10 (40% out of the 25 sera tested. Our results also showed that this protein might trigger some cross-reactions toward IgE antibodies and thus could be considered as a panallergen. Conclusions: For the first time recombinant manganese superoxide dismutase from nut source was expressed as a possible allergen. This pistachio allergen could be a possible basis for cross-reactivity with MnSOD from other sources. KEY WORDS: cloning, cross-reaction, Manganese Superoxide Dismutase (MnSOD, pistachio (Pistacia vera, recombinant allergen

Superoxide Dismutase as a Tool for the Mulacular Identification of Plant Parasitic Nematodes

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S. Molinari

2004-08-01

Full Text Available Superoxide dismutase (SOD is a constitutive family of enzymes produced by all aerobic organisms. Varying amounts of SOD activity have been found at all life stages of the most diffused plant parasitic nematodes. SOD is important to aerobic metabolism and parasitism of nematodes in that it catalyzes the first step of the neutralization of the highly toxic superoxide anion (O2 •-, which is largely produced in plant-nematode incompatible reactions. SOD has also been shown to be a significant tool to diagnose root-knot, cyst-, and longidorid nematodes. A high SOD polymorphism has been revealed by Native-Page on gradient polyacrylamide gels for Meloidogyne spp. and by isoelectrofocusing for Globodera, Xiphinema and Longidorus spp. The sensitivity of such procedures has been improved by using the PhastSystem (Amersham Biosciences, Piscata, NJ, USA, an automated equipment for electrophoresis. An accurate discrimination of species of all the nematode genera tested has been achieved and an attempt was made to group populations of the Xiphinema americanum-group and to detect Globodera rostochiensis and G. pallida pathotypes.

Paroxysmal atrial fibrillation: dynamics of the main antioxidant enzymes--superoxide dismutase and catalase.

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Negreva, Mariya N; Penev, Atanas P; Georgiev, Svetoslav Zh; Aleksandrova, Albena A

2014-01-01

Researchers have a particularly strong interest in the mechanisms implicated in the clinical manifestation of atrial fibrillation. To examine dynamically the activity of the antioxidant enzymes, superoxide dismutase and catalase in patients with paroxysmal atrial fibrillation (duration enzyme activity was determined by a spectrophotometric method. The average duration of atrial fibrillation episodes until the time of hospitalization was 8.14 hours (from 2 to 24 hours). During patient hospitalization the activity of superoxide dismutase and catalase was considerably higher compared to that of the controls (8.46 +/- 0.26 vs 5.81 +/- 0.14 U/mg Hb; 7.36 +/- 0.25 vs 4.76 +/- 0.12 E240/min/mg Hb; P catalase remained increased (5.11 +/- 0.08 vs 4.76 +/- 0.12 E240/min/mg Hb, p catalase even in the early hours of clinical manifestation of the disorder, which then slowly decreased with the restoration of sinus rhythm. Therefore, we can conclude that changes in oxidative status are closely related to the disease and are probably a part of the intimate mechanisms related to its initiation and clinical course.

Crystallization and preliminary characterization of dihydropteridine reductase from Dictyostelium discoideum

International Nuclear Information System (INIS)

Chen, Cong; Seo, Kyung Hye; Kim, Hye Lim; Zhuang, Ningning; Park, Young Shik; Lee, Kon Ho

2008-01-01

The dihydropteridine reductase from D. discoideum has been crystallized. Diffraction data were collected from a rectangular-shaped crystal to 2.16 Å resolution. Dihydropteridine reductase from Dictyostelium discoideum (dicDHPR) can produce d-threo-BH 4 [6R-(1′R,2′R)-5,6,7,8-tetrahydrobiopterin], a stereoisomer of l-erythro-BH 4 , in the last step of tetrahydrobiopterin (BH 4 ) recycling. In this reaction, DHPR uses NADH as a cofactor to reduce quinonoid dihydrobiopterin back to BH 4 . To date, the enzyme has been purified to homogeneity from many sources. In this report, the dicDHPR–NAD complex has been crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as a precipitant. Rectangular-shaped crystals were obtained. Crystals grew to maximum dimensions of 0.4 × 0.6 × 0.1 mm. The crystal belonged to space group P2 1 , with unit-cell parameters a = 49.81, b = 129.90, c = 78.76 Å, β = 100.00°, and contained four molecules in the asymmetric unit, forming two closely interacting dicDHPR–NAD dimers. Diffraction data were collected to 2.16 Å resolution using synchrotron radiation. The crystal structure has been determined using the molecular-replacement method

Superoxide scavenging activity of pirfenidone-iron complex

International Nuclear Information System (INIS)

Mitani, Yoshihiro; Sato, Keizo; Muramoto, Yosuke; Karakawa, Tomohiro; Kitamado, Masataka; Iwanaga, Tatsuya; Nabeshima, Tetsuji; Maruyama, Kumiko; Nakagawa, Kazuko; Ishida, Kazuhiko; Sasamoto, Kazumi

2008-01-01

Pirfenidone (PFD) is focused on a new anti-fibrotic drug, which can minimize lung fibrosis etc. We evaluated the superoxide (O 2 ·- ) scavenging activities of PFD and the PFD-iron complex by electron spin resonance (ESR) spectroscopy, luminol-dependent chemiluminescence assay, and cytochrome c reduction assay. Firstly, we confirmed that the PFD-iron complex was formed by mixing iron chloride with threefold molar PFD, and the complex was stable in distillated water and ethanol. Secondary, the PFD-iron complex reduced the amount of O 2 ·- produced by xanthine oxidase/hypoxanthine without inhibiting the enzyme activity. Thirdly, it also reduced the amount of O 2 ·- released from phorbor ester-stimulated human neutrophils. PFD alone showed few such effects. These results suggest the possibility that the O 2 ·- scavenging effect of the PFD-iron complex contributes to the anti-fibrotic action of PFD used for treating idiopathic pulmonary fibrosis

Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms

International Nuclear Information System (INIS)

Yagi, T.

1987-01-01

The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD). This inhibition correlated with the presence of an energy-transducing site in this segment of the respiratory chain. Where the NADH-quinone reductase segment involved an energy-coupling site (e.g., in bovine heart and rat liver mitochondria, and in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes), DCCD acted as an inhibitor of ubiquinone reduction by NADH. By contrast, where energy-coupling site 1 was absent (e.g., in Saccharomyces cerevisiae mitochondria and BacilLus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by DCCD. In the bovine and P. denitrificans systems, DCCD inhibition was pseudo first order with respect to incubation time, and reaction order with respect to inhibitor concentration was close to unity, indicating that inhibition resulted from the binding of one inhibitor molecule per active unit of NADH-ubiquinone reductase. In the bovine NADH-ubiquinone reductase complex (complex I), [ 14 C]DCCD was preferentially incorporated into two subunits of molecular weight 49,000 and 29,000. The time course of labeling of the 29,000 molecular weight subunit with [ 14 C]DCCD paralleled the time course of inhibition of NADH-ubiquinone reductase activity

Inhibition of thioredoxin reductase but not of glutathione reductase by the major classes of alkylating and platinum-containing anticancer compounds.

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Witte, Anne-Barbara; Anestål, Karin; Jerremalm, Elin; Ehrsson, Hans; Arnér, Elias S J

2005-09-01

Mammalian thioredoxin reductase (TrxR) is important for cell proliferation, antioxidant defense, and redox signaling. Together with glutathione reductase (GR) it is the main enzyme providing reducing equivalents to many cellular processes. GR and TrxR are flavoproteins of the same enzyme family, but only the latter is a selenoprotein. With the active site containing selenocysteine, TrxR may catalyze reduction of a wide range of substrates, but can at the same time easily be targeted by electrophilic compounds due to the extraordinarily high reactivity of a selenolate moiety. Here we addressed the inhibition of the enzyme by major anticancer alkylating agents and platinum-containing compounds and we compared it to that of GR. We confirmed prior studies suggesting that the nitrosourea carmustine can inhibit both GR and TrxR. We next found, however, that nitrogen mustards (chlorambucil and melphalan) and alkyl sulfonates (busulfan) efficiently inhibited TrxR while these compounds, surprisingly, did not inhibit GR. Inhibitions were concentration and time dependent and apparently irreversible. Anticancer anthracyclines (daunorubicin and doxorubicin) were, in contrast to the alkylating agents, not inhibitors but poor substrates of TrxR. We also found that TrxR, but not GR, was efficiently inhibited by both cisplatin, its monohydrated complex, and oxaliplatin. Carboplatin, in contrast, could not inhibit any of the two enzymes. These findings lead us to conclude that representative compounds of the major classes of clinically used anticancer alkylating agents and most platinum compounds may easily target TrxR, but not GR. The TrxR inhibition should thereby be considered as a factor that may contribute to the cytotoxicity seen upon clinical use of these drugs.

Ursolic acid inhibits superoxide production in activated neutrophils and attenuates trauma-hemorrhage shock-induced organ injury in rats.

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Tsong-Long Hwang

Full Text Available Neutrophil activation is associated with the development of organ injury after trauma-hemorrhagic shock. In the present study, ursolic acid inhibited the superoxide anion generation and elastase release in human neutrophils. Administration of ursolic acid attenuated trauma-hemorrhagic shock-induced hepatic and lung injuries in rats. In addition, administration of ursolic acid attenuated the hepatic malondialdehyde levels and reduced the plasma aspartate aminotransferase and alanine aminotransferase levels after trauma-hemorrhagic shock. In conclusion, ursolic acid, a bioactive natural compound, inhibits superoxide anion generation and elastase release in human neutrophils and ameliorates trauma-hemorrhagic shock-induced organ injury in rats.

The concentration of extracellular superoxide dismutase in plasma is maintained by LRP-mediated endocytosis

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Petersen, Steen V; Thøgersen, Ida B; Valnickova, Zuzana

2010-01-01

In this study, we show that human extracellular superoxide dismutase (EC-SOD) binds to low-density lipoprotein receptor-related protein (LRP). This interaction is most likely responsible for the removal of EC-SOD from the blood circulation via LRP expressed in liver tissue. The receptor recognition...

The study of the mechanism of arsenite toxicity in respiration-deficient cells reveals that NADPH oxidase-derived superoxide promotes the same downstream events mediated by mitochondrial superoxide in respiration-proficient cells

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Guidarelli, Andrea; Fiorani, Mara; Carloni, Silvia; Cerioni, Liana; Balduini, Walter; Cantoni, Orazio, E-mail: orazio.cantoni@uniurb.it

2016-09-15

We herein report the results from a comparative study of arsenite toxicity in respiration-proficient (RP) and -deficient (RD) U937 cells. An initial characterization of these cells led to the demonstration that the respiration-deficient phenotype is not associated with apparent changes in mitochondrial mass and membrane potential. In addition, similar levels of superoxide (O{sub 2}{sup .-}) were generated by RP and RD cells in response to stimuli specifically triggering respiratory chain-independent mitochondrial mechanisms or extramitochondrial, NADPH-oxidase dependent, mechanisms. At the concentration of 2.5 μM, arsenite elicited selective formation of O{sub 2}{sup .-} in the respiratory chain of RP cells, with hardly any contribution of the above mechanisms. Under these conditions, O{sub 2}{sup .-} triggered downstream events leading to endoplasmic reticulum (ER) stress, autophagy and apoptosis. RD cells challenged with similar levels of arsenite failed to generate O{sub 2}{sup .-} because of the lack of a functional respiratory chain and were therefore resistant to the toxic effects mediated by the metalloid. Their resistance, however, was lost after exposure to four fold greater concentrations of arsenite, coincidentally with the release of O{sub 2}{sup .-} mediated by NADPH oxidase. Interestingly, extramitochondrial O{sub 2}{sup .-} triggered the same downstream events and an identical mode of death previously observed in RP cells. Taken together, the results obtained in this study indicate that arsenite toxicity is strictly dependent on O{sub 2}{sup .-} availability that, regardless of whether generated in the mitochondrial or extramitochondrial compartments, triggers similar downstream events leading to ER stress, autophagy and apoptosis. - Highlights: • Mitochondrial superoxide mediates arsenite toxicity in respiration-proficient cells. • NADPH-derived superoxide mediates arsenite toxicity in respiration-deficient cells. • Arsenite causes apoptosis

Solution structure of an arsenate reductase-related protein, YffB, from Brucella melitensis, the etiological agent responsible for brucellosis

International Nuclear Information System (INIS)

Buchko, Garry W.; Hewitt, Stephen N.; Napuli, Alberto J.; Van Voorhis, Wesley C.; Myler, Peter J.

2011-01-01

B. melitensis is a NIAID Category B microorganism that is responsible for brucellosis and is a potential agent for biological warfare. Here, the solution structure of the 116-residue arsenate reductase-related protein Bm-YffB (BR0369) from this organism is reported. Brucella melitensis is the etiological agent responsible for brucellosis. Present in the B. melitensis genome is a 116-residue protein related to arsenate reductases (Bm-YffB; BR0369). Arsenate reductases (ArsC) convert arsenate ion (H 2 AsO 4 − ), a compound that is toxic to bacteria, to arsenite ion (AsO 2 − ), a product that may be efficiently exported out of the cell. Consequently, Bm-YffB is a potential drug target because if arsenate reduction is the protein’s major biological function then disabling the cell’s ability to reduce arsenate would make these cells more sensitive to the deleterious effects of arsenate. Size-exclusion chromatography and NMR spectroscopy indicate that Bm-YffB is a monomer in solution. The solution structure of Bm-YffB shows that the protein consists of two domains: a four-stranded mixed β-sheet flanked by two α-helices on one side and an α-helical bundle. The α/β domain is characteristic of the fold of thioredoxin-like proteins and the overall structure is generally similar to those of known arsenate reductases despite the marginal sequence similarity. Chemical shift perturbation studies with 15 N-labeled Bm-YffB show that the protein binds reduced glutathione at a site adjacent to a region similar to the HX 3 CX 3 R catalytic sequence motif that is important for arsenic detoxification activity in the classical arsenate-reductase family of proteins. The latter observation supports the hypothesis that the ArsC-YffB family of proteins may function as glutathione-dependent thiol reductases. However, comparison of the structure of Bm-YffB with the structures of proteins from the classical ArsC family suggest that the mechanism and possibly the function of Bm

Competitive Deprotonation and Superoxide [O₂⁻•)] Radical-Anion Adduct Formation Reactions of Carboxamides under Negative-Ion Atmospheric-Pressure Helium-Plasma Ionization (HePI) Conditions.

Science.gov (United States)

Hassan, Isra; Pinto, Spencer; Weisbecker, Carl; Attygalle, Athula B

2016-03-01

Carboxamides bearing an N-H functionality are known to undergo deprotonation under negative-ion-generating mass spectrometric conditions. Herein, we report that N-H bearing carboxamides with acidities lower than that of the hydroperoxyl radical (HO-O(•)) preferentially form superoxide radical-anion (O2(-•)) adducts, rather than deprotonate, when they are exposed to the glow discharge of a helium-plasma ionization source. For example, the spectra of N-alkylacetamides show peaks for superoxide radical-anion (O2(-•)) adducts. Conversely, more acidic amides, such as N-alkyltrifluoroacetamides, preferentially undergo deprotonation under similar experimental conditions. Upon collisional activation, the O2(-•) adducts of N-alkylacetamides either lose the neutral amide or the hydroperoxyl radical (HO-O(•)) to generate the superoxide radical-anion (m/z 32) or the deprotonated amide [m/z (M - H)(-)], respectively. For somewhat acidic carboxamides, the association between the two entities is weak. Thus, upon mildest collisional activation, the adduct dissociates to eject the superoxide anion. Superoxide-adduct formation results are useful for structure determination purposes because carboxamides devoid of a N-H functionality undergo neither deprotonation nor adduct formation under HePI conditions.

The role of quinone reductase (NQO1) and quinone chemistry in quercetin cytotoxicity

NARCIS (Netherlands)

Gliszczynska-Swiglo, A.; Woude, van der H.; Haan, de L.H.J.; Tyrakowska, B.; Aarts, J.M.M.J.G.; Rietjens, I.M.C.M.

2003-01-01

The effects of quercetin on viability and proliferation of Chinese Hamster Ovary (CHO) cells and CHO cells overexpressing human quinone reductase (CHO+NQO1) were studied to investigate the involvement of the pro-oxidant quinone chemistry of quercetin. The toxicity of menadione was significantly

A novel mercuric reductase from the unique deep brine environment of Atlantis II in the Red Sea.

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Sayed, Ahmed; Ghazy, Mohamed A; Ferreira, Ari J S; Setubal, João C; Chambergo, Felipe S; Ouf, Amged; Adel, Mustafa; Dawe, Adam S; Archer, John A C; Bajic, Vladimir B; Siam, Rania; El-Dorry, Hamza

2014-01-17

A unique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg(2+), and efficiently detoxifies Hg(2+) in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL.

Heme oxygenase attenuates angiotensin II-mediated superoxide production in cultured mouse thick ascending loop of Henle cells.

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Kelsen, Silvia; Patel, Bijal J; Parker, Lawson B; Vera, Trinity; Rimoldi, John M; Gadepalli, Rama S V; Drummond, Heather A; Stec, David E

2008-10-01

Heme oxygenase (HO)-1 induction can attenuate the development of angiotensin II (ANG II)-dependent hypertension. However, the mechanism by which HO-1 lowers blood pressure is not clear. The goal of this study was to test the hypothesis that induction of HO-1 can reduce the ANG II-mediated increase in superoxide production in cultured thick ascending loop of Henle (TALH) cells. Studies were performed on an immortalized cell line of mouse TALH (mTALH) cells. HO-1 was induced in cultured mTALH cells by treatment with cobalt protoporphyrin (CoPP, 10 microM) or hemin (50 microM) or by transfection with a plasmid containing the human HO-1 isoform. Treatment of mTALH cells with 10(-9) M ANG II increased dihydroethidium (DHE) fluorescence (an index of superoxide levels) from 35.5+/-5 to 136+/-18 relative fluorescence units (RFU)/microm2. Induction of HO-1 via CoPP, hemin, or overexpression of the human HO-1 isoform significantly reduced ANG II-induced DHE fluorescence to 64+/-5, 64+/-8, and 41+/-4 RFU/microm2, respectively. To determine which metabolite of HO-1 is responsible for reducing ANG II-mediated increases in superoxide production in mTALH cells, cells were preincubated with bilirubin or carbon monoxide (CO)-releasing molecule (CORM)-A1 (each at 100 microM) before exposure to ANG II. DHE fluorescence averaged 80+/-7 RFU/microm2 after incubation with ANG II and was significantly decreased to 55+/-7 and 53+/-4 RFU/microm2 after pretreatment with bilirubin and CORM-A1. These results demonstrate that induction of HO-1 in mTALH cells reduces the levels of ANG II-mediated superoxide production through the production of both bilirubin and CO.

Positive correlation between decreased cellular uptake, NADPH-glutathione reductase activity and adriamycin resistance in Ehrlich ascites tumor lines.

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Scheulen, M E; Hoensch, H; Kappus, H; Seeber, S; Schmidt, C G

1987-01-01

From a wild type strain of Ehrlich ascites tumor (EATWT) sublines resistant to daunorubicin (EATDNM), etoposide (EATETO), and cisplatinum (EATCIS) have been developed in vivo. Increase in survival and cure rate caused by adriamycin (doxorubicin) have been determined in female NMRI mice which were inoculated i.p. with EAT cells. Adriamycin concentrations causing 50% inhibition of 3H-thymidine (ICT) and 3H-uridine incorporation (ICU) and intracellular adriamycin steady-state concentrations (SSC) were measured in vitro. Adriamycin resistance increased and SSC decreased in the following sequence: EATWT - EATCIS - EATDNM - EATETO. When ICT and ICU were corrected for intracellular adriamycin concentrations in consideration of the different SSC (ICTc, ICUc), ICTc and ICUc still varied up to the 3.2 fold in EATCIS, EATDNM and EATETO in comparison to EATWT. Thus, in addition to different SSC other factors must be responsible for adriamycin resistance. Therefore, enzymes which may play a role in the cytotoxicity related to adriamycin metabolism (NADPH-cytochrome P-450 reductase, NADPH-glutathione reductase, NADP-glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase) were measured. In contrast to the other parameters determined, NADPH-glutathione reductase was significantly (p less than 0.01) increased up to the 3.2 fold parallel to adriamycin resistance as determined by increase in life span, cure rate, ICTc, and ICUc, respectively. It is concluded that high activities of NADPH-glutathione reductase may contribute to an increase in adriamycin resistance of malignant tumors.

Effect of pharmaceutical potential endocrine disruptor compounds on protein disulfide isomerase reductase activity using di-eosin-oxidized-glutathione.

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Danièle Klett

Full Text Available BACKGROUND: Protein Disulfide Isomerase (PDI in the endoplasmic reticulum of all cells catalyzes the rearrangement of disulfide bridges during folding of membrane and secreted proteins. As PDI is also known to bind various molecules including hormones such as estradiol and thyroxin, we considered the hypothesis that adverse effects of endocrine-disrupter compounds (EDC could be mediated through their interaction with PDI leading to defects in membrane or secreted proteins. METHODOLOGY/PRINCIPAL FINDINGS: Taking advantage of the recent description of the fluorescence self quenched substrate di-eosin-oxidized-glutathione (DiE-GSSG, we determined kinetically the effects of various potential pharmaceutical EDCs on the in-vitro reductase activity of bovine liver PDI by measuring the fluorescence of the reaction product (E-GSH. Our data show that estrogens (ethynylestradiol and bisphenol-A as well as indomethacin exert an inhibition whereas medroxyprogesteroneacetate and nortestosterone exert a potentiation of bovine PDI reductase activity. CONCLUSIONS: The present data indicate that the tested EDCs could not only affect endocrine target cells through nuclear receptors as previously shown, but could also affect these and all other cells by positively or negatively affecting PDI activity. The substrate DiE-GSSG has been demonstrated to be a convenient substrate to measure PDI reductase activity in the presence of various potential EDCs. It will certainly be usefull for the screening of potential effect of all kinds of chemicals on PDI reductase activity.

Molecular Cloning and Expression of Sequence Variants of Manganese Superoxide Dismutase Genes from Wheat

Science.gov (United States)

Reactive oxygen species (ROS) are very harmful to living organisms due to the potential oxidation of membrane lipids, DNA, proteins, and carbohydrates. Transformed E.coli strain QC 871, superoxide dismutase (SOD) double-mutant, with three sequence variant MnSOD1, MnSOD2, and MnSOD3 manganese supero...

Inclusions of amyotrophic lateral sclerosis-linked superoxide dismutase in ventral horns, liver, and kidney

DEFF Research Database (Denmark)

Jonsson, P.A.; Bergemalm, D.; Andersen, P.M.

2008-01-01

Mutant superoxide dismutases type 1 (SOD1s) cause amyotrophic lateral sclerosis by an unidentified toxic property. In a patient carrying the G127X truncation mutation, minute amounts of SOD1 were found in ventral horns using a mutant-specific antibody. Still, both absolute levels and ratios versus...

A non-invasive and rapid seed vigor biosensor based on quantitative measurement of superoxide generated by aleurone cell in intact seeds.

Science.gov (United States)

Liu, Xuejun; Gao, Caiji; Xing, Da

2009-02-15

Superoxide generated during the early imbibition is an excellent marker for evaluating seed vigor. In this paper, a new principle biosensor for non-invasive detection of seed vigor based on quantitative measurement of superoxide via selective probe 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2alpha] pyrazin-3-one (MCLA)-mediated chemiluminescence (CL) was developed. The biosensor, which used a compact single-photon counting module (SPCM) to collect the CL signal, could evaluate seed vigor in vivo. Benefiting from the high CL efficiency of MCLA reacting with superoxide and high sensitivity of the SPCM technique, the trace superoxide generated by dry seeds under storage state can be detected to achieve rapid and non-invasive determination of the seed vigor. In comparison with the traditional methods for fast measuring seed vigor based on measurement of physiological and biochemical properties, our proposed technique has significant advantages such as low cost, simplicity, convenient operation and short time consuming. To demonstrate the utility of the system, it was applied to evaluate MCLA-mediated CL of three different plant species wheat (Ze Yu No. 2), maize (Tai Gu No. 1 and 2) and rice (Jing Dao No. 21) seeds with different degrees of aging. The experimental results suggested that there was an excellent positive correlation between the seed vigor assessment from quantitative TTC-test and the detection based on MCLA-mediated CL of superoxide measurement. The new principle of seed vigor measurement is a challenge and breakthrough to conventional method of seed vigor determination and may be a potential technique of the next generation seed vigor detection.

Superoxide produced by Kupffer cells is an essential effector in concanavalin A-induced hepatitis in mice.

Science.gov (United States)

Nakashima, Hiroyuki; Kinoshita, Manabu; Nakashima, Masahiro; Habu, Yoshiko; Shono, Satoshi; Uchida, Takefumi; Shinomiya, Nariyoshi; Seki, Shuhji

2008-12-01

Although concanavalin A (Con-A)-induced experimental hepatitis is thought to be induced by activated T cells, natural killer T (NKT) cells, and cytokines, precise mechanisms are still unknown. In the current study, we investigated the roles of Kupffer cells, NKT cells, FasL, tumor necrosis factor (TNF), and superoxide in Con-A hepatitis in C57BL/6 mice. Removal of Kupffer cells using gadolinium chloride (GdCl(3)) from the liver completely inhibited Con-A hepatitis, whereas increased serum TNF and IFN-gamma levels were not inhibited at all. Unexpectedly, anti-FasL antibody pretreatment did not inhibit Con-A hepatitis, whereas it inhibited hepatic injury induced by a synthetic ligand of NKT cells, alpha-galactosylceramide. Furthermore, GdCl(3) pretreatment changed neither the activation-induced down-regulation of NK1.1 antigens as well as T cell receptors of NKT cells nor the increased expression of the CD69 activation antigen of hepatic T cells. CD68(+) Kupffer cells greatly increased in proportion in the early phase after Con-A injection; this increase was abrogated by GdCl(3) pretreatment. Anti-TNF antibody (Ab) pretreatment did not inhibit the increase of Kupffer cells, but it effectively suppressed superoxide/reactive oxygen production from Kupffer cells and the resulting hepatic injury. Conversely, depletion of NKT cells in mice by NK1.1 Ab pretreatment did suppress both the increase of CD68(+) Kupffer cells and Con-A hepatitis. Consistently, the diminution of oxygen radicals produced by Kupffer cells by use of free radical scavengers greatly inhibited Con-A hepatitis without suppressing cytokine production. However, adoptive transfer experiments also indicate that a close interaction/cooperation of Kupffer cells with NKT cells is essential for Con-A hepatitis. Superoxide produced by Kupffer cells may be the essential effector in Con-A hepatitis, and TNF and NKT cells support their activation and superoxide production.

Infusing sodium bicarbonate suppresses hydrogen peroxide accumulation and superoxide dismutase activity in hypoxic-reoxygenated newborn piglets.

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Jiang-Qin Liu

Full Text Available The effectiveness of sodium bicarbonate (SB has recently been questioned although it is often used to correct metabolic acidosis of neonates. The aim of the present study was to examine its effect on hemodynamic changes and hydrogen peroxide (H(2O(2 generation in the resuscitation of hypoxic newborn animals with severe acidosis.Newborn piglets were block-randomized into a sham-operated control group without hypoxia (n = 6 and two hypoxia-reoxygenation groups (2 h normocapnic alveolar hypoxia followed by 4 h room-air reoxygenation, n = 8/group. At 10 min after reoxygenation, piglets were given either i.v. SB (2 mEq/kg, or saline (hypoxia-reoxygenation controls in a blinded, randomized fashion. Hemodynamic data and blood gas were collected at specific time points and cerebral cortical H(2O(2 production was continuously monitored throughout experimental period. Plasma superoxide dismutase and catalase and brain tissue glutathione, superoxide dismutase, catalase, nitrotyrosine and lactate levels were assayed.Two hours of normocapnic alveolar hypoxia caused cardiogenic shock with metabolic acidosis (PH: 6.99 ± 0.07, HCO(3(-: 8.5 ± 1.6 mmol/L. Upon resuscitation, systemic hemodynamics immediately recovered and then gradually deteriorated with normalization of acid-base imbalance over 4 h of reoxygenation. SB administration significantly enhanced the recovery of both pH and HCO(3- recovery within the first hour of reoxygenation but did not cause any significant effect in the acid-base at 4 h of reoxygenation and the temporal hemodynamic changes. SB administration significantly suppressed the increase in H(2O(2 accumulation in the brain with inhibition of superoxide dismutase, but not catalase, activity during hypoxia-reoxygenation as compared to those of saline-treated controls.Despite enhancing the normalization of acid-base imbalance, SB administration during resuscitation did not provide any beneficial effects on hemodynamic recovery in

Thermophilic enzymes and their applications in biocatalysis: a robust aldo-keto reductase.

Science.gov (United States)

Willies, Simon; Isupov, Misha; Littlechild, Jennifer

2010-09-01

Extremophiles are providing a good source of novel robust enzymes for use in biocatalysis for the synthesis of new drugs. This is particularly true for the enzymes from thermophilic organisms which are more robust than their mesophilic counterparts to the conditions required for industrial bio-processes. This paper describes a new aldo-keto reductase enzyme from a thermophilic eubacteria, Thermotoga maritima which can be used for the production of primary alcohols. The enzyme has been cloned and over-expressed in Escherichia coli and has been purified and subjected to full biochemical characterization. The aldo-keto reductase can be used for production of primary alcohols using substrates including benzaldehyde, 1,2,3,6-tetrahydrobenzaldehyde and para-anisaldehyde. It is stable up to 80 degrees C, retaining over 60% activity for 5 hours at this temperature. The enzyme at pH 6.5 showed a preference for the forward, carbonyl reduction. The enzyme showed moderate stability with organic solvents, and retained 70% activity in 20% (v/v) isopropanol or DMSO. These properties are favourable for its potential industrial applications.

Resonance Raman spectra of the copper-sulfur chromophores in Achromobacter cycloclastes nitrite reductase.

Science.gov (United States)

Dooley, D M; Moog, R S; Liu, M Y; Payne, W J; LeGall, J

1988-10-15

Resonance Raman spectroscopy at ambient temperature and 77 K has been used to probe the structures of the copper sites in Achromobacter cycloclastes nitrite reductase. This enzyme contains three copper ions per protein molecule and has two principal electronic absorption bands with lambda max values of 458 and 585 nm. Comparisons between the resonance Raman spectra of nitrite reductase and blue copper proteins establish that both the 458 and 585 nm bands are associated with Cu(II)-S(Cys) chromophores. A histidine ligand probably is also present. Different sets of vibrational frequencies are observed with 457.9 nm (ambient) or 476.1 nm (77 K) excitation as compared with 590 nm (ambient) or 593 nm (77 K) excitation. Excitation profiles indicate that the 458 and 585 nm absorption bands are associated with separate [Cu(II)-S(Cys)N(His)] sites or with inequivalent and uncoupled cysteine ligands in the same site. The former possibility is considered to be more likely.

Molecular cloning and characterization of Fasciola gigantica thioredoxin-glutathione reductase.

Science.gov (United States)

Changklungmoa, Narin; Kueakhai, Pornanan; Sangpairoj, Kant; Chaichanasak, Pannigan; Jaikua, Wipaphorn; Riengrojpitak, Suda; Sobhon, Prasert; Chaithirayanon, Kulathida

2015-06-01

The Fasciola gigantica thioredoxin-glutathione reductase (FgTGR) gene is a fusion between thioredoxin reductase (TR) and a glutaredoxin (Grx) gene. FgTGR was cloned by polymerase chain reaction (PCR) from adult complementary DNA (cDNA), and its sequences showed two isoforms, i.e., the cytosolic and mitochondrial FgTGR. Cytosolic FgTGR (cytFgTGR) was composed of 2370 bp, and its peptide had no signal sequence and hence was not a secreted protein. Mitochondrial FgTGR (mitFgTGR) was composed of 2506 bp with a signal peptide of 43 amino acids; therefore, it was a secreted protein. The putative cytFgTGR and mitFgTGR peptides comprised of 598 and 641 amino acids, respectively, with a molecular weight of 65.8 kDa for cytFgTGR and mitFgTGR, with a conserved sequence (CPYC) of TR, and ACUG and CVNVGC of Grx domains. The recombinant FgTGR (rFgTGR) was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTGR). The FgTGR protein expression, estimated by indirect ELISA using the rabbit anti-rFgTGR as probe, showed high levels of expression in eggs, and 2- and 4-week-old juveniles and adults. The rFgTGR exhibited specific activities in the 5,5'-dithiobis (2-nitro-benzoic acid) (DTNB) reductase assay for TR activity and in β-hydroxyethul disulfide (HED) for Grx activity. When analyzed by immunoblotting and immunohistochemistry, rabbit anti-rFgTGR reacted with natural FgTGR at a molecular weight of 66 kDa from eggs, whole body fraction (WB) of metacercariae, NEJ, 2- and 4-week-old juveniles and adults, and the tegumental antigen (TA) of adult. The FgTGR protein was expressed at high levels in the tegument of 2- and 4-week-old juveniles. The FgTGR may be one of the major factors acting against oxidative stresses that can damage the parasite; hence, it could be considered as a novel vaccine or a drug target.

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation

OpenAIRE

Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E. M.; Jenkins, Jermaine L.; Heimiller, Chelsea; Maines, Mahin D.

2016-01-01

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1–3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T308 before S473 autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present ...

Coordination between antioxidant defences might be partially modulated by magnesium status.

Science.gov (United States)

Monteiro, Cristina Paula; Matias, Catarina Nunes; Bicho, Manuel; Santa-Clara, Helena; Laires, Maria José

2016-04-01

The aim of this study was to compare the redox balance in competitive adult swimmers against recreational practitioners, controlling for Mg intake. Fifteen, competitive swimmers and 16 recreational practitioners, all male and aged 18-25years, were recruited into the study. Oxidative and muscle damage markers, and antioxidant enzymes and non-enzymatic antioxidants were evaluated by photometry (except for thiobarbituric reactive substances (TBARS), which was assessed by fluorimetry). Controlling for the level of exercise, inverse correlations were observed for uric acid and glutathione reductase (GR) or susceptibility of red blood cells to peroxidation (RBCPx); plasma adrenaline oxidase activity (AdOx) and carotenoids; TBARS and GR or Vit E; and direct correlations were observed between AdOx and creatine kinase (CK) or TBARS; CK and superoxide dismutase activity; GR and RBCPx. Controlling for Mg intake in addition to exercise level revealed new inverse correlations: between carotenoids and TBARS or lactate, and new direct correlations between lactate and AdOx or TBARS; cortisol and AdOx, CK, lactate dehydrogenase, or methemoglobin reductase. The associations between uric acid and RBCPx; AdOx and CK or TBARS; and GR and RBCPx lost their significance. All others remained significant. These outcomes suggest that the coordination between antioxidant defences may be partially modulated by Mg, which may be the result of its ability to stabilize cell membranes and oxidation targets, such as adrenaline.

Metalloantibiotic Mn(II)-bacitracin complex mimicking manganese superoxide dismutase

International Nuclear Information System (INIS)

Piacham, Theeraphon; Isarankura-Na-Ayudhya, Chartchalerm; Nantasenamat, Chanin; Yainoy, Sakda; Ye Lei; Buelow, Leif; Prachayasittikul, Virapong

2006-01-01

Superoxide dismutase (SOD) activities of various metallobacitracin complexes were evaluated using the riboflavin-methionine-nitro blue tetrazolium assay. The radical scavenging activity of various metallobacitracin complexes was shown to be higher than those of the negative controls, e.g., free transition metal ions and metal-free bacitracin. The SOD activity of the complex was found to be in the order of Mn(II) > Cu(II) > Co(II) > Ni(II). Furthermore, the effect of bacitracin and their complexation to metals on various microorganisms was assessed by antibiotic susceptibility testing. Moreover, molecular modeling and quantum chemical calculation of the metallobacitracin complex was performed to evaluate the correlation of electrostatic charge of transition metal ions on the SOD activity

Direct 19F NMR observation of the conformational selection of optically active rotamers of the antifolate compound fluoronitropyrimethamine bound to enzyme dihydrofolate reductase

International Nuclear Information System (INIS)

Tendler, S.J.B.; Birdsall, B.; Feeney, J.; Griffin, R.J.; Stevens, M.F.G.; Roberts, G.C.K.

1988-01-01

The molucular basis of the binding of the lipophilic antifolate compound fluoronitropyrimethamine to its target enzyme dihydrofolate reductase has been investigated using a combination of 19 F NMR spectroscopy and molecular mechanical calculations 19 F NMR reveals the presence of two different conformational states for the fluoronitropyrimethamine-Lactobacillus casei enzyme complex. MM2 molecular mechanical calculations predict restricted rotation about the C5-C1 bond of the ligand and this give rise to two slowly interconverting rotamers which are an enantiomeric pair. The results of 19 F NMR spectroscopy reveal that both these isomers bind to the enzyme, with different affinities. There is no detectable interconversion of the bound rotamers themselves on the NMR timescale. The effect of the addition of co-enzyme to the sample is to reverse the preference the enzyme has for each rotamer. (author). 11 refs.; 3 figs

Monooxygenase system in Guerin’s carcinoma of rats under conditions of ω-3 polyunsaturated fatty acids administration

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M. M. Marchenko

2016-08-01

Full Text Available The aim of the study was to determine the variations of function in components of monooxygenase system (MOS of rat Guerin’s carcinoma under ω-3 polyunsaturated fatty acids (PUFAs administration. The activity of Guerin’s carcinoma microsomal NADH-cytochrome b5 reductase, the content and the rate of cytochrome b5 oxidation-reduction, the content and the rate of cytochrome Р450 oxidation-reduction have been investigated in rats with tumor under conditions of ω-3 PUFAs administration. ω-3 PUFAs supplementation before and after transplantation of Guerin’s carcinoma resulted in the increase of NADH-cytochrome b5 reductase activity and decrease of cytochrome b5 level in the Guerin’s carcinoma microsomal fraction in the logarithmic phases of carcinogenesis as compared to the tumor-bearing rats. Increased activity of NADH-cytochrome b5 reductase facilitates higher electron flow in redox-chain of MOS. Under decreased cytochrome b5 levels the electrons are transferred to oxygen, which leads to heightened generation of superoxide (O2•- in comparison to control. It was shown, that the decrease of cytochrome P450 level in the Guerin’s carcinoma microsomal fraction in the logarithmic phases of oncogenesis under ω-3 PUFAs administration may be associated with its transition into an inactive form – cytochrome P420. This decrease in cytochrome P450 coincides with increased generation of superoxide by MOS oxygenase chain.

Methylenetetrahydrofolate reductase (MTHFR) deficiency presenting as a rash.

LENUS (Irish Health Repository)

Crushell, Ellen

2012-09-01

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.

Developing a high performance superoxide dismutase based electrochemical biosensor for radiation dosimetry of thallium 201

International Nuclear Information System (INIS)

Salem, Fatemeh; Tavakoli, Hassan; Sadeghi, Mahdi; Riazi, Abbas

2014-01-01

To develop a new biosensor for measurement of superoxide free radical generated in radiolysis reaction, three combinations of SOD-based biosensors including Au/Cys/SOD, Au/GNP/Cys/SOD and Au/GNP/Cys/SOD/Chit were fabricated. In these biosensors Au, GNP, Cys, SOD and Chit represent gold electrode, gold nano-particles, cysteine, superoxide dismutase and chitosan, respectively. For biosensors fabrication, SOD, GNP, Cys and Chit were immobilized at the surface of gold electrode. Cyclic voltametry and chronoamperometry were utilized for evaluation of biosensors performances. The results showed that Au/GNP/Cys/SOD/Chit has significantly better responses compared to Au/Cys/SOD and Au/GNP/Cys/SOD. As a result, this biosensor was selected for dosimetry of ionizing radiation. For this purpose, thallium 201 at different volumes was added to buffer phosphate solution in electrochemical cell. To obtain analytical parameters of Au/GNP/Cys/SOD/Chit, calibration curve was sketched. The results showed that this biosensor has a linear response in the range from 0.5 to 4 Gy, detection limit 0.03 μM. It also has a proper sensitivity (0.6038 nA/Gy), suitable long term stability and cost effective as well as high function for radiation dosimetry. - highlights: • Our biosensor is able to measure produced superoxide radical during water radiolysis. • It has suitable linearity range, good detection limit and long term stability. • It also has proper sensitivity and high performance for low LET ionizing radiation. • The electrochemical method is as good as traditional methods for radiation dosimetry

Purification, crystallization and preliminary X-ray analysis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase of Streptococcus pneumoniae

International Nuclear Information System (INIS)

Zhang, Liping; Feng, Lingling; Zhou, Li; Gui, Jie; Wan, Jian; Hu, Xiaopeng

2010-01-01

3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of Streptococcus pneumoniae has been cloned, overexpressed and purified to homogeneity using Ni–NTA affinity chromatography. Crystals were obtained using the hanging-drop vapour-diffusion method. Class II 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases are potential targets for novel antibiotic development. In order to obtain a precise structural model for use in virtual screening and inhibitor design, HMG-CoA reductase of Streptococcus pneumoniae was cloned, overexpressed and purified to homogeneity using Ni–NTA affinity chromatography. Crystals were obtained using the hanging-drop vapour-diffusion method. A complete data set was collected from a single frozen crystal on a home X-ray source. The crystal diffracted to 2.3 Å resolution and belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 773.4836, b = 90.3055, c = 160.5592 Å, α = β = γ = 90°. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be 54.1% (V M = 2.68 Å 3 Da −1 )

Biocatalysis with thermostable enzymes: structure and properties of a thermophilic 'ene'-reductase related to old yellow enzyme.

Science.gov (United States)

Adalbjörnsson, Björn V; Toogood, Helen S; Fryszkowska, Anna; Pudney, Christopher R; Jowitt, Thomas A; Leys, David; Scrutton, Nigel S

2010-01-25

We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.

Effect of cephalandra indica against advanced glycation end products, sorbitol accumulation and aldose reductase activity in homoeopathic formulation

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Lalit Kishore

2018-01-01

Full Text Available Background: Extreme generation of free radicals leads to oxidative stress which has been apprehensive in several disease processes such as diabetic complications and vascular and neurodegenerative diseases. Objective: The present study was designed to evaluate the potential of homoeopathic preparations of Cephalandra indica L. against oxidative stress. Materials and Methods: Potencies of Cephalandra indica (mother tincture, 6C and 30C were procured from Dr. Willmar Schwabe India Pvt. Ltd. The antioxidant activity of Cephalandra indica was evaluated by employing various in vitro antioxidant methods. Results: The total phenol content was found to be 1905, 849 and 495 mg/g gallic acid equivalents in mother tincture, 6C and 30C of Cephalandra indica and total antioxidant capacity was found to be 2710, 759 and 510 μM/g ascorbic acid equivalents, respectively. Mother tincture, 6C and 30C of Cephalandra indica was found to have strong reducing power, 2,2-diphenyl-1-picrylhydrazyl radical, hydrogen peroxide, nitric oxide and superoxide radical scavenging activity. Percentage inhibition of AGEs formation by mother tincture, 6C and 30C of Cephalandra indica (10–50 μl was found to be 30.34%–91.77%, 29.98%–65.71% and 33.05%–57.75%, respectively. Mother tincture, 6C and 30C of Cephalandra indica showed inhibitory effect against sorbitol accumulation with IC50value of 26.12 μl, 203.10 μl and 897.3 μl, respectively, whereas, in aldose reductase inhibition assay, the IC50value was 32.54 μl, 175.02 μl and 834.34 μl, respectively. Conclusion: The results revealed that homoeopathic preparations of Cephalandra indica exhibit protective effect against oxidative stress.

Lethal doses of ionizing radiation versus endogenous level of superoxide dismutase

Energy Technology Data Exchange (ETDEWEB)

Lipecka, K; Domanski, T; Dniaszewska, K; Grabowska, B; Pietrowicz, D; Lindner, P; Cisowska, B; Gorski, H [Military Medical Academy, Lodz (Poland). Inst. of Occupational Medicine

1982-06-22

The stability of superoxide dismutase (SOD) as well as its activity distribution in a human population were investigated. The SOD activity level of the erythrocytes proved to be an index for the endogenous SOD activity in the whole body. In a rat population, having similar SOD frequency distribution as a human population, the mortality due to acute irradiation depended on the SOD level; after a single acute dose approximating the lethal dose (LD/sub 50/30/) the survival depended distinctly on the endogenous SOD activity level.

Functional properties and structural characterization of rice δ1-pyrroline-5-carboxylate reductase

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Giuseppe eForlani

2015-07-01

Full Text Available The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice (Oryza sativa L. for δ1-pyrroline-5-carboxylate (P5C reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in E. coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was able to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP+ were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP+ ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. Based on the structure, it was possible to identify dynamic structural differences among rice, human and bacterial enzymes.

Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-[acyl carrier protein]-reductase genes.

Science.gov (United States)

González-Thuillier, Irene; Venegas-Calerón, Mónica; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

2015-01-01

Enoyl-[acyl carrier protein]-reductases from sunflower. A major factor contributing to the amount of fatty acids in plant oils are the first steps of their synthesis. The intraplastidic fatty acid biosynthetic pathway in plants is catalysed by type II fatty acid synthase (FAS). The last step in each elongation cycle is carried out by the enoyl-[ACP]-reductase, which reduces the dehydrated product of β-hydroxyacyl-[ACP] dehydrase using NADPH or NADH. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus) seeds, two enoyl-[ACP]-reductase genes have been identified and cloned from developing seeds with 75 % identity: HaENR1 (GenBank HM021137) and HaENR2 (HM021138). The two genes belong to the ENRA and ENRB families in dicotyledons, respectively. The genetic duplication most likely originated after the separation of di- and monocotyledons. RT-qPCR revealed distinct tissue-specific expression patterns. Highest expression of HaENR1 was in roots, stems and developing cotyledons whereas that of H a ENR2 was in leaves and early stages of seed development. Genomic DNA gel blot analyses suggest that both are single-copy genes. In vivo activity of the ENR enzymes was tested by complementation experiments with the JP1111 fabI(ts) E. coli strain. Both enzymes were functional demonstrating that they interacted with the bacterial FAS components. That different fatty acid profiles resulted infers that the two Helianthus proteins have different structures, substrate specificities and/or reaction rates. The latter possibility was confirmed by in vitro analysis with affinity-purified heterologous-expressed enzymes that reduced the crotonyl-CoA substrate using NADH with different V max.

Crystal structure of conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 complexed with NADPH.

Science.gov (United States)

Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2013-11-01

Conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 is a member of the aldo-keto reductase (AKR) superfamily and reduces ketopantoyl lactone to d-pantoyl lactone in a NADPH-dependent and stereospecific manner. We determined the crystal structure of CPR-C1.NADPH complex at 2.20 Å resolution. CPR-C1 adopted a triose-phosphate isomerase (TIM) barrel fold at the core of the structure in which Thr25 and Lys26 of the GXGTX motif bind uniquely to the adenosine 2'-phosphate group of NADPH. This finding provides a novel structural basis for NADPH binding of the AKR superfamily. Copyright © 2013 Wiley Periodicals, Inc.

The Fumarate Reductase of Bacteroides thetaiotaomicron, unlike That of Escherichia coli, Is Configured so that It Does Not Generate Reactive Oxygen Species

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Zheng Lu

2017-01-01

Full Text Available The impact of oxidative stress upon organismal fitness is most apparent in the phenomenon of obligate anaerobiosis. The root cause may be multifaceted, but the intracellular generation of reactive oxygen species (ROS likely plays a key role. ROS are formed when redox enzymes accidentally transfer electrons to oxygen rather than to their physiological substrates. In this study, we confirm that the predominant intestinal anaerobe Bacteroides thetaiotaomicron generates intracellular ROS at a very high rate when it is aerated. Fumarate reductase (Frd is a prominent enzyme in the anaerobic metabolism of many bacteria, including B. thetaiotaomicron, and prior studies of Escherichia coli Frd showed that the enzyme is unusually prone to ROS generation. Surprisingly, in this study biochemical analysis demonstrated that the B. thetaiotaomicron Frd does not react with oxygen at all: neither superoxide nor hydrogen peroxide is formed. Subunit-swapping experiments indicated that this difference does not derive from the flavoprotein subunit at which ROS normally arise. Experiments with the related enzyme succinate dehydrogenase discouraged the hypothesis that heme moieties are responsible. Thus, resistance to oxidation may reflect a shift of electron density away from the flavin moiety toward the iron-sulfur clusters. This study shows that the autoxidizability of a redox enzyme can be suppressed by subtle modifications that do not compromise its physiological function. One implication is that selective pressures might enhance the oxygen tolerance of an organism by manipulating the electronic properties of its redox enzymes so they do not generate ROS.

Purification, cloning, functional expression and characterization of perakine reductase: the first example from the AKR enzyme family, extending the alkaloidal network of the plant Rauvolfia.

Science.gov (United States)

Sun, Lianli; Ruppert, Martin; Sheludko, Yuri; Warzecha, Heribert; Zhao, Yu; Stöckigt, Joachim

2008-07-01

Perakine reductase (PR) catalyzes an NADPH-dependent step in a side-branch of the 10-step biosynthetic pathway of the alkaloid ajmaline. The enzyme was cloned by a "reverse-genetic" approach from cell suspension cultures of the plant Rauvolfia serpentina (Apocynaceae) and functionally expressed in Escherichia coli as the N-terminal His(6)-tagged protein. PR displays a broad substrate acceptance, converting 16 out of 28 tested compounds with reducible carbonyl function which belong to three substrate groups: benzaldehyde, cinnamic aldehyde derivatives and monoterpenoid indole alkaloids. The enzyme has an extraordinary selectivity in the group of alkaloids. Sequence alignments define PR as a new member of the aldo-keto reductase (AKR) super family, exhibiting the conserved catalytic tetrad Asp52, Tyr57, Lys84, His126. Site-directed mutagenesis of each of these functional residues to an alanine residue results in >97.8% loss of enzyme activity, in compounds of each substrate group. PR represents the first example of the large AKR-family which is involved in the biosynthesis of plant monoterpenoid indole alkaloids. In addition to a new esterase, PR significantly extends the Rauvolfia alkaloid network to the novel group of peraksine alkaloids.

A pulse-radiolysis study of the manganese-containing superoxide dismutase from Bacillus stearothermophilus

International Nuclear Information System (INIS)

McAdam, M.E.; Fox, R.A.; Lavelle, F.; Fielden, E.M.

1977-01-01

The enzymic reaction mechanism of a manganese-containing superoxide dismutase from Bacillus stearothermophilus was studied by using pulse radiolysis. During catalysis (pH 8.9; 25 0 C), changes occurring in the kinetics of substrate disappearance and in the visible absorption of the enzyme at 480 nm established that the simple two-step mechanism found for copper- and iron-containing superoxide dismutases was not involved. At a low ratio ( 2 sup(-.) was close to exponential, whereas at much higher ratios (> 100) the observed decay was predominantly zero-order. The simplest interpretation of the results invokes a rapid one-electron oxidation-reduction cycle ('the fast cycle') and, concurrently, a slower reaction giving a form of the enzyme that is essentially unreactive towards O 2 sup(-.) but which undergoes a first-order decay to yield fully active native enzyme ('the slow cycle'). The fast cycle involved the native enzyme Esub(A) and a form of the enzyme Esub(B) which could be obtained also be treating the form Esub(A) with H 2 O 2 . Computer calculations made with such a simple model predicted behaviour in excellent agreement with the observed results. (author)

Properties of latent and thiol-activated rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and regulation of enzyme activity.

Science.gov (United States)

Dotan, I; Shechter, I

1983-10-15

The effect of the thiols glutathione (GSH), dithiothreitol (DTT), and dithioerythritol (DTE) on the conversion of an inactive, latent form (El) of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) to a catalyticaly active form (Ea) is examined. Latent hepatic microsomal HMG-CoA reductase is activated to a similar degree of activation by DTT and DTE and to a lower extent by GSH. All three thiols affect both Km and Vmax values of the enzyme toward HMG-CoA and NADPH. Studies of the effect of DTT on the affinity binding of HMG-CoA reductase to agarose-hexane-HMG-CoA (AG-HMG-CoA) resin shows that thiols are necessary for the binding of the enzyme to the resin. Removal of DTT from AG-HMG-CoA-bound soluble Ea (active enzyme) does not cause dissociation of the enzyme from the resin at low salt concentrations. Substitution of DTT by NADPH does not promote binding of soluble El (latent enzyme) to AG-HMG-CoA. The enzymatic activity of Ea in the presence of DTT and GSH indicates that these thiols compete for the same binding site on the enzyme. Diethylene glycol disulfide (ESSE) and glutathione disulfide (GSSG) inhibit the activity of Ea. ESSE is more effective for the inhibition of Ea than GSSG, causing a higher degree of maximal inhibition and affecting the enzymatic activity at lower concentrations. A method is described for the rapid conversion of soluble purified Ea to El using gel-filtration chromatography on Bio-Gel P-4 columns. These combined results point to the importance of the thiol/disulfide ratio for the modulation of hepatic HMG-CoA reductase activity.

A Rational Approach to Identify Inhibitors of Mycobacterium tuberculosis Enoyl Acyl Carrier Protein Reductase

Czech Academy of Sciences Publication Activity Database

Chhabria, M. T.; Parmar, K. B.; Brahmkshatriya, Pathik

2013-01-01

Roč. 19, č. 21 (2013), s. 3878-3883 ISSN 1381-6128 Institutional support: RVO:61388963 Keywords : mycobacterium tuberculosis * enoyl acyl carrier protein reductase * pharmacophore modeling * molecular docking * binding interactions Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 3.288, year: 2013

Superoxide radicals mediate heptatoxicity induced by the heat shock protein 90 inhibitors benzoquinone ansamycins

International Nuclear Information System (INIS)

Goldstein, S.

2011-01-01

Complete text of publication follows. Geldanamycin (GM). a benzoquinone ansamycin antibiotic, is a natural product inhibitor of the heat shock protein 90 (Hsp90) with potent and broad anticancer properties. However, its progression to clinical trials was halted due to unacceptable levels of hepatotoxicity. Consequently, numerous less toxic analogs differing only in their 17-substituent have been synthesized including 17-AAG and the water soluble 17-DMAG (Alvespimycin), which have recently entered clinical trials. The different hepatotoxicity induced by GM and its analogs may reflect the redox active properties of the quinone moiety (Q) and possibly the extent of superoxide radical formation, which may stimulate cellular oxidative injury. Q ·- + Q 2 ↔ O 2 ·- + Q. Eq. 1 is established rapidly, and its actual position is governed by E 7 (Q/Q ·- ) and E 7 (O 2 /O 2 ·- ) and the relative concentrations of Q and O 2 . Using pulse radiolysis, E 7 (Q/Q ·- ) for 17-DMAG has been determined vs. O 2 , 1,4-naphthoquinone or menadione to be -194 ± 6 mV, which is somewhat lower than E 7 (O 2 /O 2 ·- ) = -180 mV (1 M O 2 ). Eq. 1 is well to the left in the case of 1,4-benzoquinone and substitution into the ring by electron-donating or -withdrawing groups reduces or increases, respectively, E 7 (Q/Q ·- ) in a predictable manner, e.g. linearly related to the Hammett sigma value of the substituents. Hence, E 7 (Q/Q ·- ) should follow the order GM 2 is more readily reduced to O 2 ·- by GM. It is demonstrated that O 2 ·- can be efficiently trapped by Tempol during the reduction of GM, 17-AAG and 17-DMAG by NADPH catalyzed by NADPH-cytochrome P450 reductase, and that O 2 ·- formation rate, which reflects the rate of NADPH oxidation, follows the order 17-DMAG > GM > 17-AAG. In the absence of O 2 ·- scavengers, the rate of NADPH oxidation follows the order 17-DMAG > 17-AAG > GM. The order of the drug cytotoxicity toward rat primary hepatocytes, as determined by their

Zinc-Dependent Protection of Tobacco and Rice Cells From Aluminum-Induced Superoxide-Mediated Cytotoxicity

Science.gov (United States)

Lin, Cun; Hara, Ayaka; Comparini, Diego; Bouteau, François; Kawano, Tomonori

2015-01-01

Al3+ toxicity in growing plants is considered as one of the major factors limiting the production of crops on acidic soils worldwide. In the last 15 years, it has been proposed that Al3+ toxicity are mediated with distortion of the cellular signaling mechanisms such as calcium signaling pathways, and production of cytotoxic reactive oxygen species (ROS) causing oxidative damages. On the other hand, zinc is normally present in plants at high concentrations and its deficiency is one of the most widespread micronutrient deficiencies in plants. Earlier studies suggested that lack of zinc often results in ROS-mediated oxidative damage to plant cells. Previously, inhibitory action of Zn2+ against lanthanide-induced superoxide generation in tobacco cells have been reported, suggesting that Zn2+ interferes with the cation-induced ROS production via stimulation of NADPH oxidase. In the present study, the effect of Zn2+ on Al3+-induced superoxide generation in the cell suspension cultures of tobacco (Nicotiana tabacum L., cell-line, BY-2) and rice (Oryza sativa L., cv. Nipponbare), was examined. The Zn2+-dependent inhibition of the Al3+-induced oxidative burst was observed in both model cells selected from the monocots and dicots (rice and tobacco), suggesting that this phenomenon (Al3+/Zn2+ interaction) can be preserved in higher plants. Subsequently induced cell death in tobacco cells was analyzed by lethal cell staining with Evans blue. Obtained results indicated that presence of Zn2+ at physiological concentrations can protect the cells by preventing the Al3+-induced superoxide generation and cell death. Furthermore, the regulation of the Ca2+ signaling, i.e., change in the cytosolic Ca2+ ion concentration, and the cross-talks among the elements which participate in the pathway were further explored. PMID:26648960

Evidence for a Ustilago maydis Steroid 5α-Reductase by Functional Expression in Arabidopsis det2-1 Mutants1

Science.gov (United States)

Basse, Christoph W.; Kerschbamer, Christine; Brustmann, Markus; Altmann, Thomas; Kahmann, Regine

2002-01-01

We have identified a gene (udh1) in the basidiomycete Ustilago maydis that is induced during the parasitic interaction with its host plant maize (Zea mays). udh1 encodes a protein with high similarity to mammalian and plant 5α-steroid reductases. Udh1 differs from those of known 5α-steroid reductases by six additional domains, partially predicted to be membrane-spanning. A fusion protein of Udh1 and the green fluorescent protein provided evidence for endoplasmic reticulum localization in U. maydis. The function of the Udh1 protein was demonstrated by complementing Arabidopsis det2-1 mutants, which display a dwarf phenotype due to a mutation in the 5α-steroid reductase encoding DET2 gene. det2-1 mutant plants expressing either the udh1 or the DET2 gene controlled by the cauliflower mosaic virus 35S promoter differed from wild-type Columbia plants by accelerated stem growth, flower and seed development and a reduction in size and number of rosette leaves. The accelerated growth phenotype of udh1 transgenic plants was stably inherited and was favored under reduced light conditions. Truncation of the N-terminal 70 amino acids of the Udh1 protein abolished the ability to restore growth in det2-1 plants. Our results demonstrate the existence of a 5α-steroid reductase encoding gene in fungi and suggest a common ancestor between fungal, plant, and mammalian proteins. PMID:12068114

Inhibition of oxygen-dependent radiation-induced damage by the nitroxide superoxide dismutase mimic, tempol

International Nuclear Information System (INIS)

Mitchell, J.B.; DeGraff, W.; Kaufman, D.; Krishna, M.C.; Samuni, A.; Finkelstein, E.; Ahn, M.S.; Hahn, S.M.; Gamson, J.; Russo, A.

1991-01-01

Stable nitroxide radicals have been previously shown to function as superoxide dismutase (SOD)2 mimics and to protect mammalian cells against superoxide and hydrogen peroxide-mediated oxidative stress. These unique characteristics suggested that nitroxides, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), might protect mammalian cells against ionizing radiation. Treating Chinese hamster cells under aerobic conditions with 5, 10, 50, and 100 mM Tempol 10 min prior to X-rays resulted in radiation protection factors of 1.25, 1.30, 2.1, and 2.5, respectively. However, the reduced form of Tempol afforded no protection. Tempol treatment under hypoxic conditions did not provide radioprotection. Aerobic X-ray protection by Tempol could not be attributed to the induction of intracellular hypoxia, increase in intracellular glutathione, or induction of intracellular SOD mRNA. Tempol thus represents a new class of non-thiol-containing radiation protectors, which may be useful in elucidating the mechanism(s) of radiation-induced cellular damage and may have broad applications in protecting against oxidative stress

Simultaneous determination of superoxide and hydrogen peroxide in macrophage RAW 264.7 cell extracts by microchip electrophoresis with laser-induced fluorescence detection.

Science.gov (United States)

Li, Hongmin; Li, Qingling; Wang, Xu; Xu, Kehua; Chen, Zhenzhen; Gong, Xiaocong; Liu, Xin; Tong, Lili; Tang, Bo

2009-03-15

A method for the first time to simultaneously determine superoxide and hydrogen peroxide in macrophage RAW 264.7 cell extracts by microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF) was developed. 2-Chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) and bis(p-methylbenzenesulfonyl) dichlorofluorescein (FS), two probes that can be specifically derivatized by superoxide and hydrogen peroxide, respectively, were synthesized and used. Parameters influencing the derivatization and on-chip separation were optimized. With the use of a HEPES (20 mM, pH 7.4) running buffer, a 50 mm long separation channel, and a separation voltage of 1800 V, baseline separation was achieved within 48 s for the two derivatization products, DBZTC-oxide (DBO) and 2,7-dichlorofluorescein (DCF). The linearity ranges of the method were 0.08-5.0 and 0.02-5.0 microM with detection limits (signal-to-noise ratio = 3) of 10 nM (1.36 amol) and 5.6 nM (0.76 amol) for superoxide and hydrogen peroxide, respectively. The relative standard deviations (RSDs) of migration time and peak area were less than 2.0% and 5.0%, respectively. The recoveries of the cell extract samples spiked with 1.0 microM standard solutions were 96.1% and 93.0% for superoxide and hydrogen peroxide, respectively. With the use of this method, superoxide and hydrogen peroxide in phorbol myristate acetate (PMA)-stimulated macrophage RAW 264.7 cell extracts were found to be 0.78 and 1.14 microM, respectively. The method has paved a way for simultaneously determining two or more reactive oxygen species (ROS) in a biological system with high resolution.

Purification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolae

International Nuclear Information System (INIS)

Gazdag, Emerich Mihai; Cirstea, Ion Cristian; Breitling, Reinhard; Lukeš, Julius; Blankenfeldt, Wulf; Alexandrov, Kirill

2010-01-01

The structures of two new crystal forms of human Cu/Zn superoxide dismutase produced in the eukaryotic expression host L. tarentolae are reported. The rapid and inexpensive production of high-quality eukaryotic proteins in recombinant form still remains a challenge in structural biology. Here, a protein-expression system based on the protozoan Leishmania tarentolae was used to produce human Cu/Zn superoxide dismutase (SOD1) in recombinant form. Sequential integration of the SOD1 expression cassettes was demonstrated to lead to a linear increase in expression levels to up to 30 mg per litre. Chromatographic purification resulted in 90% pure recombinant protein, with a final yield of 6.5 mg per litre of culture. The protein was crystallized and the structures of two new crystal forms were determined. These results demonstrate the suitability of the L. tarentolae expression system for structural research

Neutrophil superoxide-anion generating capacity in chronic smoking: effect of long-term alpha-tocopherol therapy

NARCIS (Netherlands)

Tits, van L.; Waart, de F.; Hak-Lemmers, H.L.M.; Graaf, de J.; Demacker, P.N.; Stalenhoef, A.F.

2003-01-01

We investigated whether long-term alpha-tocopherol therapy in chronic smoking affects superoxide generating capacity of neutrophils ex vivo. To this purpose, we randomly assigned 128 male chronic smokers (37 21 pack years of smoking) to treatment with placebo (n = 64) or alpha-tocopherol (400 IU

Brain superoxide anion formation in immature rats during seizures: Protection by selected compounds

Czech Academy of Sciences Publication Activity Database

Folbergrová, Jaroslava; Otáhal, Jakub; Druga, Rastislav

2012-01-01

Roč. 233, č. 1 (2012), s. 421-429 ISSN 0014-4886 R&D Projects: GA ČR GA309/08/0292; GA ČR GAP303/10/0999 Institutional research plan: CEZ:AV0Z50110509 Keywords : immature rats * DL-homocysteic acid-induced seizures * superoxide anion * SOD mimetics * protection * Fluoro-Jade B staining * brain damage Subject RIV: FH - Neurology Impact factor: 4.645, year: 2012

Evidence that the intra-amoebal Legionella drancourtii acquired a sterol reductase gene from eukaryotes

Directory of Open Access Journals (Sweden)

Fournier Pierre-Edouard

2009-03-01

Full Text Available Abstract Background Free-living amoebae serve as a natural reservoir for some bacteria that have evolved into «amoeba-resistant» bacteria. Among these, some are strictly intra-amoebal, such as Candidatus "Protochlamydia amoebophila" (Candidatus "P. amoebophila", whose genomic sequence is available. We sequenced the genome of Legionella drancourtii (L. drancourtii, another recently described intra-amoebal bacterium. By comparing these two genomes with those of their closely related species, we were able to study the genetic characteristics specific to their amoebal lifestyle. Findings We identified a sterol delta-7 reductase-encoding gene common to these two bacteria and absent in their relatives. This gene encodes an enzyme which catalyses the last step of cholesterol biosynthesis in eukaryotes, and is probably functional within L. drancourtii since it is transcribed. The phylogenetic analysis of this protein suggests that it was acquired horizontally by a few bacteria from viridiplantae. This gene was also found in the Acanthamoeba polyphaga Mimivirus genome, a virus that grows in amoebae and possesses the largest viral genome known to date. Conclusion L. drancourtii acquired a sterol delta-7 reductase-encoding gene of viridiplantae origin. The most parsimonious hypothesis is that this gene was initially acquired by a Chlamydiales ancestor parasite of plants. Subsequently, its descendents transmitted this gene in amoebae to other intra-amoebal microorganisms, including L. drancourtii and Coxiella burnetii. The role of the sterol delta-7 reductase in prokaryotes is as yet unknown but we speculate that it is involved in host cholesterol parasitism.

Acinetobacter calcoaceticus CSY-P13 Mitigates Stress of Ferulic and p-Hydroxybenzoic Acids in Cucumber by Affecting Antioxidant Enzyme Activity and Soil Bacterial Community

Directory of Open Access Journals (Sweden)

Fenghui Wu

2018-06-01

Full Text Available Ferulic acid (FA and p-hydroxybenzoic acid (PHBA are main phenolic compounds accumulated in rhizosphere of continuously cropped cucumber, causing stress in plants. Microbial degradation of a mixture of FA and PHBA is not well understood in soil. We isolated a strain CSY-P13 of Acinetobacter calcoaceticus, inoculated it into soil to protect cucumber from FA and PHBA stress, and explored a mechanism underlying the protection. CSY-P13 effectively degraded a mixture of FA and PHBA in culture solution under conditions of 39.37°C, pH 6.97, and 21.59 g L-1 potassium dihydrogen phosphate, giving rise to 4-vinyl guaiacol, vanillin, vanillic acid, and protocatechuic acid. During FA and PHBA degradation, activities of superoxide dismutase (SOD, catalase, ascorbate peroxidase, and dehydroascorbate reductase in CSY-P13 were induced. Inoculated into cucumber-planted soil containing 220 μg g-1 mixture of FA and PHBA, CSY-P13 degraded FA and PHBA in soil, increased plant height, and decreased malonaldehyde, superoxide radical, and hydrogen peroxide levels in leaves. CSY-P13 also enhanced SOD, guaiacol peroxidase, catalase, glutathione peroxidase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase activities; increased ascorbate and glutathione contents; and elevated transcript levels of copper/zinc SOD, manganese SOD, and catalase in leaves under FA and PHBA. Moreover, CSY-P13 increased phosphatase, catalase, urease, and sucrase activities and changed bacterial richness, diversity, and community composition by high throughput sequencing in cucumber-planted soil supplemented with the mixture of FA and PHBA. So CSY-P13 degrades the mixture of FA and PHBA in soil and mitigates stress from the two phenolic compounds in cucumber by activating antioxidant enzymes, changing soil bacterial community, and inducing soil enzymes.

Cytoplasmic superoxide dismutase and catalase activity and resistance to radiation lethality in murine tumor cells

International Nuclear Information System (INIS)

Davy, C.A.; Tesfay, Z.; Jones, J.; Rosenberg, R.C.; McCarthy, C.; Rosenberg, S.O.

1986-01-01

Reduced species of molecular oxygen are produced by the interaction of ionizing radiation with aqueous solutions containing molecular oxygen. The enzymes catalase and superoxide dismutase (SOD) are thought to function in vivo as scavengers of metabolically produced peroxide and superoxide respectively. SOD has been shown to protect against the lethal effects of ionizing radiation in vitro and in vivo. The authors have investigated the relationship between the cytosolic SOD catalase content and the sensitivity to radiation lethality of a number of murine cell lines (402AX, EL-4, MB-2T3, MB-4, MEL, P-815, SAI, SP-2, and SV-3T3). K/sub i/(CN - ) for murine Cu-Zn-SOD was determined to be 6.8 x 10 -6 M. No cytosolic Mn-SOD activity was found in any of the cell lines studied. No correlation was found between the cytosolic Cu-Zn-SOD or cytosolic catalase activity and the resistance to radiation lethality or the murine cell lines studied

Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

DEFF Research Database (Denmark)

Chen, Z. W.; Jiang, C. Y.; She, Qunxin

2005-01-01

). The thio-modifying reagent N-ethylmaleimide and Zn2+ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant......Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system...... proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have...

Study on the Protective Effect of a New Manganese Superoxide Dismutase on the Microvilli of Rabbit Eyes Exposed to UV Radiation

OpenAIRE

Grumetto, Lucia; Del Prete, Antonio; Ortosecco, Giovanni; Barbato, Francesco; Del Prete, Salvatore; Borrelli, Antonella; Schiattarella, Antonella; Mancini, Roberto; Mancini, Aldo

2015-01-01

We present a study on the protective effects against UV radiation of a gel formulation containing a new recombinant form of manganese superoxide dismutase on the conjunctiva and corneal epithelia of rabbit eyes. The integrity of the microvilli of both ocular tissues has been considered as an indicator of the health of the tissues. Samples, collected by impression cytology technique, were added of 80??L of a gel formulation containing superoxide dismutase (2.0??g/mL) and irradiated with UV ray...

Pharmacogenetics of aldo-keto reductase 1C (AKR1C) enzymes.

Science.gov (United States)

Alshogran, Osama Y

2017-10-01

Genetic variation in metabolizing enzymes contributes to variable drug response and disease risk. Aldo-keto reductase type 1C (AKR1C) comprises a sub-family of reductase enzymes that play critical roles in the biotransformation of various drug substrates and endogenous compounds such as steroids. Several single nucleotide polymorphisms have been reported among AKR1C encoding genes, which may affect the functional expression of the enzymes. Areas covered: This review highlights and comprehensively discusses previous pharmacogenetic reports that have examined genetic variations in AKR1C and their association with disease development, drug disposition, and therapeutic outcomes. The article also provides information about the effect of AKR1C genetic variants on enzyme function in vitro. Expert opinion: The current evidence that links the effect of AKR1C gene polymorphisms to disease progression and development is inconsistent and needs further validation, despite of the tremendous knowledge available. Information about association of AKR1C genetic variants and drug efficacy, safety, and pharmacokinetics is limited, thus, future studies that advance our understanding about these relationships and their clinical relevance are needed. It is imperative to achieve consistent findings before the potential translation and adoption of AKR1C genetic variants in clinical practice.

Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector

OpenAIRE

Peluffo, Hugo; Acarin, Laia; Arís, Anna; González, Pau; Villaverde, Antoni; Castellano, Bernardo; González, Berta

2006-01-01

Abstract Background Superoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD) after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn S...

Evaluation of constitutive iron reductase (AtFRO2 expression on mineral accumulation and distribution in soybean (Glycine max. L

Directory of Open Access Journals (Sweden)

Marta Wilton Vasconcelos

2014-04-01

Full Text Available Iron is an important micronutrient in human and plant nutrition. Adequate iron nutrition during crop production is central for assuring appropriate iron concentrations in the harvestable organs, for human food or animal feed. The whole-plant movement of iron involves several processes, including the reduction of ferric to ferrous iron at several locations throughout the plant, prior to transmembrane trafficking of ferrous iron. In this study, soybean plants that constitutively expressed the AtFRO2 iron reductase gene were analyzed for leaf iron reductase activity, as well as the effect of this transgene's expression on root, leaf, pod wall, and seed mineral concentrations. High Fe supply, in combination with the constitutive expression of AtFRO2, resulted in significantly higher concentrations of different minerals in roots (K, P, Zn, Ca, Ni, Mg and Mo, pod walls (Fe, K, P, Cu and Ni, leaves (Fe, P, Cu, Ca, Ni and Mg and seeds (Fe, Zn, Cu and Ni. Leaf and pod wall iron concentrations increased as much as 500% in transgenic plants, while seed iron concentrations only increased by 10%, suggesting that factors other than leaf and pod wall reductase activity were limiting the translocation of iron to seeds. Protoplasts isolated from transgenic leaves had three-fold higher reductase activity than controls. Expression levels of the iron storage protein, ferritin, were higher in the transgenic leaves than in wild-type, suggesting that the excess iron may be stored as ferritin in the leaves and therefore unavailable for phloem loading and delivery to the seeds. Also, citrate and malate levels in the roots and leaves of transgenic plants were significantly higher than in wild-type, suggesting that organic acid production could be related to the increased accumulation of minerals in roots, leaves and pod walls, but not in the seeds. All together, these results suggest a more ubiquitous role for the iron reductase in whole-plant mineral accumulation and

The aldo-keto reductase AKR1B7 coexpresses with renin without influencing renin production and secretion.

Science.gov (United States)

Machura, Katharina; Iankilevitch, Elina; Neubauer, Björn; Theuring, Franz; Kurtz, Armin

2013-03-01

On the basis of evidence that within the adult kidney, the aldo-keto reductase AKR1B7 (aldo-keto reductase family 1, member 7, also known as mouse vas deferens protein, MVDP) is selectively expressed in renin-producing cells, we aimed to define a possible role of AKR1B7 for the regulation and function of renin cells in the kidney. We could confirm colocalization and corecruitment of renin and of AKR1B7 in wild-type kidneys. Renin cells in AKR1B7-deficient kidneys showed normal morphology, numbers, and intrarenal distribution. Plasma renin concentration (PRC) and renin mRNA levels of AKR1B7-deficient mice were normal at standard chow and were lowered by a high-salt diet directly comparable to wild-type mice. Treatment with a low-salt diet in combination with an angiotensin-converting enzyme inhibitor strongly increased PRC and renin mRNA in a similar fashion both in AKR1B7-deficient and wild-type mice. Under this condition, we also observed a strong retrograde recruitment of renin-expressing cell along the preglomerular vessels, however, without a difference between AKR1B7-deficient and wild-type mice. The isolated perfused mouse kidney model was used to study the acute regulation of renin secretion by ANG II and by perfusion pressure. Regarding these parameters, no differences were observed between AKR1B7-deficient and wild-type kidneys. In summary, our data suggest that AKR1B7 is not of major relevance for the regulation of renin production and secretion in spite of its striking coregulation with renin expression.

A novel mercuric reductase from the unique deep brine environment of atlantis II in the red sea

KAUST Repository

Sayed, Ahmed Anazadeh

2013-11-26

Aunique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg2+, and efficiently detoxifies Hg2+ in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A novel mercuric reductase from the unique deep brine environment of atlantis II in the red sea

KAUST Repository

Sayed, Ahmed Anazadeh; Ghazy, Mohamed A.; Ferreira, Ari José Scattone; Setú bal, Joã o Carlos; Chambergo, Felipe Santiago; Ouf, Amged; Adel, Mustafa; Dawe, Adam Sean; Archer, John A.C.; Bajic, Vladimir B.; Siam, Rania; El-Dorry, Hamza A A

2013-01-01

Aunique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg2+, and efficiently detoxifies Hg2+ in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification of a NAD(P) reductase-like protein from the thermogenic appendix of the Sauromatum guttatum inflorescence.

Science.gov (United States)

Skubatz, Hanna; Howald, William N

2013-03-01

A NAD(P) reductase-like protein with a molecular mass of 34.146 ± 34 Da was purified to homogeneity from the appendix of the inflorescence of the Sauromatum guttatum. On-line liquid chromatography/electrospray ionization-mass spectrometry was used to isolate and quantify the protein. For the identification of the protein, liquid chromatography/electrospray ionization-tandem mass spectrometry analysis of tryptic digests of the protein was carried out. The acquired mass spectra were used for database searching, which led to the identification of a single tryptic peptide. The 12 amino acid tryptic peptide (FLPSEFGNDVDR) was found to be identical to amino acid residues at the positions 108-120 of isoflavone reductase in the Arabidopsis genome. A BLAST search identified this sequence region as unique and specific to a class of NAD(P)-dependent reductases involved in phenylpropanoid biosynthesis. Edman degradation revealed that the protein was N-terminally blocked. The amount of the protein (termed RL, NAD(P) reductase-like protein) increased 60-fold from D-4 (4 days before inflorescence-opening, designated as D-day) to D-Day, and declined the following day, when heat-production ceased. When salicylic acid, the endogenous trigger of heat-production in the Sauromatum appendix, was applied to premature appendices, a fivefold decrease in the amount of RL was detected in the treated section relative to the non-treated section. About 40 % of RL was found in the cytoplasm. Another 30 % was detected in Percoll-purified mitochondria and the rest, about 30 % was associated with a low speed centrifugation pellet due to nuclei and amyloplast localization. RL was also found in other thermogenic plants and detected in Arabidopsis leaves. The function of RL in thermogenic and non-thermogenic plants requires further investigation.

Sex hormones reduce NNK detoxification through inhibition of short-chain dehydrogenases/reductases and aldo-keto reductases in vitro.

Science.gov (United States)

Stapelfeld, Claudia; Maser, Edmund

2017-10-01

Carbonyl reduction is an important metabolic pathway for endogenous and xenobiotic substances. The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, nicotine-derived nitrosamine ketone) is classified as carcinogenic to humans (IARC, Group 1) and considered to play the most important role in tobacco-related lung carcinogenesis. Detoxification of NNK through carbonyl reduction is catalyzed by members of the AKR- and the SDR-superfamilies which include AKR1B10, AKR1C1, AKR1C2, AKR1C4, 11β-HSD1 and CBR1. Because some reductases are also involved in steroid metabolism, five different hormones were tested for their inhibitory effect on NNK carbonyl reduction. Two of those hormones were estrogens (estradiol and ethinylestradiol), another two hormones belong to the gestagen group (progesterone and drospirenone) and the last tested hormone was an androgen (testosterone). Furthermore, one of the estrogens (ethinylestradiol) and one of the gestagens (drospirenone) are synthetic hormones, used as hormonal contraceptives. Five of six NNK reducing enzymes (AKR1B10, AKR1C1, AKR1C2, AKR1C4 and 11β-HSD1) were significantly inhibited by the tested sex hormones. Only NNK reduction catalyzed by CBR1 was not significantly impaired. In the case of the other five reductases, gestagens had remarkably stronger inhibitory effects at a concentration of 25 μM (progesterone: 66-88% inhibition; drospirenone: 26-87% inhibition) in comparison to estrogens (estradiol: 17-51% inhibition; ethinylestradiol: 14-79% inhibition) and androgens (14-78% inhibition). Moreover, in most cases the synthetic hormones showed a greater ability to inhibit NNK reduction than the physiologic derivatives. These results demonstrate that male and female sex hormones have different inhibitory potentials, thus indicating that there is a varying detoxification capacity of NNK in men and women which could result in a different risk for developing lung cancer. Copyright © 2017 Elsevier B